HK1232143B - Bioactive renal cells - Google Patents

Description

Bioactive kidney cells

This application is a divisional application based on a patent application with an application date of May 12, 2011, a priority date of May 12, 2010, an application number of 201180034688.X, and an invention name of “Bioactive Renal Cells”.

Field of the Invention

The present invention relates to bioactive renal cell populations or fractions that lack cellular components compared to healthy individuals but still retain therapeutic properties, methods for isolating and culturing said cell populations, and methods for using said cell populations to treat subjects in need thereof. In addition, the present invention relates to methods for using said bioactive renal cell populations to provide regenerative effects to native kidneys.

Background of the Invention

Chronic kidney disease (CKD) affects more than 19 million people in the United States and is often the result of metabolic disorders involving obesity, diabetes, and hypertension. Data surveys show that the increasing rate is attributed to the development of renal failure secondary to hypertension and non-insulin-dependent diabetes mellitus (NIDDM), two diseases that are also on the rise worldwide (United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda, MD, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007 pp 223-238). Obesity, hypertension, and poor glycemic control have all been shown to be independent risk factors for renal damage, causing glomerular and tubular damage and leading to proteinuria and other systemic detectable changes in renal filtration function (Aboushwareb, et al., World J Urol, 26: 295-300, 2008; Amann, K. et al., Nephrol Dial Transplant, 13: 1958-66, 1998). The CKD patient of 1-3 phase of progress of the drug intervention management that aims at controlling potential disease state by the change of life style and purpose, yet manage the patient of stage 4-5 by dialysis and the dosage regimen that generally comprises antihypertensive drug, erythropoiesis stimulating agent (ESA), iron and vitamin D supplement.Regenerative medicine technology can provide the next generation treatment selection of CKD.Presnell et al. WO/2010/056328 describes the nephrocyte of separation, comprises that renal tubular cell and erythropoietin (EPO) generate nephrocyte colony, and the method for separating and culturing described cell, and utilizes described cell colony treatment to have the method for experimenter of this need.There is the needs of the novel treatment mode of significantly and lasting enhancing to slow down progress and improve the quality of life of this patient colony to providing renal function.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for providing regeneration to autologous kidney. In one embodiment, the method comprises the step of contacting autologous kidney with a nephrocyte colony secretory product in vivo by enrichment. In another embodiment, the product is secreted by a nephrocyte colony that is not part of the enrichment of the construct, as described herein, for example, without the cell colony being seeded on a support. In another embodiment, the product is secreted from a nephrocyte construct comprising a nephrocyte colony of the enrichment that is directly seeded on or in the support. In another embodiment, the secretion of the product is a biological response to oxygen levels. Secretion can be induced by being lower than atmospheric oxygen levels. In another embodiment, lower oxygen levels are lower than approximately 5% oxygen.

In one embodiment, regeneration is the minimizing of epithelial-mesenchymal transition (EMT). The minimizing of EMT can be achieved by weakening TGF-β signal transduction and/or weakening plasminogen activator inhibitor-1 (PAI-1) signal transduction. In another embodiment, regeneration is the minimizing of renal fibrosis and/or the mitigation of nephritis. In some embodiments, the mitigation of inflammation can be mediated by NFκB. In another embodiment, regeneration is characterized by the differential expression of stem cell markers in autologous kidney. Expression can be the increase of marker expression in autologous kidney contacted in vivo relative to non-contacted autologous kidney.

In one aspect, the nephrocyte colony of enrichment includes one or more cell colonies, i.e. mixture, as described above. In one embodiment, colony includes the first cell colony B2, which includes the renal tubular cell colony of enrichment. In another embodiment, colony includes a mixture of the human nephrocyte with the first cell colony B2 and the second cell colony, and the second cell colony includes one or more erythropoietin (EPO) founder cells, glomerular cells and vascular cells. In one other embodiment, the second cell colony is a B4 cell colony. In another embodiment, the second cell colony is a B3 cell colony.

In one embodiment, mixture also includes the 3rd cell colony with one or more erythropoietin (EPO) founder cells, glomerular cells and vascular cells. In another embodiment, described 3rd cell colony is B4 cell colony. In another embodiment, described 3rd cell colony is B3 cell colony.

In all embodiments, the B2 cell population has a density of about 1.045 g/mL to about 1.052 g/mL. In all embodiments, the B4 cell population has a density of about 1.063 g/mL to about 1.091 g/mL. In all embodiments, the B3 cell population has a density of about 1.052 g/ml to about 1.063 g/ml.

In all embodiments, the enriched renal cell population may be non-autologous to the native kidney. In all embodiments, the enriched renal cell population may be non-autologous to the native kidney.

In all embodiments, the products include paracrine factors, endocrine factors, juxtacrine factors, RNA, vesicles, microvesicles, exosomes, and any combination thereof. In one other embodiment, the vesicles include one or more secreted products selected from paracrine factors, endocrine factors, juxtacrine factors, and RNA. In another embodiment, the products are secreted from a renal cell construct comprising an enriched renal cell population seeded directly on or in a scaffold.

In all embodiments, the scaffold may comprise a biocompatible material. In all embodiments, the biocompatible material may be a hydrogel.

In one embodiment, the present invention provides a method for assessing whether a patient with kidney disease (KD) is responsive to treatment with a therapeutic agent. The method may include the step of determining or detecting the amount of vesicles or their luminal contents in a test sample obtained from a KD patient treated with the therapeutic agent, as compared to or relative to the amount of vesicles in a control sample, wherein a higher or lower amount of vesicles or their luminal contents in the test sample compared to the amount of vesicles or their luminal contents in the control sample indicates that the treated patient is responsive to treatment with the therapeutic agent. The vesicles may be of kidney origin. The test sample may comprise urine. The vesicles may comprise a biomarker, which may be a miRNA. The therapeutic agent may comprise an enriched renal cell population.

Specifically, the present invention includes the following embodiments:

Embodiment 1. A method of providing a regenerative effect to an autologous kidney, the method comprising contacting the autologous kidney in vivo with a product secreted by an enriched population of renal cells.

Embodiment 2. The method of embodiment 1, wherein the product comprises a paracrine factor.

Embodiment 3. The method of embodiment 1, wherein the product comprises an endocrine factor.

Embodiment 4. The method of embodiment 1, wherein the product comprises a juxtacrine factor.

Embodiment 5. The method of embodiment 1, wherein the product comprises vesicles.

Embodiment 6. The method of embodiment 5, wherein the vesicles comprise microvesicles.

Embodiment 7. The method of embodiment 5, wherein the vesicles comprise exosomes.

Embodiment 8. The method of any one of embodiments 5-7, wherein the vesicles comprise secretory products selected from paracrine factors, endocrine factors, juxtacrine factors, and RNA.

Embodiment 9. The method of embodiment 1, wherein the product is secreted by a renal cell construct comprising an enriched renal cell population seeded directly on or in a scaffold.

Embodiment 10. The method of embodiment 9, wherein the scaffold comprises a biocompatible material.

Embodiment 11. The method of embodiment 10, wherein the biocompatible material is a hydrogel.

Embodiment 12. The method of embodiment 1 or 9, wherein secretion of the product is a biological response to oxygen levels.

Embodiment 13. The method of embodiment 12, wherein secretion is induced by subatmospheric oxygen levels.

Embodiment 14. The method of embodiment 13, wherein the lower oxygen level is less than about 5% oxygen.

Embodiment 15. The method of embodiment 1 or 9, wherein the regenerative effect is a reduction in epithelial-mesenchymal transition (EMT).

Embodiment 16. The method of embodiment 15, wherein the reduction of epithelial-mesenchymal transition (EMT) is performed by attenuating TGF-β signaling.

Embodiment 17. The method of embodiment 1 or 9, wherein the regenerative effect is a reduction in renal fibrosis and/or a reduction in nephritis.

Embodiment 18. The method of embodiment 1 or 9, wherein the regeneration is characterized by differential expression of stem cell markers in the native kidney.

Embodiment 19. A method according to embodiment 1 or 9, wherein the population comprises a first cell population B2 comprising an enriched population of tubular cells.

Embodiment 20. A method according to embodiment 1 or 9, wherein the population comprises a mixture of human kidney cells comprising a first cell population B2 and a second cell population, wherein B2 comprises a population enriched for tubular cells and wherein the second cell population comprises one or more of erythropoietin (EPO)-producing cells, glomerular cells and vascular cells.

Embodiment 21. The method of embodiment 19 or 20, wherein the B2 cell population has a density of about 1.045 g/mL to about 1.052 g/mL.

Embodiment 22. A method according to embodiment 20, wherein the second cell population is a B4 cell population having a density of about 1.063 g/mL to about 1.091 g/mL.

Embodiment 23. A method according to embodiment 20, wherein the second cell population is a B3 cell population having a density of about 1.052 g/ml to about 1.063 g/ml.

Embodiment 24. A method according to any one of embodiments 21-23, wherein the mixture further comprises a third cell population, wherein the third cell population comprises one or more of erythropoietin (EPO)-producing cells, glomerular cells, and vascular cells.

Embodiment 25. The method of embodiment 24, wherein the third population is a B4 cell population having a density of about 1.063 g/mL to about 1.091 g/mL.

Embodiment 26. A method according to embodiment 24, wherein the third cell population is a B3 cell population having a density of about 1.052 g/ml to about 1.063 g/ml.

Embodiment 27. A method according to embodiment 1 or 9, wherein the enriched renal cell population is non-autologous to the native kidney.

Embodiment 28. A method according to embodiment 1 or 9, wherein the enriched renal cell population is autologous to the native kidney.

Embodiment 29. A method for assessing whether a kidney disease (KD) patient responds to treatment with a therapeutic agent, the method comprising detecting the amount of vesicles or their luminal contents in a test sample obtained from a KD patient treated with the therapeutic agent, compared to or relative to the amount of vesicles or their luminal contents in a control sample, wherein a higher or lower amount of vesicles or their luminal contents in the test sample compared to the amount of vesicles or their luminal contents in the control sample indicates a response of the patient to treatment with the therapeutic agent.

Embodiment 30. The method of embodiment 29, wherein the vesicles are of kidney origin.

Embodiment 31. The method of embodiment 29 or 30, wherein the vesicles comprise a biomarker.

Embodiment 32. The method of embodiment 31, wherein the biomarker is miRNA.

Embodiment 33. A method according to embodiment 29, wherein the therapeutic agent comprises an enriched population of renal cells.

Embodiment 34. The method of embodiment 29, wherein the test sample comprises urine.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the enrichment of erythropoietin-producing cell fractions from freshly isolated kidney tissue using either multilayer discontinuous gradient technology (A-left panel) or single layer mixed gradient technology (B-right panel). Both methods resulted in the partial depletion of non-erythropoietin-producing cell components (primarily renal tubular cells) from the erythropoietin band, which was expressed as 1.025 g/mL to 1.035 g/mL.

Figure 2 shows step gradients of "normoxic" (21% oxygen) and "hypoxic" (2% oxygen) rodent cultures harvested separately and applied in parallel to the same step gradient.

Figure 3 shows step gradients of "normoxic" (21% oxygen) and "anoxic" (2% oxygen) canine cultures harvested separately and used in parallel on the same step gradient.

FIG4 shows the histopathological characteristics of HK17 and HK19 samples.

Figure 5 shows high content analysis (HCA) of albumin transport in human NKA cells defining regions of interest (ROI).

FIG6 shows a quantitative comparison of albumin transport by NKA cells derived from non-CKD and CKD kidneys.

FIG. 7 depicts comparative analysis of marker expression between the B2 subfraction, which is enriched for tubular cells, and the B4 subfraction, which is depleted of tubular cells.

FIG8 depicts comparative functional analysis between the B2 subfraction enriched in tubular cells and the B4 subfraction depleted in tubular cells.

FIG9 shows the expression of SOX2 mRNA in host tissues after treatment of 5/6NX rats.

FIG10 is a Western blot showing the time course of expression of CD24, CD133, UTF1, SOX2, NODAL, and LEFTY.

FIG11 depicts the time course of the regenerative response index (RRI).

FIG12 provides a schematic diagram of the preparation and analysis of UNFX-conditioned medium.

Figures 13A-C show that conditioned medium from UNFX cultures affects multiple cellular processes in vitro that are potentially relevant to regenerative outcomes. Figure 13A shows that UNFX-conditioned medium attenuates TNF-α-mediated NF-κB activation. Figure 13B shows that UNFX-conditioned medium enhances the pro-angiogenic behavior of HUVEC cell cultures. Figure 13C shows that UNFX-conditioned medium attenuates the fibrotic pathway of epithelial cells. Figure 13D depicts the positive feedback loop established by TGFβ1 and plasminogen activator inhibitor-1 (PAI-1).

14A-B show Western blot analysis demonstrating attenuation of fibrotic pathways in mesangial cells.

Figures 15A-C show that conditioned medium from UNFX contains secreted vesicles. Figure 15A depicts secreted vesicles, which are bilipid structures (red) containing cytoplasmic-derived internal components (green). Figures 15B-C show FACS sorting.

Figure 16A shows a Western blot where total protein was prepared and assayed for PAI-1 and b-actin. Figure 16B depicts the microRNA, miR-30b-5p.

Figure 17A-C is presented at the representative immunohistochemical image of PAI-1 in Lewis rat kidney after delivering bioactive nephrocytes after experience nephrectomy.Figure 17D shows the comparison of PAI-1 expression in untreated nephrectomized rats (red squares), nephrectomized rats (blue circles) and control animals (green triangles).Figure 17E shows the representative Western blot analysis of kidney samples collected 3 and 6 months after treatment.Figure 17F is presented at the nuclear localization that reduces NFκB p65 in 2 hours of exposure of NKA conditioned medium.Figure 17G describes the classical activation of TNFα to NFkB pathway.

Figures 18A-B show nuclear localization of the NFκB p65 subunit in animals with (A) progressive CKD caused by 5/6 nephrectomy and (B) non-progressive renal insufficiency caused by unilateral nephrectomy. Figures 18C-D show (C) Western blot analysis of NFκB p65 in extracts of kidney tissue from Lewis rats that have undergone 5/6 nephrectomy; and (D) electrophoretic mobility shift assay (EMSA) on the extracts. Figure 18E shows immunohistochemical detection of the NFκB p65 subunit in tissue obtained from Lewis rats with established CKD that received intrarenal injections of NKA (Panel A) or non-bioactive renal cells (Panel B).

19A-C show in vivo evaluation of the biomaterial at 1 and 4 weeks post-implantation.

Figures 20A-D show live/dead staining of NKA constructs. Figures 20E-G show transcriptomic profiling characterization of NKA constructs.

Figures 21A-B show secretomic profiling of NKA constructs.

Figures 22A-B show proteomic profiling characterization of NKA constructs.

Figures 23A-C show confocal microscopy of NKA constructs.

24A-B show in vivo evaluation of NKA Constructs at 1 and 4 weeks post-implantation.

Figures 25A-D show in vivo evaluation of NKA Constructs 8 weeks after implantation.

FIG26 shows that conditioned medium from NKA constructs attenuates TGF-β-induced EMT of HK2 cells in vitro.

FIG. 27 depicts a method for exposing cells to hypoxia during treatment.

Figure 28 shows that after exposure to 2% oxygen, the following phenomena were observed: altered distribution of cells across the density gradient and increased overall post-gradient yield.

Figure 29A depicts an assay developed to visualize in vitro repair of renal tubular monolayers. Figure 29B shows the results of quantitative image analysis (BD Pathway 855 BioImager). Figure 29C shows that cells induced by 2% oxygen are more specialized for repair of renal tubular epithelial monolayers.

Figure 30A depicts an assay developed to observe in vitro repair of renal tubular monolayers. Figure 30B shows that cells induced with 2% oxygen enhance migration and wound repair compared to uninduced (21% oxygen) cells. Figure 30C plots the percentage of migrated cells versus migration time.

Figure 31A shows that osteopontin is secreted by renal tubular cells and is upregulated in response to injury (osteopontin immunocytochemistry: Hoechst nuclear stain (blue), osteopontin (red), 10x). Osteopontin is upregulated in established renal tubular cell monolayers in response to injury, as shown by immunofluorescence (Figure 31A) and ELISA (Figure 31B).

Figure 32A shows that the migratory response of the cells is mediated in part by osteopontin (green = migrating cells (5x)). Figure 32B shows that neutralizing antibodies (NAbs) to osteopontin reduce the migratory response of kidney cells by 50%.

FIG33 shows that hypoxia induction regulates the expression of tissue remodeling genes in cells.

FIG34 depicts a putative mechanism for hypoxic enhancement of cellular bioactivity leading to kidney regeneration.

FIG35 shows detection of microvesicles by Western blotting.

Detailed Description of the Invention

The present invention relates to a heterogeneous mixture or fraction of bioactive renal cells (BRC), and methods for separating and culturing the cells, and methods for treating a subject in need thereof using BRC and/or a construct containing BRC formed from a scaffold utilizing BRC inoculation as described herein. Bioactive renal cells can be isolated renal cells, including renal tubular cells and erythropoietin (EPO)-producing renal cells. A BRC cell colony can include a renal tubular and EPO-producing cell colony of enrichment. BRC can be derived from the renal cell part of a healthy individual or they themselves are the renal cell fractions of a healthy individual. In addition, the invention provides renal cell fractions obtained from unhealthy individuals, which can lack certain cellular components when compared with the corresponding renal cell fractions of a healthy individual, but still maintain therapeutic properties. The present invention also provides a therapeutically active cell colony lacking a cellular component compared to a healthy individual, and in one embodiment, can be isolated and amplified from an autologous source in various disease states.

The present invention also relates to methods of providing a regenerative effect to an autologous kidney by contacting the autologous kidney with a product secreted by renal cells in vivo, and methods of preparing the secreted product. The present invention also relates to the use of markers to determine the presence of renal regeneration after treatment using the methods described herein.

definition

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Principles of Tissue Engineering , 3rd edition (edited by R. Lanza, R. Langer, & J. Vacanti), 2007 provides a general guide to many of the terms used in this application for those skilled in the art. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. In fact, the present invention is in no way limited to such methods and materials.

As used herein, the term "cell colony" refers to a number of cells obtained by directly isolating from an appropriate tissue source, typically from a mammal. The cell colony separated can subsequently be cultured in vitro. Those skilled in the art will appreciate the various methods for isolating and culturing the cell colony of the present invention and the different numbers of cells suitable for use in the cell colony of the present invention. The cell colony can be an unfractionated heterologous cell colony derived from the kidney. For example, a heterologous cell colony can be isolated from a renal biopsy or from intact renal tissue. Alternatively, the heterologous cell colony can be derived from an in vitro culture of mammalian cells set up from a renal biopsy or intact renal tissue. An unfractionated heterologous cell colony can also be referred to as a non-enriched cell colony.

The term "autologous kidney" means a kidney from a living subject. The subject can be healthy or unhealthy. An unhealthy subject may have kidney disease.

The term "regenerative effect" refers to an effect that provides a benefit to the native kidney. The effect may include, but is not limited to, a reduction in the degree of damage to the native kidney or an improvement in the restoration or stabilization of native renal function. Renal damage may take the form of fibrosis, inflammation, glomerular hypertrophy, etc., and is associated with the subject's renal disease.

As used herein, the term "mixture" refers to a combination of two or more isolated enriched cell populations derived from an unfractionated heterogeneous cell population. According to certain embodiments, the cell population of the present invention is a kidney cell population.

"Enriched" cell colony or preparation refers to a cell colony derived from a starting nephrocyte colony (e.g., an unfractionated heterologous cell colony) that comprises a cell type that has a percentage greater than that of a specific cell type in the starting colony. For example, the first, second, third, fourth, fifth, etc. target cell colonies of the starting nephrocyte colony can be enriched. As used herein, the terms "cell colony," "cell preparation," and "cell prototype" are used interchangeably.

In one aspect, as used herein, term " enrichment " cell colony refers to the cell colony (for example, from the cell suspension of the mammalian nephrocyte of renal biopsy or cultivation) deriving from initial nephrocyte colony, and it can comprise the cell that can generate EPO than the percentage ratio of the cell that can generate EPO in initial colony greater than the percentage ratio.For example, term " B4 " is the cell colony deriving from initial nephrocyte colony, and it comprises EPO founder cell, messangial cell and vascular cell of greater percentage ratio compared with initial colony.Cell colony of the present invention can enrich one or more cell types and consume one or more other cell types therein.For example, the EPO founder cell colony of enrichment can enrich interstitial fibroblast, and consumes renal tubular cell and collecting duct epithelial cell, interstitial fibroblast and renal tubular cell in the initial cell colony that the cell colony of described enrichment is derived from relative to the cell colony of non-enrichment. In all embodiments referencing an enriched EPO or "B4" population, the enriched cell population is a heterogeneous cell population comprising cells that can produce EPO in an oxygen-regulated manner (as demonstrated by oxygen-regulatable EPO expression from an endogenous endogenous EPO gene).

In another aspect, a cell population that is enriched for a particular cell type, such as vascular cells, glomerular cells, or endocrine cells, at a greater percentage than the percentage of that cell type in the starting population can also lack or be deficient in one or more particular cell types, such as vascular cells, glomerular cells, or endocrine cells, compared to a starting kidney cell population derived from a healthy individual or subject. For example, the term "B4'" or "B4prime", in one aspect, refers to a cell population derived from a starting kidney cell population that lacks or is deficient in one or more cell types, such as vascular cells, glomerular cells, or endocrine cells (depending on the disease state of the starting sample), compared to a healthy individual. In one embodiment, the B4' cell population is derived from a subject with chronic kidney disease. In one embodiment, the B4' cell population is derived from a subject with focal segmental glomerulosclerosis. In another embodiment, the B4' cell population is derived from a subject with autoimmune glomerulonephritis. In another aspect, B4' is a cell population derived from a starting cell population comprising all cell types, such as vascular cells, glomerular cells, or endocrine cells, which is subsequently depleted of one or more cell types, such as vascular cells, glomerular cells, or endocrine cells, or which is subsequently lacking the cell types. In another aspect, B4' is a cell population derived from a starting cell population comprising all cell types, such as vascular cells, glomerular cells, or endocrine cells, which is subsequently enriched for one or more specific cell types, such as vascular cells, renal tubular cells, or endocrine cells. In one embodiment, the B4' cell colony can be enriched in vascular cells but consumes glomerular cells and/or endocrine cells. In another embodiment, the B4' cell colony can be enriched in glomerular cells but consumes vascular cells and/or endocrine cells. In another embodiment, the B4' cell colony can be enriched in endocrine cells but consumes blood vessels and/or glomerular cells. In another embodiment, the B4' cell colony can be enriched in vascular cells and endocrine cells but consumes glomerular cells. In another embodiment, the B4' cell colony can be enriched in vascular cells and endocrine cells but consumes glomerular cells. In a preferred embodiment, the B4' cell colony alone or mixed with another enriched cell colony (e.g., B2 and/or B3) retains therapeutic properties. For example, the B4' cell colony is described in Examples 7-9 herein.

In another aspect, the cell colony of enrichment can also refer to the cell colony deriving from the starting nephrocyte colony discussed above, and described cell colony comprises the cell expressing one or more renal tubular cell markers of the percentage ratio greater than the percentage ratio of the cell expressing one or more renal tubular cell markers in the starting colony.For example, term " B2 " refers to the cell colony deriving from the starting nephrocyte colony, and described cell colony comprises the renal tubular cell of larger percentage ratio compared with the starting cell colony.In addition, can comprise some epithelial cells from collecting duct system for the cell colony (or " B2 ") of expressing one or more renal tubular cell markers enrichment.Although make the cell colony of the cell enrichment for expressing one or more renal tubular cell markers (or " B2 ") relatively consume EPO founder cell, messangial cell and vascular cell, the colony of enrichment can comprise this type of cell (EPO founder cell, messangial cell and vascular cell) of smaller percentage ratio compared with the starting colony.Generally, make heterologous cell colony consume one or more cell types, so that the cell colony of consumption comprises the cell type of smaller ratio relative to the cell type comprised in the heterologous cell colony before consumption.The cell type that can be consumed is the nephrocyte of any type. For example, in certain embodiments, the cell types that can be consumed include cells with large granularity of the collecting duct and tubular system (referred to as "B1") with a density of < about 1.045 g/ml. In certain other embodiments, the cell types that can be consumed include debris and small cells with low granularity and viability (referred to as "B5") with a density of > about 1.095 g/ml. In some embodiments, all of the following cells of a cell population that is relatively enriched for tubular cells are consumed: "B1," "B5," oxygen-regulatable EPO-expressing cells, glomerular cells, and vascular cells.

As used herein, the term "hypoxic" culture conditions refers to culture conditions in which cells are subjected to a reduction in the effective oxygen level relative to standard culture conditions in which cells are cultured at atmospheric oxygen levels (about 21%). Non-hypoxic conditions herein refer to normal or normoxic culture conditions.

As used herein, the term "oxygen-regulatable" refers to the ability of a cell to regulate gene expression (either up- or down-regulate) based on the amount of oxygen available to the cell. "Hypoxia-inducible" refers to the upregulation of gene expression in response to a decrease in oxygen tension (regardless of pre-induction or initial oxygen tension).

As used herein, the term "biomaterial" refers to a natural or synthetic biocompatible material suitable for introduction into living tissue. Natural biomaterials are materials produced by living systems. Synthetic biomaterials are materials not produced by living systems. The biomaterials disclosed herein can be a combination of natural and synthetic biocompatible materials. For example, as used herein, biomaterials include, for example, polymer matrices and scaffolds. Those skilled in the art will appreciate that biomaterials can be configured in a variety of forms, such as liquid hydrogel suspensions, porous foams, and can include one or more natural or synthetic biocompatible materials.

As used herein, the term "anemia" refers to the inability of the EPO producing cells of the experimenter to produce sufficient functional EPO proteins, and/or the insufficient release of EPO proteins to systemic circulation, and/or the inability of the immature erythrocytes in the bone marrow to respond to the deficiency of the number of red blood cells and/or the reduction of hemoglobin levels caused by the EPO proteins. The experimenter suffering from anemia cannot maintain the dynamic balance (erythroid homeostasis) of red blood cells. Generally, anemia can occur due to the decline or loss (e.g., chronic renal failure) of renal function, the anemia associated with relative EPO deficiency, the anemia associated with congestive heart failure, the anemia associated with myelosuppression therapy such as chemotherapy or antiviral therapy (e.g., AZT), the anemia associated with non-myeloid cancer, the anemia associated with viral infection such as HIV, and chronic diseases such as autoimmune diseases (e.g., rheumatoid arthritis), liver disease, and multiple organ system failure.

The term "EPO deficiency" refers to any condition or disorder treatable with an erythropoietin receptor agonist (eg, recombinant EPO or an EPO analog), including anemia.

As used herein, the term "nephropathy" refers to a condition related to any stage or degree of acute or chronic renal failure, which can cause the kidney to filter blood and remove excess fluid, electrolytes and waste from the blood. Nephropathy also includes endocrine dysfunction such as anemia (erythropoietin deficiency) and mineral imbalance (vitamin D deficiency). Nephropathy can begin with the kidney or can be secondary to a variety of conditions including, but not limited to, heart failure, hypertension, diabetes, autoimmune diseases or liver disease. Nephropathy can be the condition of chronic renal failure developed after acute damage to the kidney. For example, the renal damage produced by anemia and/or exposure to poisons can cause acute renal failure; incomplete recovery of acute renal damage can lead to the development of chronic renal failure.

The term "treatment" refers to therapeutic treatment and preventive or preventive measures for nephropathy, anemia, EPO deficiency, tubular transport defect or glomerular filtration defect, wherein the purpose is to reverse, prevent or slow down (mitigate) the targeted disease. People in need of treatment include those who have suffered from nephropathy, anemia, EPO deficiency, tubular transport defect or glomerular filtration defect and those who are prone to nephropathy, anemia, EPO deficiency, tubular transport defect or glomerular filtration defect, or those who will prevent their nephropathy, anemia, EPO deficiency, tubular transport defect or glomerular filtration defect. As used herein, the term "treatment" includes stabilization and/or improvement of renal function.

As used herein, the term "in vivo contact" refers to direct in vivo contact between a secretory product of an enriched renal cell population (or a mixture or construct comprising renal cells/renal cell fractions) and an autologous kidney. Direct in vivo contact can be natural paracrine, endocrine, or juxta-crine. The secretory product can be a heterologous population of different products described herein.

As used herein, the term "ribonucleic acid" or "RNA" refers to a chain of nucleotide units wherein each unit is composed of a nitrogenous base, a ribose sugar, and a phosphate. RNA may be single-stranded or double-stranded. RNA may be part of or associated with a vesicle. A vesicle may be an exosome. RNA includes, but is not limited to, mRNA, rRNA, small RNA, snRNA, snoRNA, microRNA (miRNA), small interfering RNA (siRNA), and non-coding RNA. RNA is preferably human RNA.

The term "construct" refers to one or more cell colonies deposited on the support or matrix composed of one or more synthetic or naturally occurring biocompatible materials or on or in the matrix. Available biomaterials composed of one or more synthetic or naturally occurring biocompatible polymers, proteins or peptides are coated with one or more cell colonies, or described cell colony can be deposited on described biomaterial, embedded therein, adhere thereto, be seeded therein, or be captured therein. One or more cell colonies can be combined with biomaterial or support or matrix in vitro or in vivo. Generally, one or more biocompatible materials selected for forming support/biomaterial instruct, promote or allow at least one formation multicellular three-dimensional tissue of the cell colony thereon. One or more biomaterials for generating constructs also can be selected in order to instruct, promote or allow the cellular component of construct or construct to dispersion and/or integration of endogenous host tissue, or instruct, promote or allow the cellular component of construct or construct to survive, transplant, tolerate or functional operation.

The term "marker" or "biomarker" generally refers to a DNA, RNA, protein, carbohydrate or glycolipid-based molecular marker whose expression or presence in a cultured cell population can be detected by standard methods (or methods disclosed herein) and is consistent with one or more cells being cells of a particular type in the cultured cell population. A marker can be a polypeptide expressed by a cell or an identifiable physical location on a chromosome such as a gene, a restriction endonuclease recognition site, or a nucleic acid encoding a polypeptide expressed by an autologous cell (e.g., mRNA). A marker can be an expression region of a gene called a "gene expression marker," or some segments of DNA that do not have a known coding function. A biomarker can be of cell origin, such as a secretory product.

The terms "differentially expressed genes", "differential gene expression" and their synonyms, which are used interchangeably, refer to genes whose expression in a first cell or cell population is activated to a higher or lower level relative to its expression in a second cell or cell population. The term also includes genes whose expression is activated to a higher or lower level at different stages over time during the first or second cell culture passage process. It should also be understood that differentially expressed genes can be activated or inhibited at the nucleic acid level or protein level, or the genes can be subjected to alternative splicing to result in different polypeptide products. Such differences can be confirmed by changes in, for example, the mRNA level, surface expression, secretion or other distribution of the polypeptide. Differential gene expression can include a comparison of expression between two or more genes or their gene products, or a comparison of the ratio of expression between two or more genes or their gene products, or even a comparison of two differentially processed products of the same gene, which are different between the first and second cells. Differential expression includes, for example, quantitative and qualitative differences in the temporal or cellular expression pattern of a gene or its expression product between a first cell and a second cell. For the purposes of the present invention, "differential gene expression" is considered to exist when there is a difference between the expression of a given gene in a first cell and a second cell. There may be differential expression of a marker in cells from the patient before administration of the cell population, admixture, or construct (first cells) relative to expression in cells from the patient after administration (second cells).

The terms "inhibit," "downregulate," "underexpress," and "reduce" are used interchangeably and mean that the expression of a gene encoding one or more proteins or protein subunits, or the level of its RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits is reduced relative to one or more controls, e.g., one or more positive and/or negative controls. Underexpression can occur in cells from a patient before administration of a cell population, mixture, or construct relative to cells from a patient after administration.

The terms "upregulate" or "overexpress" are used to refer to the expression of genes encoding one or more proteins or protein subunits, or the level of their RNA molecules or equivalent RNA molecules, or the activity of one or more proteins or protein subunits, relative to one or more controls, such as one or more positive and/or negative controls. Overexpression occurs in cells from a patient after administration of a cell population, mixture, or construct relative to cells from a patient before administration.

The term "subject" should refer to any single human subject, including patients suitable for treatment who are experiencing or have experienced one or more signs, symptoms or other indicators of nephropathy, anemia or EPO deficiency. Such subjects include but are not limited to newly diagnosed or previously diagnosed and currently experiencing the reproduction or recurrence of nephropathy, anemia or EPO deficiency or subjects at risk of the disease, regardless of the cause of disease. The subject may have previously been treated for nephropathy, anemia or EPO deficiency, or may not have been treated in this way.

The term "patient" refers to any individual animal for whom treatment is desired, more preferably a mammal (including such non-human animals as dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep and non-human primates). Most preferably, the patient herein is a human.

The term "sample" or "patient sample" or "biological sample" generally refers to any biological sample obtained from a subject or patient, body fluid, body tissue, cell line, tissue culture, or other source. The term includes biopsies, such as kidney biopsies. The term includes cultured cells, such as cultured mammalian kidney cells. Methods for obtaining biopsies and cultured cells from mammals are well known in the art. If the term "sample" is used alone, it still means that the "sample" is a "biological sample" or a "patient sample," i.e., the terms are used interchangeably.

The term "test sample" refers to a sample from a subject that has been treated by the methods of the present invention. The test sample can be derived from a variety of sources of mammalian subjects, including but not limited to blood, semen, serum, urine, bone marrow, mucus, tissue, etc.

The term "control" or "control sample" refers to a negative or positive control in which a negative or positive result is expected to help associate with the result of a test sample. Suitable controls for the present invention include, but are not limited to, samples of indicators characterized by known display characteristics of normal erythrocyte system homeostasis, samples characterized by known display characteristics of anemia, samples obtained from known subjects who are not anemic, and samples obtained from subjects who are known to be anemic. Other controls suitable for the method of the present invention include, but are not limited to, samples derived from subjects treated with known pharmaceutical agents (e.g., recombinant EPO or EPO analogs) for regulating erythropoiesis. In addition, controls can be samples obtained from subjects before utilizing the method of the present invention for treatment. Other suitable controls can be test samples obtained from subjects known to suffer from any type or stage of ephrosis and samples from subjects known to not suffer from any type or stage of ephrosis. Controls can be controls of normal health matching. Those skilled in the art will appreciate that other controls are suitable for the present invention.

"Regenerative prognosis," "regenerative prognosis," or "regenerative prognosis" generally refers to a prediction or projection of the likely regenerative process or outcome of administration or implantation of a cell population, admixture, or construct described herein. For regenerative prognosis, the prediction or projection can be informed by one or more of the following: enhancement of a functional kidney following implantation or administration, development of a functional kidney following implantation or administration, development of enhanced renal function or capacity following implantation or administration, and expression of certain markers by the native kidney following implantation or administration.

"Regenerated kidney" refers to an autologous kidney after implantation or administration of a cell colony, mixture, or construct as described herein. The regenerated kidney is characterized by various indicators, including but not limited to the development of the function or ability of the autologous kidney, the enhancement of the function or ability of the autologous kidney, and the expression of certain markers in the autologous kidney. It will be appreciated by those skilled in the art that other indicators may be applicable to characterize the regenerated kidney.

Cell population

Previously, in U.S. application No. 12/617,721, filed on November 12, 2009 (incorporating the disclosure thereof in its entirety into this paper), described a heterogeneous population of isolated nephrocytes and mixtures thereof for treating nephropathy, i.e., providing stabilization and/or enhancement and/or regeneration of renal function (enriched with specific bioactive components or cell types and/or consumed specific inactive or undesirable components or cell types for the population). The present invention provides isolated nephrocyte fractions that lack cellular components but still maintain therapeutic properties, i.e., provide stabilization and/or enhancement and/or regeneration of renal function, compared to healthy individuals. The cell colonies, cell fractions, and/or mixtures of cells described herein can be derived from healthy individuals, individuals suffering from nephropathy, or subjects described herein.

Bioactive cell populations

In one aspect, the present invention is based on the surprising discovery that certain subfractions of a heterogeneous population of renal cells enriched for their bioactive components and depleted of inactive or undesirable components provide superior therapeutic and regenerative outcomes compared to the starting population. For example, the bioactive components of the invention, such as B2, B4, and B3, depleted of inactive or undesirable components (e.g., B1 and B5), alone or in combination, provide unexpected stabilization and/or enhancement and/or regeneration of renal function.

In another aspect, the present invention is based on surprising discovery: consume or lack the specific subfraction B4 of one or more cell types such as vascular cells, endocrine cells or endothelial cells, i.e. B4 ', alone or when mixed with other biologically active subfractions such as B2 and/or B3, keep therapeutic properties, such as stabilization and/or enhancement and/or regeneration of renal function. In preferred embodiments, the biologically active cell colony is B2. In certain embodiments, the B2 cell colony is mixed with B4 or B4 '. In other embodiments, the B2 cell colony is mixed with B3. In other embodiments, the B2 cell colony is mixed with B3 and B4 or the specific cell components of B3 and/or B4.

The B2 cell population is characterized by the expression of tubular cell markers selected from one or more of the following markers: megaprotein, cubilin, hyaluronan synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family member, member A3 (Aldh1a3) and calpain-8 (Capn8), and the collecting duct marker aquaporin-4 (Aqp4). B2 is larger and more granulated than B3 and/or B4, having a buoyant density of about 1.045 g/ml to about 1.063 g/ml (rodent), about 1.045 g/ml to 1.052 g/ml (human), and about 1.045 g/ml to about 1.058 g/ml (canine).

The B3 cell population is characterized by expression of vascular, glomerular, and proximal tubular markers in some EPO-producing cells, and has an intermediate size and granularity compared to B2 and B4, resulting in a buoyant density of about 1.063 g/ml to about 1.073 g/ml (rodent), about 1.052 g/ml to about 1.063 g/ml (human), and about 1.058 g/ml to about 1.063 g/ml (canine). B3 is characterized by the expression of markers selected from one or more of the following markers: aquaporin 7 (Aqp7), FXYD domain-containing ion transport regulator 2 (Fxyd2), solute carrier family 17 (sodium phosphate), member 3 (Slc17a3), solute carrier family 3, member 1 (Slc3a1), claudin 2 (Cldn2), napsin A aspartic peptidase (Napsa), solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a2), alanyl (membrane) aminopeptidase (Anpep), transmembrane protein 27 (Tmem27), acyl-CoA synthase intermediate chain family member 2 (Acsm2), glutathione peroxidase 3 (Gpx3), fructose-1,6-bisphosphatase 1 (Fbp1), and alanine-glyoxylate aminotransferase 2 (Agxt2). B3 is also characterized by expression of the vascular marker platelet endothelial cell adhesion molecule (Pecam) and the glomerular marker podocin (Podn).

The B4 cell population is characterized by the expression of a vascular marker panel comprising one or more of the following: PECAM, VEGF, KDR, HIF1a, CD31, CD146; a glomerular marker panel comprising one or more of the following: Podocin (Podn) and Nephrin (Neph); and an enrichment of the oxygen-regulated EPO-enriched population compared to unfractionated (UNFX) B2 and B3. B4 is also characterized by the expression of one or more of the following markers: chemokine (C-X-C motif) receptor 4 (Cxcr4), endothelin receptor type B (Ednrb), collagen type V, alpha 2 (Col5a2), cadherin 5 (Cdh5), plasminogen activator, tissue (Plat), angiopoietin 2 (Angpt2), kinase insert domain protein receptor (Kdr), secreted protein, acidic cysteine-rich osteonectin (Sparc), serglycan (Srgn), TIMP metallopeptidase inhibitor 3 (Timp3), Wilms' tumor 1 (Wt1), wingless type MMTV integration site family, member 4 (Wnt4), regulator of G protein signaling 4 (Rgs4), platelet endothelial cell adhesion molecule (Pecam), and erythropoietin (Epo). B4 was also characterized by smaller, less granulated cells than B2 or B3, with a buoyant density of about 1.073 g/ml to about 1.091 g/ml (rodent), about 1.063 g/ml to about 1.091 g/mL (human and canine).

The B4' cell population is defined as having a buoyant density of 1.063 g/mL to 1.091 g/mL and expressing one or more of the following markers: PECAM, vEGF, KDR, HIF1a, podocin, phenylephrine, EPO, CK7, CK8/18/19. In one embodiment, the B4' cell population is characterized by the expression of a vascular marker panel comprising one or more of the following markers: PECAM, vEGF, KDR, HIF1a, CD31, CD146. In another embodiment, the B4' cell population is characterized by the expression of the endocrine marker EPO. In one embodiment, the B4' cell population is characterized by the expression of a glomerular marker panel comprising one or more of the following markers: Podocin (Podn) and phenylephrine (Neph). In certain embodiments, the B4' cell population is characterized by the expression of a vascular marker panel comprising one or more of the following markers: PECAM, vEGF, KDR, HIF1a and the expression of the endocrine marker EPO. In another embodiment, B4' is further characterized by smaller, less granulated cells compared to B2 or B3, having a buoyant density of about 1.073 g/ml to about 1.091 g/ml (rodent), about 1.063 g/ml to about 1.091 g/mL (human and canine).

In one aspect, the invention provides B4 ' colonies of the enrichment of the separation of human kidney cells with a density of 1.063g/mL to 1.091g/mL, comprising at least one of erythropoietin (EPO) founder cells, vascular cells and glomerular cells. In one embodiment, the B4 ' cell colony is characterized by the expression of vascular markers. In certain embodiments, the B4 ' cell colony is not characterized by the expression of glomerular markers. In some embodiments, the B4 ' cell colony is capable of oxygen-regulated erythropoietin (EPO) expression.

In one embodiment, the B4' cell population does not include a B2 cell population comprising renal tubular cells having a density of 1.045 g/mL to 1.052 g/mL. In another embodiment, the B4' cell population does not include a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density of <1.045 g/ml. In another embodiment, the B4' cell population does not include a B5 cell population comprising debris and small cells having a density of >1.091 g/ml.

In one embodiment, the B4' cell population does not include a B2 cell population comprising renal tubular cells having a density of 1.045 g/mL to 1.052 g/mL; a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density of <1.045 g/ml; and a B5 cell population comprising debris and small cells having low granularity and viability having a density of >1.091 g/ml. In some embodiments, the B4' cell population may be derived from a subject with renal disease.

In one aspect, the present invention provides a mixture of human kidney cells comprising a first cell population B2 (which comprises an isolated enriched tubular cell population) with a density of 1.045 g/mL to 1.052 g/mL and a second cell population B4' (which comprises erythropoietin (EPO) producing cells and vascular cells but has consumed glomerular cells) with a density of about 1.063 g/mL to 1.091 g/mL, wherein the mixture does not include a B1 cell population of large granular cells comprising the collecting duct and tubular system with a density of <1.045 g/ml, or a B5 cell population comprising debris and small cells with low granularity and vitality with a density of >1.091 g/ml. In certain embodiments, the B4' cell population is characterized by the expression of vascular markers. In one embodiment, the B4' cell population is not characterized by the expression of glomerular markers. In certain embodiments, B2 also includes collecting duct epithelial cells. In one embodiment, the mixture of cells is capable of receptor-mediated albumin absorption. In another embodiment, the mixture of cells can express erythropoietin (EPO) regulated by oxygen. In one embodiment, the mixture includes cells expressing HAS-2 that can generate and/or stimulate the generation of high molecular weight species of hyaluronic acid (HA) in vitro and in vivo. In all embodiments, the first and second cell colonies can be derived from renal tissue or cultured renal cells.

In one embodiment, the mixture is capable of providing a regenerative stimulus upon in vivo delivery. In other embodiments, the mixture is capable of reducing the decline of tubular filtration, tubular absorption, urine production and/or endocrine function upon in vivo delivery, stabilizing or enhancing said function. In one embodiment, the B4' cell colony is derived from a subject with nephropathy.

In one aspect, the invention provides B4 ' colonies comprising the separation of enriched human kidney cells comprising at least one of erythropoietin (EPO) founder cells, vascular cells and glomerular cells having a density of 1.063 g/mL to 1.091 g/mL. In one embodiment, the B4 ' cell colony is characterized by the expression of vascular markers. In certain embodiments, the B4 ' cell colony is not characterized by the expression of glomerular markers. The glomerular marker not expressed can be podocin (see Example 7). In some embodiments, the B4 ' cell colony is capable of oxygen-adjustable erythropoietin (EPO) expression.

In one embodiment, the B4' cell population does not include a B2 cell population comprising renal tubular cells having a density of 1.045 g/mL to 1.052 g/mL. In another embodiment, the B4' cell population does not include a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density of <1.045 g/ml. In another embodiment, the B4' cell population does not include a B5 cell population comprising debris and small cells having a density of >1.091 g/ml.

In one embodiment, the B4' cell population does not include a B2 cell population comprising tubular cells having a density of 1.045 g/mL to 1.052 g/mL; a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density of <1.045 g/ml; and a B5 cell population comprising debris and small cells with low granularity and viability having a density of >1.091 g/ml.

In some embodiments, B4 ' cell colony can be derived from a subject suffering from nephropathy. In one aspect, the invention provides a mixture of human kidney cells comprising a first cell colony B2 (which comprises the enriched renal tubular cell colony separated) with a density of 1.045g/mL to 1.052g/mL and a second cell colony B4 ' (which comprises erythropoietin (EPO) producing cells and vascular cells but consumes glomerular cells) with a density of 1.063g/mL to 1.091g/mL, wherein the mixture does not include the B1 cell colony of large granular cells comprising collecting duct and renal tubular system with a density of <1.045g/ml, or the B5 cell colony comprising debris and small cells with low granularity and viability with a density of >1.091g/ml. In certain embodiments, B4 ' cell colony is characterized by the expression of vascular markers. In one embodiment, B4 ' cell colony is not characterized by the expression of glomerular markers. In certain embodiments, B2 also includes collecting duct epithelial cells. In one embodiment, the mixture of cells can carry out receptor-mediated albumin absorption. In another embodiment, the mixture of cells is capable of expressing oxygen-adjustable erythropoietin (EPO). In one embodiment, the mixture comprises cells expressing HAS-2 that are capable of generating and/or stimulating high molecular weight species of hyaluronic acid (HA) in vitro and in vivo. In all embodiments, the first and second cell populations can be derived from renal tissue or cultured renal cells.

In one embodiment, the mixture is capable of providing a regenerative stimulus upon in vivo delivery. In other embodiments, the mixture is capable of reducing the decline of glomerular filtration, tubular absorption, urine production and/or endocrine function, stabilizing or enhancing the function upon in vivo delivery. In one embodiment, the B4' cell colony is derived from a subject suffering from nephropathy.

In a preferred embodiment, the mixture comprises B2 in combination with B3 and/or B4. In another preferred embodiment, the mixture comprises B2 in combination with B3 and/or B4'. In other preferred embodiments, the mixture consists of or consists essentially of (i) B2 in combination with B3 and/or B4; or (ii) B2 in combination with B3 and/or B4'.

The mixture comprising the B4' cell population may comprise a B2 and/or B3 cell population also obtained from a non-healthy subject. The non-healthy subject may be the same subject from which the B4' fraction was obtained. In contrast to the B4' cell population, the B2 and B3 cell populations obtained from non-healthy subjects are generally not deficient in one or more specific cell types compared to the starting kidney cell population derived from a healthy individual.

Hyaluronic acid production through B2 and B4

Hyaluronan (also referred to as hyaluronic acid or hyaluronate) is a glycosaminoglycan (GAG), which is composed of non-sulfated disaccharide units, especially regular repeats of N-acetylglucosamine and glucuronic acid. The scope of its molecular weight can be 400 daltons (disaccharides) to more than one million daltons. It is found to exist with variable amounts in all tissues such as skin, cartilage and eyes and in the body fluids of most (if not all) adult animals. It is particularly abundant in early embryos. By hyaluronan, the space produced by general GAG in fact allows it to work in cell migration, cell attachment, in wound repair process, organogenesis, immune cell adhesion, activation of intracellular signal transduction and tumor metastasis. These effects are mediated by the specific protein and proteoglycan in conjunction with hyaluronan. Cell motility and immune cell adhesion are mediated by cell surface receptors RHAMM (Receptor for Hyaluronan-Mediated Motility; Hardwick et al., 1992) and CD44 (Jalkenan et al., 1987; Miyake et al., 1990). Hyaluronan is synthesized directly on the inner membrane of the cell surface, and as it synthesizes, the growing polymer is squeezed through the membrane to the outside of the cell. Synthesis is mediated by a single protein enzyme, hyaluronan synthase (HAS), whose gene family consists of at least 3 members.

Hyaluronan has recently been shown to interact with CD44, and such interaction may, among other things, recruit non-resident cells such as mesenchymal stem cells (MSCs) to damaged renal tissue and enhance renal regeneration (Kidney International (2007) 72, 430-441).

Unexpectedly, it has been found that B2 and B4 cell preparations can express the species of higher molecular weight hyaluronic acid (HA) in vitro and in vivo by the effect of hyaluronan synthase-2 (HAS-2)-a marker that is more particularly rich in B2 cell populations. It has been shown that in the 5/6Nx model, the treatment of B2 is utilized to reduce fibrosis, with the generation of expected high molecular weight HA in the tissue of strong expression of HAS-2 in vivo and treatment. It is worth noting that leaving the untreated 5/6Nx model leads to fibrosis, limited HAS-2 is detected and almost no high molecular weight HA is generated. Without wishing to be bound by theory, it is assumed that the anti-inflammatory high molecular weight species of HA generated mainly by B2 (and to a certain extent by B4) synergize with the cell preparation in the reduction of renal fibrosis and in the help of renal regeneration. Therefore, the present invention includes delivering the cell prototype of the present invention in a biomaterial comprising hyaluronic acid. The present invention further relates to a biomaterial component that provides a regenerative stimulus by directly generating or by stimulating the generation of implanted cells.

In one aspect, the invention provides a heterologous population of EPO-generating nephrocytes isolated for treating a subject in need thereof, for example, nephropathy, anemia, and/or EPO deficiency. In one embodiment, the cell colony is derived from a renal biopsy. In another embodiment, the cell colony is derived from intact renal tissue. In another embodiment, the cell colony is derived from an in vitro culture of mammalian nephrocytes set up from a renal biopsy or intact renal tissue. In all embodiments, these colonies are unfractionated cell colonies, also referred to herein as non-enriched cell colonies.

In yet another aspect, the invention provides the erythropoietin (EPO) of separation and generates nephrocyte colony, and its further enrichment is so that the ratio of EPO founder cell in the colony of enrichment is greater than the ratio of EPO founder cell in original or initial cell colony.In one embodiment, the EPO founder cell fraction of enrichment comprises the interstitial fibroblast and the renal tubular cell of larger proportion relative to the interstitial fibroblast comprised in the initial colony of not enrichment and renal tubular cell and smaller proportion of renal tubular cell.In certain embodiments, the EPO founder cell fraction of enrichment comprises the renal glomerular cell and vascular cell and the collecting duct cell of larger proportion relative to the renal glomerular cell comprised in the initial colony of not enrichment and renal tubular cell and smaller proportion of renal tubular cell.In such embodiment, these colonies are referred to as " B4 " cell colony in this article.

In yet another aspect, the invention provides the EPO generation nephrocyte colony mixed with one or more other nephrocyte colonies.In one embodiment, the EPO founder cell colony is the first cell colony such as B4 that has been enriched for EPO founder cells.In another embodiment, the EPO founder cell colony is the first cell colony such as B2 that is not enriched for EPO founder cells.In another embodiment, the first cell colony is mixed with the second nephrocyte colony.In some embodiments, the second cell colony is enriched for renal tubular cells, which can be proved by the existence of the phenotype of renal tubular cells.In another embodiment, the phenotype of renal tubular cells can be shown by the existence of a renal tubular cell marker.In another embodiment, the phenotype of renal tubular cells can be shown by the existence of one or more renal tubular cell markers. Renal tubular cell markers include, but are not limited to, megaprotein, cuboprotein, hyaluronan synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8). In another embodiment, the first cell colony is mixed with at least one of several types of nephrocytes (including but not limited to interstitial-derived cells, renal tubular cells, collecting duct-derived cells, glomerulus-derived cells and/or cells derived from blood or vascular system). EPO generates nephrocyte colony and can include B4 or B4', such as B2+B3+B4/B4', present in a mixture with B2 and/or B3, or in a cell colony enriched.

In one aspect, the EPO of the present invention generates kidney cell colony and is characterized in that EPO expresses and the biological response to oxygen, so that the reduction of the oxygen tension of culture system causes the induction of EPO expression.In one embodiment, EPO founder cell colony enrichment EPO founder cell.In one embodiment, when making cell undergo the reduction of available oxygen level in culture system (compared with the cell cultivated under the normal atmosphere (～ 21%) level of available oxygen) condition culture cell colony, EPO expression is induced.In one embodiment, the EPO founder cell cultivated under lower oxygen condition expresses higher level of EPO relative to the EPO founder cell cultivated under normal oxygen condition.Generally, culturing cell under the level of available oxygen (also referred to as hypoxic culture condition) reduced means the level of oxygen reduced relative to culturing cell under the normal atmosphere level of available oxygen (also referred to as normal or normoxic culture condition).In one embodiment, hypoxic cell culture condition is included in culturing cell under the oxygen of approximately less than 1%, approximately less than 2%, approximately less than 3%, approximately less than 4% oxygen or approximately less than 5% oxygen. In another embodiment, normal or normoxic culture conditions comprise culturing cells in about 10% oxygen, about 12% oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen, about 20% oxygen, or about 21% oxygen.

In one other embodiment, obtain the induction of EPO or increase expression and can be by culturing cells under about less than 5% available oxygen and comparing EPO expression level with the cell cultured under atmospheric (about 21%) oxygen to observe the induction of EPO or increase expression.In another embodiment, obtain the induction of EPO in the culture of the cell that can express EPO by method, the method described includes wherein culturing cells under atmospheric oxygen (about 21%) for a period of time and wherein reducing available oxygen level and culturing the second culturing period of the same cells under about less than 5% available oxygen.In another embodiment, the EPO expression of response hypoxic condition is regulated by HIF1 α.It will be appreciated by those skilled in the art that other oxygen operation culture conditions known in the art can be used for the cells described herein.

In one aspect, the EPO of enrichment generates mammalian cell colony and is characterized in that the biological response (for example, EPO expresses) to perfusion condition.In one embodiment, perfusion condition includes short-term, intermittent or continuous fluid flow (perfusion).In one embodiment, when intermittently or continuously circulate or stir the culture medium of wherein cultured cells in the mode that power is transferred to cell by flow, EPO expression is mechanically induced.In one embodiment, with them as three-dimensional structure, be present in the material that provides framework and/or space for the formation of such three-dimensional structure or the mode on it, cultivate the cell of short-term, intermittent or continuous fluid flow.In one embodiment, by cell culture on porous beads and utilize rocking platform, platform or rotating flask running along track to be experienced intermittent or continuous fluid flow.In another embodiment, by cell culture on three-dimensional support and be placed in the device with fixed support, fluid orientation flows through or passes support.It will be appreciated by those skilled in the art that other perfusion culture conditions known in the art can be used for cell described herein.

Inactive cell population

As described herein, the present invention is based, in part, on the surprising discovery that certain fractions of a heterogeneous population of renal cells enriched for bioactive components and depleted for inactive or undesirable components provide superior therapeutic and regenerative outcomes compared to the starting population. In preferred embodiments, the B1 and/or B5 cell populations of the cell populations of the present invention are depleted. For example, B1 and/or B5 of the following cell populations can be depleted: a mixture of two or more of B2, B3, and B4'; or an enriched cell population of B2, B3, and B4'.

The B1 cell population contained large granular cells of the collecting duct and tubular system and had a buoyant density of less than about 1.045 g/m. The B5 cell population consisted of debris and small cells with low granularity and viability and had a buoyant density greater than about 1.091 g/ml.

Methods for isolating and culturing cell populations

The present invention, in one aspect, provides methods for separating and isolating renal cell components, such as enriched cell populations, for therapeutic use, including treatment of renal disease, anemia, EPO deficiency, tubular transport deficiency, and glomerular filtration deficiency. In one embodiment, the cell population is isolated from freshly digested, i.e., mechanically or enzymatically digested, renal tissue or from heterogeneous in vitro cultures of mammalian renal cells.

It has been unexpectedly discovered that a heterogeneous mixture of nephrocytes cultured in hypoxic culture conditions before separation on a density gradient provides an increased distribution and composition of cells in B4 (including B4 ') and B2 and/or B3 fractions. For nephrocytes isolated from diseased and non-diseased kidneys, an enrichment of oxygen-dependent cells in B4 and B2 was observed. Without wishing to be bound by theory, this may be attributed to one or more of the following phenomena: 1) selective survival, death or proliferation of specific cell components during the hypoxic culture phase; 2) changes in the particle size and/or size of cells cultured in response to hypoxia, thereby affecting changes in buoyant density and subsequent positioning during density gradient separation; and 3) changes in cell gene/protein expression in response to the hypoxic culture phase, thereby resulting in differential characteristics of cells within any given gradient fraction. Thus, in one embodiment, a cell population enriched in tubular cells, such as B2, is hypoxia-resistant.

Exemplary techniques for separating and isolating cell colonies of the present invention include separations based on the differential specific gravity of the different cell types included in the target population in a density gradient. The specific gravity of any given cell type can be affected by the degree of intracellular granularity, the volume of intracellular water and other factors. In one aspect, the invention provides optimal gradient conditions for separating cell preparations of the present invention such as B2 and B4 (including B4 ') across multiple species including but not limited to humans, dogs and rodents. In a preferred embodiment, a density gradient is used to obtain a colony, i.e., the B2 cell colony, of the renal tubular cell fraction of the novel enrichment derived from a heterologous colony of nephrocytes. In one embodiment, a density gradient is used to obtain a colony, i.e., the B4 cell colony, of the EPO founder cell fraction of the novel enrichment derived from a heterologous colony of nephrocytes. In other embodiments, a density gradient is used to obtain the subpopulation of renal tubular cells, glomerular cells and endothelial cells of the kidney of enrichment. In one embodiment, EPO founder cells and renal tubular cells are separated from red blood cells and cell debris. In one embodiment, EPO is generated that renal glomerulus and vascular cells are separated from other cell types and from red blood cells and cell fragments, and simultaneously concomitantly the subpopulation of renal tubular cells and collecting duct cells are separated from other cell types and from red blood cells and cell fragments. In another embodiment, endocrine cells, renal glomerular cells and/or vascular cells are separated from other cell types and from red blood cells and cell fragments, and simultaneously concomitantly the subpopulation of renal tubular cells and collecting duct cells are separated from other cell types and from red blood cells and cell fragments.

The present invention is based on certain key features described below, and generates novel cell populations by, in part, using an Axis-Shield density gradient medium containing 60% of the non-ionic iodinated compound iodixanol in water. However, those skilled in the art will recognize that any density gradient or other method, such as immunological separation using cell surface markers known in the art (including the essential characteristics for isolating the cell populations of the present invention), can be used in accordance with the present invention. Those skilled in the art will also recognize that the same cell characteristics that facilitate the separation of cell subpopulations by density gradients (size and granularity) can be exploited to separate cell subpopulations by flow cytometry (forward scatter = a reflection of size by a flow cytometer, and side scatter = a reflection of granularity). Importantly, density gradient medium should have low toxicity to specific target cells. Although density gradient medium should have low toxicity to specific target cells, the present invention relates to the use of gradient medium that functions in the selection process of target cells. Without wishing to be bound by theory, the cell populations of the present invention recovered using a gradient including iodixanol appear to be resistant to iodixanol, as there is a considerable loss of cells between the loading and recovery steps, suggesting that exposure to iodixanol under gradient conditions leads to the elimination of certain cells. The cells that appear on the specific bands after the iodixanol gradient are resistant to any adverse effects of iodixanol and/or density gradient exposure. Therefore, the invention further relates to other contrast media that are mild to moderate nephrotoxins in the separation and/or selection of cell colonies of the present invention. In addition, the density gradient medium should not be combined with proteins in human plasma or adversely affect the key functions of the target cells.

In another aspect, the present invention provides methods for enriching and/or depleting renal cell types using fluorescence activated cell sorting (FACS). In one embodiment, renal cell types can be enriched and/or depleted using a BD FACSAria ^™ or equivalent technology.

In another aspect, the present invention provides methods for enriching and/or depleting renal cell types using magnetic cell sorting. In one embodiment, renal cell types can be enriched and/or depleted using the Miltenyi system or equivalent techniques.

In yet another aspect, the invention provides the method for the three-dimensional culture of nephrocyte colony.In one aspect, the invention provides the method by continuous perfusion culture cell colony.In one embodiment, by three-dimensional culture and continuous perfusion culture cell colony when compared with the cell colony of static culture, show stronger cellularity (cellularity) and interconnectivity (interconnectivity).In another embodiment, by three-dimensional culture and continuous perfusion culture cell colony when compared with the static culture of such cell colony, show more EPO expression and the expression of the urinary tubules related gene such as e-cadherin that increases.

In another embodiment, the cell population cultured by continuous perfusion exhibits higher levels of glucose and glutamine consumption when compared to the cell population cultured statically.

As described herein (including Example 3), hypoxic or anoxic conditions can be used in the methods of preparing cell populations of the present invention. However, the methods of the present invention can be used without a hypoxic conditioning step. In one embodiment, normoxic conditions can be used.

Those skilled in the art will appreciate that other isolation and culture methods known in the art can be used with the cells described herein.

Biomaterials (polymer matrices or scaffolds)

As Bertram et al., described in the application 20070276507 (incorporated herein by reference in its entirety) published by the U.S., polymer matrix or support can be shaped into many desired configurations to satisfy many complete systems, geometry or spatial limitations. In one embodiment, matrix of the present invention or support can be three-dimensional and can be shaped to make the size and shape that is suitable for organ or tissue structure. For example, in using polymer support to treat nephropathy, anemia, EPO deficiency, renal tubular transport defect or glomerular filtration defect, three-dimensional (3-D) matrix can be used. The 3-D support of multiple difference molding can be used. Naturally, polymer matrix can be shaped with different sizes and shapes so that the patient of different sizes is suitable. Polymer matrix can be shaped to adapt to the special needs of patient in other ways. In another embodiment, polymer matrix or support can be a biocompatible multipolymer support. Scaffolds can be formed from a variety of synthetic or naturally occurring materials including, but not limited to, open-cell polylactic acid cellulose ethers, cellulose, cellulosic esters, fluorinated polyethylene, phenol, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polyarylethernitrile, polyester, polyestercarbonate, polyether, polyetheretherketone, polyetherimide, polyetherketone, polyethylenesulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylenesulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polysulfide, polytriazole, polyurethane, polyethylene, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or copolymers or physical mixtures thereof. The scaffold configuration can be varied from a liquid hydrogel suspension to a soft porous scaffold to a rigid, shape-retaining porous scaffold.

Hydrogels can be formed from a variety of polymeric materials and used in a variety of biomedical applications. Hydrogels can be physically described as three-dimensional networks of hydrophilic polymers. Depending on the type of hydrogel, they contain varying percentages of water but are completely insoluble in water. Despite their high water content, hydrogels are able to bind large volumes of liquid due to the presence of hydrophilic residues. Hydrogels can swell fully without changing their gel-like structure. Depending on the nature of the polymers used and other specialized equipment for the product, the basic physical characteristics of the hydrogel can be specifically modified.

Preferably, hydrogel is made up of polymer, biologically derived material, synthetically produced material or its combination, and the material is biologically inert and physiologically compatible with mammalian tissue.Hydrogel material preferably does not induce inflammatory response.The example of other materials that can be used to form hydrogel includes (a) modified alginate, (b) by being exposed to the polysaccharide (such as gellan gum and carrageenan) that can be gelled by monovalent cation, (c) is very sticky liquid or has thixotropy and by the slow evolution of structure over time past the polysaccharide (such as, hyaluronic acid) and (d) polymer hydrogel precursor (such as, polyethylene oxide-polypropylene glycol block copolymer and protein) that form gel.U.S. Patent No.6,224,893B1 provides the various polymers that are applicable to prepare the chemical property of hydrogel according to the present invention and this type of polymer.

The scaffold or biomaterial features can enable cells to adhere to the scaffold or biomaterial and interact with it, and/or provide a porous space in which cells can be captured. In one embodiment, the porous scaffold of the present invention or biomaterial allow one or more colonies or mixtures of cells to be added or deposited on the biomaterial configured as a porous scaffold (e.g., by the attachment of cells) and/or in the pores of the scaffold (e.g., by the capture of cells). In another embodiment, the scaffold or biomaterial allow or promote interactions between cells and/or between cells and biomaterial within the scaffold to form a construct described herein.

In one embodiment, the biomaterial used according to the present invention is composed of the hyaluronic acid (HA) existing in the form of a hydrogel, which comprises a size from 5.1kDA to> 2x10 ⁶ HA molecules of kDa scope. In another embodiment, the biomaterial used according to the present invention is composed of the hyaluronic acid existing in the form of a porous foam, which also comprises a size from 5.1kDA to> 2x10 ⁶ HA molecules of kDa scope. In another embodiment, the biomaterial used according to the present invention is composed of a foam based on polylactic acid (PLA), which has an open-cell structure and a pore size of approximately 50 microns to approximately 300 microns. In another embodiment, specific cell colonies, preferably B2 but also B4, directly provide and/or stimulate the synthesis of the high molecular weight hyaluronic acid by hyaluronan synthase-2 (HAS-2), particularly after implantation in the kidney.

Those skilled in the art will appreciate that other types of synthetic or naturally occurring materials known in the art can be used to form the scaffolds described herein.

In one aspect, the present invention provides a construct described herein made from the above-mentioned scaffold or biomaterial.

Construct

In one aspect, the invention provides the implantable constructs of the cell colony described herein that one or more ephrosis, anemia or EPO that are used to treat the experimenter of this need are lacked.In one embodiment, construct is made up of biocompatible material or biomaterial, the support or matrix that is made up of one or more synthetic or naturally occurring biocompatible materials and one or more by adhering to and/or capturing and being deposited on the surface of support or being embedded in cell colony described herein or cell mixture therein.In certain embodiments, construct is made up of biomaterial and one or more biomaterial components coated, be deposited on biomaterial, be deposited therein, be attached thereto, be captured in it, be embedded in it or with its cell colony described herein or cell mixture in combination.Any cell colony described herein, including cell colony or its mixture of enrichment, can be used in combination with matrix to form construct.

In another embodiment, the cell component of the deposited cell colony or construct is a first kidney cell colony enriched with oxygen-adjustable EPO-producing cells. In another embodiment, the first kidney cell colony also includes glomeruli and vascular cells except the oxygen-adjustable EPO-producing cells. In one embodiment, the first kidney cell colony is a B4 'cell colony. In another embodiment, the cell component of the deposited cell colony or construct includes a first kidney cell colony enriched with a second kidney cell colony. In some embodiments, the oxygen-adjustable EPO-producing cells of the second cell colony are not enriched. In another embodiment, the second cell colony is enriched with renal tubular cells. In another embodiment, the second cell colony is enriched with renal tubular cells, and the second cell colony includes collecting duct epithelial cells. In other embodiments, the tubular cells are characterized by the expression of one or more tubular cell markers, which may include, but are not limited to, megaprotein, cuboprotein, hyaluronan synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8).

In one embodiment, the cell population deposited on or combined with a biomaterial or scaffold to form a construct of the present invention is derived from a variety of sources, such as autologous sources. Non-autologous sources are also suitable for use, including but not limited to allogeneic or isogenic (autologous or isogenic) sources.

Those skilled in the art will appreciate that there are several suitable methods for depositing a cell population onto a biomaterial or otherwise combining a cell population with a biomaterial to form a construct.

In one aspect, construct of the present invention is applicable to the method for use described herein. In one embodiment, construct is suitable for use to a subject who needs any etiology of nephropathy, anemia or the EPO deficiency of any etiology. In other embodiments, construct is suitable for use to a subject who needs the enhancing or recovery of erythrocyte homeostasis. In another embodiment, construct is suitable for use to a subject who needs enhanced renal function.

In another aspect, the present invention provides a construct for implantation into a subject in need of enhanced renal function, comprising: a) a biomaterial comprising one or more biocompatible synthetic polymers or naturally occurring proteins or peptides; and

b) a mixture of mammalian kidney cells derived from a subject with a kidney disease, comprising a first cell population B2 comprising an isolated, enriched renal tubular cell population having a density of 1.045 g/mL to 1.052 g/mL and a second cell population B4' comprising erythropoietin (EPO)-producing cells and vascular cells but depleted of glomerular cells having a density of 1.063 g/mL to 1.091 g/mL, the cell mixture being coated with a biomaterial, or deposited on or in a biomaterial, captured therein, suspended therein, embedded therein and/or otherwise combined therewith. In certain embodiments, the mixture does not include a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density of <1.045 g/ml, or a B5 cell population comprising debris and small cells having low granularity and viability having a density of >1.091 g/ml.

In one embodiment, the construct comprises a B4' cell population characterized by expression of vascular markers. In some embodiments, the B4' cell population is not characterized by expression of glomerular markers. In certain embodiments, the mixture is capable of oxygen-regulated erythropoietin (EPO) expression. In all embodiments, the mixture can be derived from mammalian kidney tissue or cultured renal cells.

In one embodiment, construct includes a three-dimensional (3-D) porous biomaterial configured to be suitable for mixture capture and/or attachment. In another embodiment, construct includes a biomaterial configured to be suitable for embedding, attachment, suspension or coating mammalian cells in a liquid or semi-liquid gel. In another embodiment, construct includes a biomaterial constructed to exist in a hydrogel form by being composed of the hyaluronic acid (HA) of a predominantly high molecular weight species. In another embodiment, construct includes a biomaterial constructed to exist in a porous form by being composed of the hyaluronic acid of a predominantly high molecular weight species. In another embodiment, construct includes a biomaterial composed of a foam based on polylactic acid with a hole of approximately 50 microns to approximately 300 microns. In another embodiment, construct includes one or more cell colonies that can derive from a renal sample that is autologous for a subject whose renal function needs to be enhanced. In certain embodiments, the sample is a renal biopsy. In some embodiments, the subject suffers from nephropathy. In other embodiments, the cell colony derives from a non-autologous renal sample. In one embodiment, construct provides a dynamic balance of red blood cells.

secretory products

In one other aspect, the present invention relates to a mixture secretory product from a renal cell colony of enrichment or an enrichment renal cell colony, as described herein. In one embodiment, the product includes one or more of the following substances: paracrine factors, endocrine factors, adjacent secretory factors, and vesicles. The vesicles may include one or more of the following substances: paracrine factors, endocrine factors, adjacent secretory factors, microvesicles, exosomes, and RNA. The secretory product may also include a product that is not in the microvesicle, including but not limited to paracrine factors, endocrine factors, adjacent secretory factors, and RNA. For example, extracellular miRNA (Wang et al., Nuc Acids Res 2010, 1-12 doi: 10.1093/nar/gkq601, July 7, 2010) has been detected in the outer portion of the vesicle. The secretory product may also be referred to as a cell-derived product, such as a cell-derived vesicle.

In another embodiment, the secreted product can be part of a vesicle derived from a renal cell. The vesicle can be capable of delivering factors to other destinations. In one embodiment, the vesicle is a secreted vesicle. Several types of secreted vesicles are involved, including but not limited to exosomes, microvesicles, exosomes, membrane particles, exosome-like vesicles, and apoptotic vesicles (Thery et al. 2010. Nat. Rev. Immunol. 9: 581-593). In one embodiment, the secreted vesicle is an exosome. In another embodiment, the secreted vesicle is a microvesicle. In another embodiment, the secreted vesicle contains or includes one or more cellular components. The components can be one or more of the following components: membrane lipids, RNA, proteins, metabolites, cytoplasmic components, and any combination thereof. In a preferred embodiment, the secreted vesicle includes, consists of, or is essentially composed of, microRNA. Preferably, the miRNA is a human miRNA. In one embodiment, the one or more miRNAs are selected from miR-30b-5p, miR-449a, miR-146a, miR-130a, miR-23b, miR-21, miR-124, and miR-151. In another embodiment, the one or more miRNAs can be selected from the group consisting of let-7a-1, let-7a-2, let-7a-3, let-7b, let-7c, let-7d, let-7e, let-7f-1, let-7f-2, let-7g, let-7i, mir-1-1, mir-1-2, mir-7-1, mir-7-2, mir-7-3, mir-9-1, mir-9-2, mir-9-3, mir-10a, mir-10b, mir-15a, mir-15b, mir-16-1, mir-16-2, mir-17, mir-18a, mir-18b, mir-19a, mir-19b-1, mir-19b-2, mir-20a, mir-20b, mir-21, mir-22 ,mir-23a,mir-23b,mir-23c,mir-24-1,mir-24-2,mir-25,mir-26a-1,mir-26a-2,mir-26 b, mir-27a, mir-27b, mir-28, mir-29a, mir-29b-1, mir-29b-2, mir-29c, mir-30a, mir-30b, mir-30c-1, mir-30c-2, mir-30d, mir-30e, mir-31, mir-32, mir-33a, mir-33b, mir-34a, mir-34b, mir-34c, mir-92a-1, mir-92a-2, mir-92b, mir-93, mir-95, mir-96, mir-98, mir-99a mir-99b, mir-100, mir-101-1, mir-101-2, mir-103-1, mir-103-1-as, mir-103-2, mir-103-2-as, mir-105-1, mir-105-2, mir-106a, mir-106b, mir-107, mir-122, mi r-124-1, mir-124-2, mir-124-3, mir-125a, mir-125b-1, mir-125b-2, mir-126, mir-127, mir-128-1, mir-128-2, mir-129-1, mir-129-2, mir-130a, mir-130b, mir-13 2. mir-132, mir-133a-1, mir-133a-2, mir-133b, mir-134, mir-135a-1, mir-135a-2, mir-135b, mir-136MI101351120, mir-137, mir-138-1, mir-138-2, mir-139, mir -140, mir-141, mir-142, mir-143, mir-144, mir-145, mir-146a, mir-146b, mir-147, mir-147b, mir-148a, mir-148b, mir-149, mir-150, mir-151, mir-152, mir-153-1 , mir-153-2, mir-154, mir-155, mir-181a-1, mir-181a-2, mir-181b-1, mir-181b-2, mir-181c, mir-181d, mir-182, mir-183, mir-184, mir-185, mir-186, mir-187, m ir-188, mir-190, mir-190b, mir-191, mir-192, mir-193a, mir-193b, mir-194-1, mir-194-2, mir-195, mir-196a-1, mir-196a-2, mir-196b, mir-197, mir-198, mir-19 9a-1, mir-199a-2, mir-199b, mir-200a, mir-200b, mir-200c, mir-202, mir-203, mir-204, mir-205, mir-206, mir-208a, mir-208b, mir-210, mir-211, mir-212, mir- 214, mir-215, mir-216a, mir-216b, mir-217, mir-218-1, mir-218-2, mir-219-1, mir-219-2, mir-221, mir-222, mir-223, mir-224, mir-296, mir-297, mir-298, mir-2 99. mir-300, mir-301a, mir-301b, mir-302a, mir-302b, mir-302c, mir-302d, mir-302e, mir-302f, mir-320a, mir-320b-1, mir-320b-2, mir-320c-1, mir-320c-2, mi r-320d-1,mir-320d-2,mir-320e,mir-323,mir-323b,mir-324,mir-325,mir-326,mir-328,mir-329-1,mir-329-2,mir-330,mir-331,mir-335,mir-337,mir-338,m ir-339, mir-340, mir-342, mir-345, mir-346, mir-361, mir-362, mir-363, mir-365-1, mir-365-2, mir-367, mir-369, mir-370, mir-37, mir-372, mir-373, mir-374a ,mir-374b,mir-374c,mir-375,mir-376a-1,mir-376a-2,mir-376b,mir-376c,mir-377,mir-378,mir-378b,mir-378c,mir-379,mir-380,mir-381,mir-382,mir-38 3. mir-384, mir-409, mir-410, mir-411, mir-412, mir-421, mir-422a, mir-423, mir-424, mir-425, mir-429, mir-431, mir-432, mir-433, mir-448, mir-449a, mir-44 9b, mir-449c, mir-450a-1, mir-450a-2, mir-450b, mir-451, mir-452, mir-454, mir-455, mir-466, mir-483, mir-484, mir-485, mir-486, mir-487a, mir-487b, mir-48 8. mir-489, mir-490, mir-491, mir-492, mir-493, mir-494, mir-495, mir-496, mir-497, mir-498, mir-499, mir-500a, mir-500b, mir-501, mir-502, mir-503, mir-50 4. mir-505, mir-506, mir-507, mir-508, mir-509-1, mir-509-2, mir-509-3, mir-510, mir-511-1, mir-511-2, mir-512-1, mir-512-2, mir-513a-1, mir-513a-2, mir-5 13b, mir-513c, mir-514-1, mir-514-2, mir-514-3, mir-514b, mir-515-1, mir-515-2, mir-516a-1, mir-516a-2, mir-516b-1, mir-516b-2, mir-517a, mir-517b, mir- 517c, mir-518a-1, mir-518a-2, mir-518b, mir-518c, mir-518d, mir-518e, mir-518f, mir-519a-1, mir-519a-2, mir-519b, mir-519c, mir-519d, mir-519e, mir-520a, mir-520b, mir-520c, mir-520d, mir-520e, mir-520f, mir-520g, mir-520h, mir-521-1, mir-521-2, mir-522, mir-523, mir-524, mir-525, mir-526a-1, mir-526a-2, m ir-526b, mir-527, mir-532, mir-539, mir-541, mir-542, mir-543, mir-544, mir-544b, mir-545, mir-548a-1, mir-548a-2, mir-548a-3, mir-548aa-1, mir-548aa-2, m ir-548b, mir-548c, mir-548d-1, mir-548d-2, mir-548e, mir-548f-1, mir-548f-2, mir-548f-3, mir-548f-4, mir-548f-5, mir-548g, mir-548h-1, mir-548h-2, mir- 548h-3, mir-548h-4, mir-548i-1, mir-548i-2, mir-548i-3, mir-548i-4, mir-548j, mir-548k, mir-548l, mir-548m, mir-548n, mir-548o, mir-548p, mir-548s, mir-5 48t, mir-548u, mir-548v, mir-548w, mir-548x, mir-548y, mir-548z, mir-549, mir-550a-1, mir-550a-2, mir-550b-1, mir-550b-2, mir-551a, mir-551b, mir-552, mi r-553, mir-554, mir-555, mir-556, mir-557, mir-558, mir-559, mir-561, mir-562, mir-563, mir-564, mir-566, mir-567, mir-568, mir-569, mir-570, mir-571, mir-5 72. mir-573, mir-574, mir-575, mir-576, mir-577, mir-578, mir-579, mir-580, mir-581, mir-582, mir-583, mir-584, mir-585, mir-586, mir-587, mir-588, mir-589 ,mir-590,mir-591,mir-592,mir-593,mir-595,mir-596,mir-597,mir-598,mir-599,mir-600,mir-601,mir-602,mir-603,mir-604,mir-605,mir-606,mir-607,mi r-608, mir-609, mir-610, mir-611, mir-612, mir-613, mir-614, mir-615, mir-616, mir-617, mir-618, mir-619, mir-620, mir-621, mir-622, mir-623, mir-624, mir- 625, mir-626, mir-627, mir-628, mir-629, mir-630, mir-631, mir-632, mir-633, mir-634, mir-635, mir-636, mir-637, mir-638, mir-639, mir-640, mir-641, mir-642 a, mir-642b, mir-643, mir-644, mir-645, mir-646, mir-647, mir-648, mir-649, mir-650, mir-651, mir-652, mir-653, mir-654, mir-655, mir-656, mir-657, mir-658 , mir-659, mir-660, mir-661, mir-662, mir-663, mir-663b, mir-664, mir-665, mir-668, mir-670, mir-671, mir-675, mir-676, mir-708, mir-711, mir-718, mir-720, m ir-744, mir-758, mir-759, mir-760, mir-761, mir-762, mir-764, mir-765, mir-766, mir-767, mir-769, mir-770, mir-802, mir-873, mir-874, mir-875, mir-876, mir- 877, mir-885, mir-887, mir-888, mir-889, mir-890, mir-891a, mir-891b, mir-892a, mir-892b, mir-920, mir-921, mir-922, mir-924, mir-933, mir-934, mir-935, mir -936, mir-937, mir-938, mir-939, mir-940, mir-941-1, mir-941-2, mir-941-3, mir-941-4, mir-942, mir-942, mir-943, mir-944, mir-1178, mir-1179, mir-1180, mi r-1181, mir-1182, mir-1183, mir-1184-1, mir-1184-2, mir-1184-3, mir-1185-1, mir-1185-2, mir-1193, mir-1197, mir-1200, mir-1202, mir-1203, mir-1204, mir-1 205, mir-1206, mir-1207, mir-1208, mir-1224, mir-1225, mir-1226, mir-1227, mir-1228, mir-1229, mir-1231, mir-1233-1, mir-1233-2, mir-1234, mir-1236, mir- 1237, mir-1238, mir-1243, mir-1244-1, mir-1244-2, mir-1244-3, mir-1245, mir-1246, mir-1247, mir-1248, mir-1249, mir-1250, mir-1251, mir-1252, mir-1253, mi r-1254, mir-1255a, mir-1255b-1, mir-1255b-2, mir-1256, mir-1257, mir-1258, mir-1260, mir-1260b, mir-1261, mir-1262, mir-1263, mir-1264, mir-1265, mir-12 66. mir-1267, mir-1268, mir-1269, mir-1270-1, mir-1270-2, mir-1271, mir-1272, mir-1273, mir-1273c, mir-1273d, mir-1273e, mir-1274a, mir-1274b, mir-1275, m ir-1276, mir-1277, mir-1278, mir-1279, mir-1280, mir-1281, mir-1282, mir-1283-1, mir-1283-2, mir-1284, mir-1285-1, mir-1285-2, mir-1286, mir-1287, mir-1 288.mir-1289-1,mir-1289-2,mir-1290,mir-1291,mir-1292,mir-1293,mir-1294,mir-1295,mir-1296,mir-1297,mir-1298,mir-1299,mir-1301,mir-1302-1,mir -1302-10, mir-1302-11, mir-1302-2, mir-1302-3, mir-1302-4, mir-1302-5, mir-1302-6, mir-1302-7, mir-1302-8, mir-1302-9, mir-1303, mir-1304, mir-1305, mi r-1306, mir-1307, mir-1321, mir-1322, mir-1323, mir-1324, mir-1468, mir-1469, mir-1470, mir-1471, mir-1537, mir-1538, mir-1539, mir-1825, mir-1827, mir-19 08.mir-1909,mir-1910,mir-1911,mir-1912,mir-1913,mir-1914,mir-1915,mir-1972-1,mir-1972-2,mir-1973,mir-1976,mir-2052,mir-2053,mir-2054,mir-2 110.mir-2113,mir-2114,mir-2115,mir-2116,mir-2117,mir-2276,mir-2277,mir-2278,mir-2355,mir-2861,mir-2909,mir-3065,mir-3074,mir-3115,mir-3116- 1. mir-3116-2, mir-3117, mir-3118-1, mir-3118-2, mir-3118-3, mir-3118-4, mir-3118-5, mir-3118-6, mir-3119-1; mir-3119-2, mir-3120, mir-3121, mir-3122, m m ir-3137, mir-3138, mir-3139, mir-3140, mir-3141, mir-3142, mir-3143, mir-3144, mir-3145, mir-3146, mir-3147, mir-3148, mir-3149, mir-3150, mir-3151, mir- 3152, mir-3153, mir-3154, mir-3155, mir-3156-1, mir-3156-2, mir-3156-3, mir-3157, mir-3158-1, mir-3158-2, mir-3159, mir-3160-1, mir-3160-2, mir-3161, mir -3162, mir-3163, mir-3164, mir-3165, mir-3166, mir-3167, mir-3168, mir-3169, mir-3170, mir-3171, mir-3173, mir-3174, mir-3175, mir-3176, mir-3177, mir-31 78. mir-3179-1, mir-3179-2, mir-3179-3, mir-3180-1, mir-3180-2, mir-3180-3, mir-3180-4, mir-3180-5, mir-3181, mir-3182, mir-3183, mir-3184, mir-3185, mir -3186, mir-3187, mir-3188, mir-3189, mir-3190, mir-3191, mir-3192, mir-3193, mir-3194, mir-3195, mir-3196, mir-3197, mir-3198, mir-3199-1, mir-3199-2, mi r-3200, mir-3201, mir-3202-1, mir-3202-2, mir-3605, mir-3606, mir-3607, mir-3609, mir-3610, mir-3611, mir-3612, mir-3613, mir-3614, mir-3615, mir-3616, mi r-3617, mir-3618, mir-3619, mir-3620, mir-3621, mir-3622a, mir-3622b, mir-3646, mir-3647, mir-3648, mir-3649, mir-3650, mir-3651, mir-3652, mir-3653, mir -3654, mir-3655, mir-3656mir-3657, mir-3658, mir-3659, mir-3660, mir-3661, mir-3662, mir-3663, mir-3664, mir-3665, mir-3666, mir-3667, mir-3668, mir-3669 ,mir-3670,mir-3670,mir-3671,mir-3671,mir-3673,mir-3673,mir-3675,mir-3675,mir-3676,mir-3663,mir-3677,mir-3678,mir-3679,mir-3680,mir-3681,mi r-3682, mir-3683, mir-3684, mir-3685, mir-3686, mir-3687, mir-3688, mir-3689a, mir-3689b, mir-3690, mir-3691, mir-3692, mir-3713, mir-3714, mir-3907, mir- 3908, mir-3909, mir-3910-1, mir-3910-2, mir-3911, mir-3912, mir-3913-1, mir-3913-2, mir-3914-1, mir-3914-2, mir-3915, mir-3916, mir-3917, mir-3918, mir- 3919, mir-3920, mir-3921, mir-3922, mir-3923, mir-3924, mir-3925, mir-3926-1, mir-3926-2, mir-3927, mir-3928, mir-3929, mir-3934, mir-3935, mir-3936, mir- 3937, mir-3938, mir-3939, mir-3940, mir-3941, mir-3942, mir-3943, mir-3944, mir-3945, mir-4251, mir-4252, mir-4253, mir-4254, mir-4255, mir-4256, mir-425 7. mir-4258, mir-4259, mir-4260, mir-4261, mir-4262, mir-4263, mir-4264, mir-4265, mir-4266, mir-4267, mir-4268, mir-4269, mir-4270, mir-4271, mir-4272, mi m ir-4287, mir-4288, mir-4289, mir-4290, mir-4291, mir-4292, mir-4293, mir-4294, mir-4295, mir-4296, mir-4297, mir-4298, mir-4299, mir-4300, mir-4301, mir-4 302, mir-4303, mir-4304, mir-4305, mir-4306, mir-4307, mir-4308, mir-4309, mir-4310, mir-4311, mir-4312, mir-4313, mir-4314, mir-4315-1, mir-4315-2, mir-4 316, mir-4317, mir-4318, mir-4319, mir-4320, mir-4321, mir-4322, mir-4323, mir-4324, mir-4325, mir-4326, mir-4327, mir-4328; mir-4329, mir-4329 and mir-4330.

The present invention relates to the cell-derived or secretory miRNA that can be obtained from cell colony described herein or construct.In one embodiment, one or more individual miRNA can be used to provide regeneration to autologous kidney.The combination of individual miRNA is applicable to providing such effect.Exemplary combination includes following two or more kinds: miR-21, miR-23a, miR-30c, miR-1224, miR-23b, miR-92a, miR-100, miR-125b-5p, miR-195, miR-10a-5p and any combination thereof.Another exemplary combination includes following two or more kinds: miR-30b-5p, miR-449a, miR-146a, miR-130a, miR-23b, miR-21, miR-124, miR-151 and any combination thereof.In one embodiment, the combination of miRNA can include 2,3,4,5,6,7,8,9,10 or more individual miRNAs. Those skilled in the art will appreciate that other miRNAs and combinations of mirRNAs may be suitable for use in the present invention.Additional sources of miRNAs include miRBase at http://mirbase.org , which is hosted and maintained by the School of Life Sciences at the University of Manchester.

In one embodiment, the secretory product includes a paracrine factor. Generally, a paracrine factor is a molecule synthesized by a cell that can diffuse over a short distance to induce or effect a change in a neighboring cell, i.e., a paracrine interaction. Diffusible molecules are referred to as paracrine factors.

In another embodiment, the present invention relates to a composition of one or more isolated renal cell-derived secretory vesicles, as described herein. One skilled in the art will appreciate that various types of compositions comprising secreted vesicles will be suitable.

In another aspect, the present invention provides a method for preparing renal cell secretory products such as vesicles. In one embodiment, the method includes the step of providing a renal cell population (including a mixture of one or more enriched renal cell populations). In another embodiment, the method also includes the step of culturing the population under appropriate conditions. The conditions may be hypoxic conditions. In another embodiment, the method also includes the step of isolating the secretory product from the renal cell population. The secreted vesicles can be obtained from the cell culture medium of the cell population. In another embodiment, the renal cells are characterized by vesicle production and/or secretion that is biologically responsive to oxygen levels (so that a reduction in the oxygen tension of the culture system leads to induction of vesicle production and/or secretion). In one embodiment, when the cell population is cultured under conditions in which the cells are subjected to a decrease in the effective oxygen level in the culture system compared to a cell population cultured under normal atmospheric (~21%) levels of effective oxygen, vesicle production and/or secretion are induced. In one embodiment, the cell population cultured under lower oxygen conditions produces and/or secretes higher levels of vesicles relative to the cell population cultured under normal oxygen conditions. Typically, culturing cells under reduced levels of available oxygen (also referred to as hypoxic culture conditions) means that the level of reduced oxygen is reduced relative to culturing cells under normal atmospheric levels of available oxygen (also referred to as normal or normoxic culture conditions). In one embodiment, hypoxic cell culture conditions include culturing cells under about less than 1% oxygen, about less than 2% oxygen, about less than 3% oxygen, about less than 4% oxygen, or about less than 5% oxygen. In another embodiment, normal or normoxic culture conditions include culturing cells under about 10% oxygen, about 12% oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen, about 20% oxygen, or about 21% oxygen. In a preferred embodiment, the method includes providing for separating exosomes and/or microvesicles from kidney cells.

In one embodiment, the product is secreted from the kidney cells.The product may be secreted by kidney cells that are not on the scaffold (eg, cells that are not part of the constructs described herein).

In another embodiment, the product is secreted by renal cells that have been seeded onto a scaffold, such as a construct.The construct comprises one or more enriched renal cell populations or mixtures thereof that are seeded directly onto or into the scaffold.

In another aspect, the invention provides an in vitro method for the therapeutic efficacy of a nephrocyte colony for screening/optimizing/monitoring one or more enrichments and a mixture or construct comprising the cell colony. In one embodiment, the method includes providing a step of one or more test colonies, test mixtures or test constructs ("test article"). In another embodiment, the method includes culturing the test article under appropriate conditions, as described herein. In another embodiment, the method includes collecting a cell culture medium from the cultured test article. The culture medium is called "conditioned medium" and is expected to include a product secreted by the nephrocytes of the test article.

In other aspects, conditioned medium can be used to perform one or more in vitro assays to test the biotherapeutic efficacy of the test article. In one embodiment, conditioned medium can be subjected to an epithelial-mesenchymal transition (EMT) assay. The assay can test for EMT induced by TGFβ1. Example 15 provides an exemplary protocol for this assay.

In another embodiment, RNA (e.g., by PCR-based assay) and/or vesicle or exosome (e.g., by FACS) are detected in conditioned medium. In another embodiment, signal transduction pathway assays are performed in conditioned medium, such as immune response (e.g., NFκB), fibrosis response (PAI-1), and angiogenesis. Examples 12-14 provide exemplary schemes for such assays.

How to use

In one aspect, the invention provides a method for treating nephropathy, anemia or EPO deficiency in a subject in need thereof using a mixture of nephrocyte colonies and nephrocytes as described herein. In one embodiment, the method includes administering to the subject a composition comprising a first nephrocyte colony enriched in EPO founder cells. In another embodiment, the first cell colony is enriched in EPO founder cells, glomerular cells and vascular cells. In one embodiment, the first cell colony is a B4 ' cell colony. In another embodiment, the composition may also include one or more other nephrocyte colonies. In one embodiment, other cell colonies are a second cell colony not enriched in EPO founder cells. In another embodiment, other cell colonies are a second cell colony not enriched in EPO founder cells, glomerular cells or vascular cells. In another embodiment, the composition also includes a mixture of nephrocyte colonies or nephrocytes, and the cell colony or mixture are deposited in a biomaterial, deposited thereon, embedded therein, coated thereon or captured therein to form an implantable construct (for treating a disease or illness described herein) as described herein. In one embodiment, the cell populations are used alone or in combination with other cells or biomaterials (eg, hydrogels, porous scaffolds, or natural or synthetic peptides or proteins) to stimulate regeneration in acute or chronic disease states.

In another aspect, the effective treatment of nephropathy, anemia or EPO deficiency in a subject can be observed by various indicators of erythropoiesis and/or renal function. In one embodiment, indicators of red blood cell homeostasis include but are not limited to hematocrit (HCT), hemoglobin (HB), mean corpuscular hemoglobin (MCH), red blood cell count (RBC), reticulocyte count (reticulocyte number), reticulocyte %, mean cell volume (MCV) and red blood cell distribution width (RDW). In another embodiment, indicators of renal function include but are not limited to serum albumin, albumin-globulin ratio (A/G ratio), serum phosphorus, serum sodium, kidney size (measurable by ultrasound), serum calcium, phosphorus-calcium ratio, serum potassium, proteinuria, urine creatinine, serum creatinine, blood urea nitrogen (BUN), cholesterol level, triglyceride level and glomerular filtration rate (GFR). In addition, several indicators of general health and well-being include but are not limited to weight gain or loss, survival, blood pressure (mean systemic blood pressure, diastolic or systolic blood pressure) and physical endurance performance.

In another embodiment, effective treatment is demonstrated by the stabilization of one or more indicators of renal function. The stabilization of renal function is demonstrated by comparing the change of the indicator of the experimenter treated by the method of the present invention with the same indicator of the experimenter not treated by the method of the present invention. Alternatively, the stabilization of renal function can be demonstrated by comparing the change of the indicator of the experimenter treated by the method of the present invention with the same indicator of the same experimenter before treatment. The change of the first indicator can be an increase or decrease in value. In one embodiment, the treatment provided by the present invention may include the stabilization of the blood urea nitrogen (BUN) level of the experimenter, wherein the BUN level observed in the experimenter is lower than the experimenter with similar disease state not treated by the method of the present invention. In another embodiment, the treatment may include the stabilization of the serum creatinine level of the experimenter, wherein the serum creatinine level observed in the experimenter is lower than the experimenter with similar disease state not treated by the method of the present invention. In another embodiment, the treatment includes the stabilization of the hematocrit (HCT) level of the experimenter, wherein the HCT level observed in the experimenter is lower than the experimenter with similar disease state not treated by the method of the present invention. In another embodiment, treatment comprises stabilization of the subject's red blood cell (RBC) levels, wherein the RBC levels observed in the subject are higher than in subjects with a similar disease state who have not been treated using the methods of the invention. It will be appreciated by those skilled in the art that one or more additional indices described herein or known in the art may be measured to determine the effective treatment of the subject's nephropathy.

In another aspect, the present invention relates to a method for providing a dynamic balance of red blood cells in a subject in need thereof. In one embodiment, the method comprises the following steps: (a) administering nephrocyte colonies such as B2 or B4 ', or a mixture of nephrocytes such as B2/B4 ' and/or B2/B3, as described herein to the subject; and (b) determining the level of erythropoiesis index relative to the index level in the control in a biological sample from the subject, wherein the difference (i) of the index level represents that the subject responds to administering step (a), or (ii) represents the dynamic balance of the red blood cells of the subject. In another embodiment, the method comprises the following steps: (a) administering a composition comprising a mixture of nephrocyte colonies or nephrocytes described herein to the subject; and (b) determining the level of erythropoiesis index relative to the index level in the biological sample from the subject, wherein the difference (i) of the index level represents that the subject responds to administering step (a), or (ii) represents the dynamic balance of the red blood cells of the subject. In another embodiment, the method includes the following steps: (a) providing a biomaterial or biocompatible polymer scaffold; (b) depositing a renal cell population or renal cell mixture of the present invention on or within the biomaterial or scaffold in a manner described herein to form an implantable construct; (c) implanting the construct into a subject; and (d) determining in a biological sample of the subject whether the level of an erythropoiesis indicator is different from the level of the indicator in a control, wherein the difference in the level of the indicator (i) indicates that the subject is responsive to the administration of step (a), or (ii) indicates the dynamic balance of the subject's red blood cells.

In another aspect, the present invention relates to a method for providing stabilization of renal function and restoration of red blood cell homeostasis to a subject in need thereof, wherein the subject has renal dysfunction and anemia and/or EPO deficiency. In one embodiment, the method comprises the step of administering a renal cell population or renal cell mixture as described herein, wherein the cell population or mixture comprises at least one of the following cell types: tubular-derived cells, glomerular-derived cells, interstitial-derived cells, collecting duct-derived cells, interstitial tissue-derived cells, or cells derived from the vasculature. In another embodiment, the population or mixture comprises EPO-producing cells and renal tubular epithelial cells, renal tubular cells identified by at least one of the following markers: megaprotein, cuboprotein, hyaluronan synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase family 3, member B1 (Aldh3b1), aldehyde dehydrogenase family 1, member A3 (Aldh1a3), and calpain-8 (Capn8). In this embodiment, treatment of the subject can be evidenced by an increase in at least one indicator of erythropoiesis accompanied by an improvement in at least one indicator of renal function compared to an untreated subject or the subject's pre-treatment indicator.

In one aspect, the invention provides a method of (i) treating nephropathy, anemia or EPO deficiency by administering a renal cell colony enriched in EPO producer cells or a mixture of renal cells comprising a cell colony enriched in EPO producer cells as described herein; (ii) stabilizing renal function, (iii) restoring the dynamic balance of red blood cells or (iv) any combination thereof, wherein the beneficial effect of administration is greater than the effect of administering a cell colony not enriched in EPO producer cells. In another embodiment, the enriched cell colony provides elevated levels of serum blood urea nitrogen (BUN). In another embodiment, the enriched cell colony improves the retention of protein in serum. In another embodiment, the enriched cell colony provides elevated levels of serum cholesterol and/or triglycerides. In another embodiment, the enriched cell colony provides elevated levels of vitamin D. In one embodiment, the enriched cell colony provides a phosphorus-calcium ratio that is increased compared to a non-enriched cell colony. In another embodiment, the enriched cell colony provides elevated levels of hemoglobin compared to a non-enriched cell colony. In other embodiments, the enriched cell colony provides elevated levels of serum creatinine compared to a non-enriched cell colony. In another embodiment, the enriched cell colony provides an elevated hematocrit compared to the non-enriched cell colony. In other embodiments, the enriched cell colony provides an elevated red blood cell count (RBC#) compared to the non-enriched cell colony. In one embodiment, the elevated hematocrit returns to 95% normal healthy level. In other embodiments, the enriched cell colony provides an increased reticulocyte count compared to the non-enriched cell colony. In other embodiments, the enriched cell colony provides an increased reticulocyte percentage compared to the non-enriched cell colony. In other embodiments, the enriched cell colony provides an elevated red blood cell volume distribution width (RDW) compared to the non-enriched cell colony. In another embodiment, the enriched cell colony provides an elevated hemoglobin compared to the non-enriched cell colony. In another embodiment, the enriched cell colony provides an erythropoietic response in the bone marrow so that bone marrow cellularity is close to normal and the bone marrow: erythrocyte system ratio is close to normal.

In another aspect, the present invention provides a method for (i) treating kidney disease, anemia or EPO deficiency by administering an enriched cell population; (ii) stabilizing renal function, (iii) restoring red blood cell homeostasis, or (iv) any combination thereof, wherein the beneficial effects of administering a renal cell population or a mixture of renal cell populations described herein are characterized by improved red blood cell homeostasis (when compared to the beneficial effects provided by administering recombinant EPO (rEPO)). In one embodiment, the population or mixture, when administered to a subject in need thereof, provides improved red blood cell homeostasis (as measured by hematocrit, hemoglobin, or RBC#) (when compared to administering recombinant EPO protein). In one embodiment, the population or mixture, when administered, provides elevated levels of hematocrit, RBC, or hemoglobin (when compared to recombinant EPO) that are no more than about 10% lower or higher than the hematocrit of a control. In other embodiments, a single dose or delivery of a population or mixture, when administered, provides an improvement in the dynamic balance of red blood cells (as measured by an increase in hematocrit, hemoglobin, or RBC#) in the treated subject for a period of time that significantly exceeds the time for which a single dose or delivery of recombinant EPO protein provides an improvement in the dynamic balance of red blood cells. In another embodiment, the population or mixture, when administered, does not result in a hematocrit, hemoglobin, or RBC# greater than about 110% of the normal level of a matched healthy control at the doses described herein. In other embodiments, the population or mixture, when administered at the doses described herein, provides a better dynamic balance of red blood cells (as measured by hematocrit, hemoglobin, or RBC#) than recombinant EPO protein delivered at the doses described herein. In another embodiment, recombinant EPO is delivered at a dose of about 100 IU/kg, about 200 IU/kg, about 300 IU/kg, about 400 IU/kg, or about 500 IU/kg. Those skilled in the art will appreciate that other dosages of recombinant EPO known in the art may be appropriate.

Another embodiment of the present invention relates to the use of at least one cell population described herein (including enriched cell populations and mixtures thereof), or an implantable construct described herein, or a secreted product described herein for the preparation of a medicament for treating kidney disease, anemia or EPO deficiency in a subject in need thereof, providing red blood cell homeostasis in a subject in need thereof, enhancing renal function in a subject in need thereof, or providing a regenerative effect to an autologous kidney.

Another embodiment of the present invention relates to the use of specific enriched cell populations (described herein), based on the selection of specific cell subpopulations (based on specific validated therapeutic properties), for the treatment of kidney diseases with specific etiologies.

In another aspect, the present invention provides a method for treating kidney disease in a subject in need thereof, comprising administering to the subject a composition comprising a mixture of mammalian kidney cells, the cell mixture comprising a first cell population B2 comprising an isolated enriched renal tubular cell population having a density of 1.045 g/mL to 1.052 g/mL, and a second cell population B4' comprising erythropoietin (EPO)-producing cells and vascular cells but depleted of glomerular cells having a density of 1.063 g/mL to 1.091 g/mL, wherein the mixture does not include a B1 cell population of large granular cells of the collecting duct and tubular system having a density of <1.045 g/ml, or a B5 cell population comprising debris and small cells having low granularity and vitality having a density of >1.091 g/ml. In certain embodiments, the method comprises determining in a test sample of a subject that the level of a renal function index is different from the index level of a control, wherein the difference in the index level indicates a reduction in the decline of one or more renal functions of the subject, a stabilization of the function, or an enhancement of the function. In one embodiment, the B4 ' cell colony for the method is characterized by the expression of vascular markers. In certain embodiments, the B4 ' cell colony for the method is not characterized by the expression of glomerular markers. In one embodiment, the mixture of cells for the method can express oxygen-adjustable erythropoietin (EPO). In certain embodiments, nephropathy to be treated by the methods of the present invention is accompanied by erythropoietin (EPO) deficiency. In certain embodiments, EPO deficiency is anemia. In some embodiments, EPO deficiency or anemia are secondary to the renal failure of the experimenter. In some other embodiments, EPO deficiency or anemia are secondary to the disease selected from chronic renal failure, primary EPO deficiency (primary EPO deficiency), chemotherapy or antiviral therapy, non-myeloid cancer, HIV infection, liver disease, heart failure, rheumatoid arthritis or multiple organ system failure. In certain embodiments, the composition for the method also comprises a biomaterial containing one or more biocompatible synthetic polymers and/or naturally occurring proteins or peptides, wherein the biomaterial is coated with a mixture, the mixture is deposited on or in the biomaterial, captured in, suspended in, embedded in, and/or combined with the mixture in addition. In certain embodiments, the mixture used in the method of the present invention derives from mammalian kidney tissue or cultivated mammalian kidney cells. In other embodiments, the mixture derives from an autologous kidney sample for a subject in need thereof. In one embodiment, the sample is a renal biopsy. In other embodiments, the mixture used in the method of the present invention derives from a non-autologous kidney sample.

In another aspect, the present invention provides use of the cell populations, mixtures thereof, or implantable constructs of the present invention for the preparation of a medicament for treating kidney disease, anemia, or EPO deficiency in a subject in need thereof.

In another aspect, the present invention provides a method for regenerating an autologous kidney in a subject in need thereof. In one embodiment, the method comprises administering or implanting a cell colony, mixture, or construct as described herein to the subject. The regenerated autologous kidney may be characterized by a number of indicators, including but not limited to the development of the function or ability of the autologous kidney, the enhancement of the function or ability of the autologous kidney, and the expression of certain markers in the autologous kidney. In one embodiment, the function or ability developed or enhanced can be observed based on various indicators of the dynamic balance of red blood cells and renal function described above. In another embodiment, the regenerated kidney is characterized by the differential expression of one or more stem cell markers. The stem cell marker may be one or more of the following markers: SRY (sex determining region Y)-box 2 (Sox2); undifferentiated embryonic cell transcription factor (UTF1); Nodal homolog (NODAL) from mouse; prominin 1 (PROM1) or CD133 (CD133); CD24 and any combination thereof. In another embodiment, the expression of the stem cell marker is upregulated compared to the control.

Cell colonies described herein (including enriched cell colonies and mixtures thereof and constructs comprising the cell colonies) can be used to provide regeneration to autologous kidney. The effect can be provided by the cell itself and/or by secretory products from the cell. The feature of regeneration can be one or more of the following aspects: reduction of epithelial-mesenchymal cell transition (which can be carried out by the weakening of TGF-β signal transduction); reduction of renal fibrosis; alleviation of nephritis; differential expression of stem cell markers in autologous kidney; migration of implanted cells and/or autologous cells to renal damage such as renal tubular damage, transplantation of implanted cells in the position of renal damage such as renal tubular damage; stabilization of one or more indicators of renal function (as described herein); recovery of the dynamic balance of erythrocytes (as described herein); and any combination thereof.

Methods for monitoring regeneration

In another aspect, the invention provides a prognostic method for monitoring the regeneration of autologous kidney after cell colony, mixture or construct described herein is administered or implanted into a subject. In one embodiment, the method includes the step of detecting the level of marker expression in a test sample and a control sample obtained from the subject, wherein compared with the control sample, the higher expression level of the marker in the test sample indicates the regeneration of the autologous kidney of the subject. In another embodiment, the method includes the expression of one or more stem cell markers in the detection sample. The stem cell marker can be selected from Sox2, UTF1, NODAL, CD133, CD24 and any combination thereof. The detection step can include measuring the expression of the stem cell marker in the test sample and being raised or higher relative to the control sample, wherein higher expression level indicates the regeneration of the autologous kidney of the subject. In another embodiment, the mRNA expression of the stem cell marker is detected. In other embodiments, mRNA expression can be detected by PCR-based methods, such as qRT-PCR. In situ hybridization can also be used to detect mRNA expression.

In another embodiment, the expression of a stem cell marker polypeptide is detected. In another embodiment, the expression of the polypeptide is detected using an anti-stem cell marker reagent. In another embodiment, the reagent is an anti-marker antibody. In another embodiment, the expression of the stem cell marker polypeptide is detected using immunohistochemistry or Western blotting.

Those skilled in the art will appreciate other methods for detecting mRNA and/or polypeptide expression of a marker.

In one embodiment, the detecting step is performed after the step of obtaining a test sample from the subject. In another embodiment, the test sample is kidney tissue.

In one other aspect, the invention provides the purposes of the surrogate marker of the regeneration of mark such as stem cell marker as autologous kidney.Such mark can be used independently or with the assessment (for example, erythrocyte dynamic balance and the index of renal function) of whether developed or enhanced regeneration based on function or ability.Monitoring surrogate marker can also be used as the prognostic index of regeneration in the time course of regeneration.

In another aspect, the invention provides a method for evaluating the prognosis of a patient after implanting or administering a cell colony, mixture, or construct described herein. In one embodiment, the method comprises the following steps: detecting the level of marker expression in a test sample obtained from the subject; (b) determining the expression level of the marker in the test sample relative to the marker expression level (or control reference value) of a control sample; and (c) predicting the regeneration prognosis of the patient based on the determination of the marker expression level, wherein a higher level of marker expression in the test sample compared to the control sample (or control reference value) predicts the regeneration in the subject.

In another aspect, the invention provides a method for evaluating the prognosis of a patient after implanting or administering a cell colony, mixture or construct described herein. In one embodiment, the method comprises the following steps: (a) obtaining a biological sample of the patient; and (b) detecting the expression of stem cell markers in the biological sample, wherein the expression of stem cell markers indicates the regeneration of the patient's autologous kidney. In some embodiments, the expression of the stem cell markers increased relative to a control sample (or control reference value) patient biological sample indicates the regeneration in the subject. In some embodiments, the expression of the stem cell markers reduced relative to a control sample (or control reference value) patient sample indicates the regeneration in the subject. The patient sample can be a test sample, including a biopsy. The patient sample can be a body fluid, such as blood or urine.

In one other aspect, the invention provides the prognostic method for the regeneration of autologous kidney that is used to or monitor after the experimenter is implanted by cell colony, mixture or construct described herein, wherein non-invasive method is used.As a substitute for biopsy tissue, the regeneration result in the experimenter for treatment can be received from the inspection assessment of body fluid such as urine.Have been found that the microvesicle obtained from the urine-derived microvesicle in experimenter's source comprises some components, includes but is not limited to specific proteins and the miRNA of the nephrocyte colony that finally derives from the treatment influence of cell colony of the present invention.This type of component can comprise the factor that participates in stem cell replication and differentiation, apoptosis, inflammation and immunomodulation.The time analysis of the expression pattern of the miRNA/ protein that microvesicle is combined allows continuous monitoring to accept the regeneration result in the kidney of the experimenter of cell colony of the present invention, mixture or construct.Embodiment 17 has described the exemplary scheme of the analysis of urine for experimenter.

Such kidney-derived vesicles and/or the luminal contents of kidney-derived vesicles excreted into the subject's urine can be analyzed for biomarkers indicative of regenerative outcome.

In one embodiment, the present invention provides a method for assessing whether a patient with kidney disease (KD) is responsive to treatment with a therapeutic agent. The method can include the step of determining or detecting the amount of vesicles or their luminal contents in a test sample obtained from a KD patient treated with the therapeutic agent, as compared to or relative to the amount of vesicles in a control sample, wherein a higher or lower amount of vesicles or their luminal contents in the test sample compared to the amount of vesicles or their luminal contents in the control sample indicates that the treated patient is responsive to treatment with the therapeutic agent.

The present invention also provides methods for monitoring the efficacy of treating a KD patient with a therapeutic agent. In one embodiment, the method includes the step of determining or detecting the amount of vesicles in a test sample obtained from a KD patient treated with the therapeutic agent, as compared to or relative to the amount of vesicles or their luminal contents in a control sample, wherein a higher or lower amount of vesicles or their luminal contents in the test sample compared to the amount of vesicles or their luminal contents in the control sample indicates efficacy of treating the KD patient with the therapeutic agent.

The present invention also provides methods for identifying an agent as a therapeutic agent effective for treating kidney disease (KD) in a patient subpopulation. In one embodiment, the method includes the step of correlating the efficacy of the agent with the presence of a number of vesicles in a sample from the patient subpopulation compared to the amount of vesicles or their luminal contents in a sample obtained from a control sample, wherein a higher or lower amount of vesicles or their luminal contents in the sample from the patient subpopulation compared to the amount of vesicles or their luminal contents in the control sample indicates that the agent is effective for treating KD in the patient subpopulation.

The present invention provides a method for identifying a patient subpopulation for which an agent is effective for treating kidney disease (KD). In one embodiment, the method includes the step of determining a correlation between the efficacy of the agent and the presence of a number of vesicles or their luminal contents in a sample from the patient subpopulation (compared to the amount of vesicles or their luminal contents in a sample obtained from a control sample), wherein a higher or lower amount of vesicles in the sample from the patient subpopulation compared to the amount of vesicles or their luminal contents in the control sample indicates that the agent is effective for treating KD in the patient subpopulation.

The determining or detecting step may include analyzing the amount of miRNA or other secreted product that may be present in the test sample (see Example 17).

The non-invasive prognostic method may include the step of obtaining a urine sample from the subject before and/or after administration or implantation of the cell population, mixture, or construct described herein. Standard techniques, including but not limited to centrifugation to remove unwanted debris (Zhou et al. 2008. Kidney Int. 74(5): 613-621; Skog et al., U.S. Published Patent Application No. 20110053157, each of which is incorporated herein by reference in its entirety), can be used to separate vesicles and other secreted products from the urine sample.

The present invention relates to a non-invasive method for detecting regenerative outcomes in a subject after treatment. The method comprises detecting vesicles or their lumen contents in the urine of the treated subject. The lumen contents may be one or more miRNAs. Detection of combinations or panels of individual miRNAs may be applicable to such prognostic methods. Exemplary combinations include two or more of the following: miR-24, miR-195, miR-871, miR-30b-5p, miR-19b, miR-99a, miR-429, let-7f, miR-200a, miR-324-5p, miR-10a-5p, and any combination thereof. In one embodiment, the combination of miRNAs may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more individual miRNAs. Those skilled in the art will appreciate that other miRNAs and combinations of miRNAs may be applicable to such prognostic methods. Sources of other miRNAs include miRBase at http://mirbase.org , which is hosted and maintained by the School of Life Sciences at the University of Manchester.

Those skilled in the art will appreciate that, in addition to the cell populations and constructs described herein, the prognostic methods for detecting regeneration may also be applicable to subjects treated with other therapeutic agents known in the art.

In some embodiments, the determining step comprises using a software program executed by a suitable processor to (i) measure the differential level of marker expression (or vesicles/vesicle contents) in the test sample and the control; and/or (ii) analyze the data obtained from measuring the differential level of marker expression in the test sample and the control. Suitable software and processors are well known in the art and commercially available. The program can be embodied in software stored on a tangible medium such as a CD-ROM, floppy disk, hard drive, DVD, or memory associated with the processor, but those skilled in the art will understand that the entire program or portions thereof can alternatively be executed by a device other than a processor and/or embodied in firmware and/or dedicated hardware in a known manner.

After the assay step, the measurement results, findings, diagnoses, predictions, and/or treatment recommendations are typically recorded and, for example, communicated to a technician, physician, and/or patient. In certain embodiments, a computer can be used to communicate such information to a party, such as a patient and/or attending physician. In some embodiments, the assay can be performed or the assay results analyzed in a country or jurisdiction different from the country or jurisdiction to which the results or diagnosis were communicated.

In a preferred embodiment, after the determination is completed and the prognosis and/or prediction is generated, the prognosis, prediction and/or treatment recommendations based on the level of marker expression measured in the test subject with the differential level of marker expression are communicated to the subject as quickly as possible. The results and/or relevant information can be communicated to the subject by the subject's treating physician. Alternatively, the results can be communicated directly to the test subject by any means of communication, including writing, electronic communication forms such as email or telephone. A computer can be used, for example, in the case of email communication, to help communicate. In certain embodiments, a communication containing the results of the prognostic test and/or the conclusions drawn from the test and/or the treatment recommendations based on the test can be generated and automatically delivered to the subject using a combination of computer hardware and software familiar to those skilled in the art of telecommunications. An example of a healthcare communication system is described in U.S. Patent No. 6,283,761; however, the present invention is not limited to methods utilizing this particular communication system. In certain embodiments of the methods of the present invention, all or some of the method steps, including the determination of the sample, the prognosis and/or prediction of regeneration, and the communication of the determination results or prognosis, can be performed in different (e.g., foreign) jurisdictions.

In another aspect, the prognostic methods described herein provide a party with information regarding the regenerative success of an implant or administration.

In all embodiments, the methods of providing a regenerated kidney to a subject in need of such treatment as described herein can include a post-implantation step of prognostic assessment of regeneration, as described above.

Methods and routes of administration

The cell preparations and/or constructs of the present invention may be administered alone or in combination with other biologically active components.

A therapeutically effective amount of a renal cell population or mixture of renal cell populations described herein can vary widely from the amount of cells that is safely accepted by a subject to the minimum amount of cells necessary to treat a renal disease, such as stabilization, reduced rate-of-decline, or enhancement of one or more renal functions. In certain embodiments, the methods of the invention provide for administration of a renal cell population or a mixture of renal cell populations described herein at a dose of about 10,000 cells/kg, about 20,000 cells/kg, about 30,000 cells/kg, about 40,000 cells/kg, about 50,000 cells/kg, about 100,000 cells/kg, about 200,000 cells/kg, about 300,000 cells/kg, about 400,000 cells/kg, about 500,000 cells/kg, about 600,000 cells/kg, about 700,000 cells/kg, about 800,000 cells/kg, about 900,000 cells/kg, about 1.1 x 10 6 cells/kg, about 1.2 x 10 6 cells/kg, about 1.3 x 10 6 cells/kg, about 1.4 x 10 6 cells/kg, about 1.6 x 10 6 cells/kg, about 1.7 x 10 6 cells/kg, about 1.8 x 10 6 cells/kg, about 1.9 x 10 6 cells/kg, about 2.3 x 10 6 cells/kg, about 2.4 x 10 6 cells/kg, about 2.5 x 10 6 cells/kg, about 2.6 x 10 6 cells/kg, about 2.7 x 10 ⁶ cells/kg, about 2.8 x 10 6 cells/kg, about 2.9 x 10 ⁶ cells/kg, about 3.4 x 10 ⁶ cells/kg, about 1.4x10 ⁶ cells/kg, about 1.5x10 ⁶ cells/kg, about 1.6x10 ⁶ cells/kg, about 1.7x10 ⁶ cells/kg, about 1.8x10 ⁶ cells/kg, about 1.9x10 ⁶ cells/kg, about 2.1x10 ⁶ cells/kg, about 2.1x10 ⁶ cells/kg, about 1.2x10 ⁶ cells/kg, about 2.3x10 ⁶ cells/kg, about 2.4x10 ⁶ cells/kg, about 2.5x10 ⁶ cells/kg, about 2.6x10 ⁶ cells/kg, about 2.7x10 ⁶ cells/kg, about 2.8x10 ⁶ cells/kg, about 2.9x10 ⁶ cells/kg, about 3x10 ⁶ cells/kg, about 3.1x10 ⁶ cells/kg, about 3.2x10 ⁶ cells/kg, about 3.3x10 ⁶ cells/kg, about 3.4x10 ⁶ cells/kg, about 3.5x10 ^{6 cells/kg, about 3.6x10 6 cells} /kg, about 3.7x10 ⁶ cells/kg, about 3.8x10 ⁶ cells/kg, about 3.9x10 ⁶ cells/kg, about 4x10 ⁶ cells/kg, about 4.1x10 ⁶ cells/kg, about 4.2x10 ⁶ cells/kg, about 4.3x10 ⁶ cells/kg, about 4.4x10 ⁶ cells/kg, about 4.5x10 ⁶ cells/kg, about 4.6x10 ⁶ cells/kg, about 4.7x10 ^{In some} embodiments, the dosage ^of the cell given to the experimenter can be a single dose or a single dose plus ^an additional dose. In other embodiments, the dosage can be provided by means of the construct described herein. In other embodiments, the dosage of the cell given to the experimenter can be calculated based on the renal mass or functional renal mass estimated.

The nephrocyte colony described herein or its mixture can be suspended in a pharmaceutically acceptable carrier or excipient for the treatment of an effective amount. Such carriers include but are not limited to basal medium plus 1% serum albumin, saline, buffered saline, glucose, water, collagen, alginate, hyaluronic acid, fibrin glue, polyethylene glycol, polyvinyl alcohol, carboxymethyl cellulose and combinations thereof. The preparation should be suitable for the mode of administration. Therefore, the invention provides nephrocyte colony or its mixture, such as B2 cell colony, alone or in admixture with B3 and/or B4 or B4' cell colony, for the manufacture of a medicament for the treatment of a subject's nephropathy. In some embodiments, the medicament also comprises a recombinant polypeptide such as a growth factor, a chemokine or a cytokine. In other embodiments, the medicament comprises a cell colony derived from human kidney. Any variant of the method described herein provided can be used to separate, derive or enrich the cells for the manufacture of the medicament.

According to conventional method, nephrocyte preparation or its mixture or composition are formulated as the pharmaceutical composition suitable for the human use.Usually, for intravenous administration, intra-arterial administration or the composition used in the kidney capsule, for example, is the solution in sterile isotonic buffered aqueous solution.If necessary, composition can also include local anesthetic to alleviate any pain on the injection site.Usually, can be separated or mixed together with unit dose form (for example, as the concentrate of the cryopreservation in the ampoule of the amount of the container such as labeling active agent) provide composition.When will be by infusion administration composition, can utilize the infusion bottle containing sterile pharmaceutical grade water or saline to disperse it.When by injection administration composition, can provide the sterile water for injection of an ampoule or saline, so that before using, mix composition.

Pharmaceutically acceptable carriers are determined in part by the specific composition to be administered and by the specific method for administering the composition. Therefore, there are many suitable formulations of pharmaceutical compositions (see, e.g., Alfonso RGennaro (ed.), Remington: The Science and Practice of Pharmacy, formerly Remington's Pharmaceutical Sciences, 20th edition, Lippincott, Williams & Wilkins, 2003, incorporated herein by reference in their entirety). Pharmaceutical compositions are typically formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

One aspect of the present invention also provides a pharmaceutical preparation comprising a renal cell population of the present invention, such as a B2 cell population alone or in combination with a B3 and/or B4 or B4' cell preparation, and a pharmaceutically acceptable carrier. In some embodiments, the preparation comprises 10 ⁴ to 10 ⁹ mammalian kidney-derived cells.

In one aspect, the present invention provides a method of providing one or more cell colonies described herein to a subject in need thereof. In one embodiment, the source of the cell colony can be autologous or allogeneic, isogenic (autologous or isogenic) and any combination thereof. In the case where the source is not autologous, the method may include administering an immunosuppressant. Suitable immunosuppressants include but are not limited to azathioprine, cyclophosphamide, mizoribine, cyclosporine, tacrolimus hydrate, chlorambucil, lobenzarit disodium, auranofin, alprostadil, guperlimus hydrochloride, biosynsorb, muromonab, alefacil, pentostatin, daclizumab, sirolimus, mycophenolate mofetil (mycophenolate mofetil) mofetil), eflonomide, basiliximab, dornase alfa, bindarid, cladribine, pimecrolimus, iloleukin, cilizumab, efalizumab, everolimus, animus, gavilimumab, faramoxumab, clofarabine, rapamycin, cilizumab, saireito, LDP-03, CD4, SR-43551, SK&F-106615, IDEC-114, IDEC-131, FTY-720, TSK-204, LF-080299, A-86281, A-802715, GVH-313, HMR-1279, Z D-7349, IPL-423323, CBP-1011, MT-1345, CNI-1493, CBP-2011, J-695, LJP-920, L-732531, ABX-RB2, AP-1903, IDPS, BMS-205820, BMS-224818, CTLA4-Ig, ER-49890, ER-38925, ISAtx-247, RDP-58, PNU-156804, LJP-1082, TMC-95A, TV-4710, PTR-262-MG, and AGI-1096 (see U.S. Pat. No. 7,563,822). Other suitable immunosuppressive drugs will be appreciated by those skilled in the art.

The therapeutic methods of the present invention include delivering the isolated renal cell colony or its mixture into an individual. In one embodiment, direct administration of the cells to the desired beneficial position is preferred. In one embodiment, the cell preparation of the present invention or its mixture can be delivered to an individual in a delivery vehicle.

The experimenter of this need can also be by by autologous kidney and from one or more enrichment nephrocyte colonies and/or mixture or the product body contact of the construct secretion that comprises described cell colony treat.Can realize the step that autologous kidney is contacted with secretion product in vivo by using/using one group (a population of) from cell culture medium such as secretion product of conditioned medium or by implanting cell colony and mixture or the construct that can secrete product in vivo.The step of contacting in vivo provides regeneration to autologous kidney.

According to this specification, many methods for administering cells and/or secretory products to a subject are obvious to those skilled in the art. Such technology includes injection of cells into the target position of a subject. Cells and/or secretory products can be inserted into a delivery device or vehicle, which is introduced by injection into or implanted into a subject. In certain embodiments, the delivery vehicle can include natural materials. In certain other embodiments, the delivery vehicle can include synthetic materials. In one embodiment, the delivery vehicle provides a structure that simulates or appropriately fits into an organ structure. In other embodiments, the delivery vehicle is fluid-like in nature. Such a delivery device can include a tube, such as a catheter, for injecting cells and fluid into the body of the recipient. In a preferred embodiment, the tube additionally has a needle, such as a syringe, by which the cells of the present invention can be introduced into a subject at a desired position. In some embodiments, a mammalian kidney-derived cell colony is formulated to utilize a catheter (wherein the term "catheter" is intended to include any various tubular systems for delivering substances to a blood vessel) to be administered into a blood vessel. Alternatively, the cells can be inserted into or onto a biomaterial or scaffold (including but not limited to textiles such as braids, fabrics, braids, meshes and non-wovens, perforated films, sponges and foams, and beads such as solid or porous beads, microparticles, nanoparticles, etc. (e.g., Cultispher-S gelatin beads - Sigma)). Cells for delivery can be prepared in a variety of different forms. For example, the cells can be suspended in a solution or gel. The cells can be mixed with a pharmaceutically acceptable carrier or diluent in which the cells of the present invention remain viable. Pharmaceutically acceptable carriers and diluents include saline, buffered aqueous solutions, solvents and/or dispersion media. The uses of such carriers and diluents are well known in the art. The solution is preferably sterile and fluid, and is generally isotonic. Preferably, the solution is stable under the conditions of manufacture and storage and resists contamination by microorganisms such as bacteria and fungi by using, for example, parahydroxybenzoic acid, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Those skilled in the art will appreciate that delivery vehicles for delivering the cell populations and mixtures thereof of the present invention may include a combination of the above-mentioned features.

The mode of administration of the B2 cell colony of the nephrocyte colony of separation such as independently or with B4 ' and/or B3 mixing includes but is not limited to systemic, intrarenal (such as, renal parenchyma), intravenous or intraarterial injection and the injection of the tissue directly to the active position of expectation.Other modes of administration to be used according to the present invention include by direct laparotomy, by direct laparoscopy, transabdominal or percutaneous single or multiple injections.Other modes of administration to be used according to the present invention include such as retrograde infusion and ureteropelvic infusion.Surgery application method includes that one-step method such as but not limited to partial nephrectomy and construct implantation, partial nephrectomy, partial patellar resection, utilizing omentum (omentum) ± peritoneum (peritoneum) vascularization, multifocal biopsy needle track (multifocal biopsy needle track) (cone or cone to cylinder) and renal pole-like replacement (renal pole-like replacement), and two-step method includes such as for replanting (replanting) organoid-internal bioreactor (organoid-internal bioreactor). In one embodiment, a mixture of cells is delivered simultaneously by the same route. In another embodiment, each cell composition comprising a controlled mixture is delivered separately to a specific location or delivered to a specific location simultaneously or in a time-controlled manner by one or more methods described herein.

The appropriate cell implantation dose for humans can be determined based on existing information regarding the cells' activities, such as EPO production, or can be extrapolated from dosing studies conducted in preclinical studies. Based on in vitro culture and in vivo animal studies, the amount of cells can be quantified and used to calculate the appropriate dose of implanted material. Alternatively, the patient can be monitored to determine whether additional implants can be performed or whether the implanted material can be reduced accordingly.

One or more other components may be added to the cell populations and mixtures thereof of the invention, including selected extracellular matrix components, such as one or more types of collagen or hyaluronic acid, and/or growth factors, platelet-rich plasma, and drugs known in the art.

Those skilled in the art will appreciate the various formulations and administration methods suitable for the secretion products described herein.

Reagent test kit

The present invention also includes a test kit, which includes a polymer matrix of the present invention and support and related materials, and/or cell culture medium and instructions for use. Instructions for use can include, for example, instructions for culturing cells or administering cells and/or cell products. In one embodiment, the invention provides a test kit including support described herein and instructions. In another embodiment, the test kit includes reagents for detecting marker expression, reagents for the use of medicaments (reagents for use of the agent) and instructions for use. The test kit can also be used for measuring the purpose of the regeneration prognosis of the autologous kidney of the experimenter after implanting or administering cell colony, mixture or construct described herein. The test kit can also be used for measuring the biotherapy efficacy of cell colony, mixture or construct described herein.

Report

The method of the present invention, when implemented for commercial purposes, generally produces a report or summary of the regeneration prognosis. The method of the present invention will generate a report including any application or implantation of the cell colony, mixture or construct described herein and a forecast of the possible process or result of regeneration. The report may include information about any indicator relevant to the prognosis. The method and report of the present invention may also include storing the report in a database. Alternatively, the method may also generate a record of the experimenter and fill in the record data in the database. In one embodiment, the report is a paper report, in another embodiment, the report is an auditory report (auditorial report), and in another embodiment, the report is an electronic record. The expected report is provided to a doctor and/or patient. The acceptance of the report may also include establishing a network connection to a server computer including data and reports and requesting data and reports from the server. The method provided by the invention may also be fully or partially automated.

All patents, patent applications, and literature references cited in this specification are incorporated herein by reference in their entirety.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Example

Example 1 - Isolation and Characterization of Bioresponsive Renal Cells

Cases of idiopathic progressive chronic kidney disease (CKD) with anemia in adult male pigs (Sus scrofa) provide fresh diseased kidney tissue for evaluation and characterization of cellular composition by direct comparison with age-matched normal pig kidney tissue. Histological examination of kidney tissue during harvest confirmed nephropathy characterized by severe chronic diffuse interstitial fibrosis and crescentic glomerulonephritis with multifocal fibrosis. Clinical chemistry confirmed azotemia (increase in blood urea nitrogen and serum creatinine) and mild anemia (slight reduction in hematocrit and reduced hemoglobin levels). Cells were isolated from diseased and normal kidney tissue, amplified, and characterized. As shown in FIG1 of Presnell et al. WO/2010/056328 (incorporated herein by reference in its entirety), Gomori's Trichrome staining highlighted the fibrosis (blue staining indicated by arrows) of diseased kidney tissue compared with normal kidney tissue. Functional renal cells expressing cubulin and capable of receptor-mediated albumin transport were propagated from normal and diseased renal tissue. Cells expressing erythropoietin (EPO) were also present in culture and maintained through multiple passages and freeze/thaw cycles. In addition, molecular analysis confirmed that EPO-expressing cells from normal and diseased tissue responded to hypoxic conditions in vitro through HIF1α-driven induction of EPO and other hypoxia-regulated gene targets including vEGF. Cells were isolated from porcine kidney tissue by enzymatic digestion using collagenase + dispase, and the cells were also isolated in a separate experiment by simple mechanical digestion and explant culture. In the second generation, explant-derived cell cultures containing EPO-expressing cells were subjected to atmospheric (21%) and different hypoxic (<5%) culture conditions to determine whether exposure to hypoxia culminated in the upregulation of EPO gene expression. As in rodent cultures (see Example 3), normal pigs showed oxygen-dependent expression and regulation of the EPO gene. Surprisingly, despite the uremic/anemic state of CKD pigs (hematocrit <34, creatinine> 9.0), EPO-expressing cells can be easily isolated and propagated from tissues, and the expression of the EPO gene is still regulated by hypoxia, as shown in Figure 2 of Presnell et al. WO/2010/056328 (incorporated herein in its entirety by reference). As shown in Figure 3 of Presnell et al. WO/2010/056328 (incorporated herein in its entirety by reference), the cells in the propagated culture showed the ability to self-organize into tubular structures. As shown in Figure 4 of Presnell et al. WO/2010/056328 (incorporated herein in its entirety by reference), the presence of functional tubular cells in culture (at passage 3) was confirmed by observing receptor-mediated uptake of FITC-conjugated albumin by the cultured cells. Green dots (indicated by thin white arrows) represent fluorescein-conjugated albumin endocytosed by the tubular cell-specific receptors megaprotein and cuboprotein, indicating that the protein is reabsorbed by functional tubular cells. Blue staining (indicated by thick white arrows) is Hoescht-stained nuclei. Taken together, these data indicate that functional tubular and endocrine cells can be isolated and propagated from porcine kidney tissue, even in kidney tissue that has been severely damaged by CKD. Furthermore, these findings support the advancement of autologous cell-based therapeutic products for the treatment of CKD.

In addition, from normal adult human kidney enzymatic separation EPO founder cell (as shown in embodiment 1).As shown in Fig. 5 of the people such as Presnell WO/2010/056328 (being incorporated herein by reference in their entirety), separation method causes after separation than more relative EPO expression in initial tissue.As shown in Fig. 6 of the people such as Presnell WO/2010/056328 (being incorporated herein by reference in their entirety), may in culture, maintain people EPO founder cell, retain EPO gene expression.Plastics at planar tissue culture treatment or with some extracellular matrix such as fibronectin or collagen coated plastics, cultivate/breeding human cell, and find that all cells support EPO expression in the past over time.

Example 2 - Isolation and Enrichment of Specific Bioreactive Renal Cells

Isolation of renal cells: Briefly, batches of 10 kidneys from 2-week-old male Lewis rats were obtained from a commercial supplier (Hilltop Lab Animals Inc.) and loaded overnight in Viaspan storage medium at a temperature of approximately 4°C. All steps described herein were performed in a biosafety kitchen (BSC) to maintain sterility. The kidneys were washed three times in Hank's balanced salt solution (HBSS) to rinse off the Viaspan storage medium. After the third wash, the remaining renal capsule and any remaining stromal tissue were removed. Microdissection techniques were also used to remove the renal calyces. The kidneys were then finely minced into a pulp using a sterile scalpel. The pulp was then transferred to a 50ml conical bottom centrifuge tube and weighed. A small amount of RNA was collected, placed in a sterile 1.5ml microcentrifuge tube free of RNase, and snap frozen in liquid nitrogen. After freezing, the samples were then transferred to a -80 degree freezer until analysis. The tissue weight of 10 juvenile kidneys was approximately equal to 1 gram. Based on the weight of the batch, the digestion medium was adjusted to deliver 20 ml of digestion medium per 1 gram of tissue. The digestion buffer used for this method contained 4 units of Dispase 1 (Stem Cell Tech), 300 units/ml of Collagenase Type IV (Worthington), and 5 mM _CaCl2 (Sigma) in HBSS.

An appropriate volume of pre-warmed digestion buffer is added to the tube, which is then sealed and placed in a 37°C incubator on a shaker for 20 minutes. This first digestion step removes many red blood cells and enhances digestion of the remaining tissue. After 20 minutes, the tube is removed and placed in a BSC. The tissue is allowed to settle at the bottom of the tube, and the supernatant is removed. The remaining tissue is then replenished with fresh digestion buffer equal to the starting volume. The tube is again placed in a 37°C incubator on a shaker for another 30 minutes.

After 30 minutes, the digestion mixture was transferred through a 70 μm cell strainer (BD Falcon) with a pipette into an equal volume of neutralization buffer (DMEM w/10% FBS) to terminate the digestion reaction. The cell suspension was subsequently washed by centrifugation at 300 x g for 5 minutes. After centrifugation, the pellet was resuspended in 20 ml of KSFM culture medium, and samples were obtained for cell counting and viability assessment using the trypan blue exclusion method. After calculating the cell count, 1 million cells were collected to extract RNA, the cells were washed in PBS, and then quickly frozen in liquid nitrogen. The remaining cell suspension was placed in 50 ml of KSFM culture medium and washed again by centrifugation at 300 x g for 5 minutes. After washing, the cell pellet was resuspended at a concentration of 15 million cells/ml of KSFM.

5 milliliters of kidney cell suspensions were subsequently added to 5ml 30% (w/v) in a 15ml conical centrifuge tube (BD Falcon), followed by mixing by inversion 6 times. This formed a final mixture of 15% (w/v). After inversion, the tube was carefully layered with 1mL PBS. The tube was centrifuged at 800xg for 15 minutes without a brake. After centrifugation, the tube was taken out and a cell band (cell band) was formed on the top of the mixed gradient. There was also a precipitation comprising red blood cells, dead cells and a small group of living cells (including some small less granular cells, some erythropoietin-producing cells, some renal tubular cells and some endothelial cells). The band was carefully taken out using a pipette and transferred to another 15ml conical centrifuge tube. The gradient medium was pipetted by suction and the precipitation was collected by refloating in 1ml KSFM. The band cells and pellet cells were then reassembled and resuspended in at least three dilutions of the collected band volume using KSFM. Washing was performed by centrifugation at 300 x g for 5 minutes. After washing, the cells were resuspended in 20 ml of KSFM and a sample was collected for cell counting. After calculating the cell count using trypan blue exclusion, 1 million cells were collected for RNA extraction, washed in PBS, and then snap-frozen in liquid nitrogen.

Pre-culture 'clean-up' to enhance the viability and culture performance of specific bioactive renal cells using density gradient separation : In order to generate a clean, viable cell population for cultivation, a cell suspension is first generated as described in "kidney cell separation" above. As an optional step and as a method for purifying the initial preparation, a total of 100 million cells suspended in sterile isotonic buffer are thoroughly mixed with an equal volume of 30% (thereby generating a final 15% w/v Optiprep solution) prepared from a stock solution of 60% (w/v) iodixanol at room temperature in a 1:1 ratio and thoroughly mixed by inverting 6 times. After mixing, 1 ml of PBS buffer is carefully layered on top of the mixed cell suspension. The gradient tube is then carefully loaded into a centrifuge to ensure proper balance. The gradient tube is centrifuged at 800 x g for 15 minutes at 25 ° C without a brake. The purified cell population (containing viable, functional collecting duct cells, tubular cells, endocrine cells, glomerular cells, and vascular cells) was fractionated between 6% and 8% (w/v), corresponding to a density of 1.025-1.045 g/mL. Other cells and debris settled to the bottom of the tube.

Kidney cell culture : The combined cell strips and pellets were then plated in a tissue culture treated triple flask (Nunc T500) or equivalent at a cell concentration of 30,000 cells/cm2 in 150 ml of a 50:50 mixture of DMEM (high glucose)/KSFM (containing 5% (v/v) FBS, 2.5 μg EGF, 25 mg BPE, 1X ITS (insulin/transferrin/sodium selenite medium supplement) and antibiotics/antimycotics). The cells were cultured for 2-3 days in a humidified 5% CO2 incubator, providing the cells with an atmospheric oxygen level of 21%. After two days, the medium was changed and the cultures were placed in a CO2/nitrogen multigas humidified incubator (Sanyo) at 2% oxygen level for 24 hours. After 24 hours of incubation, the cells were washed with 60 ml of 1XPBS and then removed with 40 ml of 0.25% (w/v) trypsin/EDTA (Gibco). After removal, the cell suspension was neutralized with an equal volume of KSFM containing 10% FBS. The cells were then washed by centrifugation at 300 x g for 10 minutes. After washing, the cells were resuspended in 20 ml of KSFM, transferred to a 50 ml conical tube, and the sample was collected for cell counting. After determining the viable cell count using trypan blue exclusion, 1 million cells were collected for RNA extraction, washed in PBS, and then quickly frozen in liquid nitrogen. The cells were washed again in PBS and collected by centrifugation at 300 x g for 5 minutes. The washed cell pellet was resuspended in KSFM at a concentration of 37.5 million cells/ml.

Use density discontinuous gradient to separate and enrich specific biological activity nephrocyte: use the density discontinuous gradient prepared by the iodixanol (Optiprep) from multiple concentration w/v to be mainly composed of renal tubular cells but the nephrocyte of the culture of the small subpopulation that comprises other cell types (collecting tubule, renal glomerulus, blood vessel and endocrine) is divided into their component subpopulation.Culture is placed in anoxic environment and reached 24 hours, then gathered in the crops and be used for gradient.By in aseptic 15mL conical tube, making 4 kinds of different density media layering on each other's top, the solution with the highest density is placed in the bottom and the solution with the lowest density is placed in the top and generates discontinuous gradient.Cell is applied to the top of discontinuous gradient, centrifugal then, this causes colony to be separated into multiple bands based on size and granularity.

In brief, densities of 7, 11, 13, and 16% (60% w/v iodixanol) were prepared using KFSM medium as a diluent. For example, for 50 ml of 7% (w/v), 5.83 ml of a stock solution of 60% (w/v) iodixanol was added to 44.17 ml of KSFM medium and thoroughly mixed by inversion. A sterile L/S 16 Tygon peristaltic pump (Master Flex L/S) connected to a sterile capillary via tubing was set to a flow rate of 2 ml per minute, and 2 ml of each of the four solutions were loaded into a sterile 15 ml conical tube, starting with a 16% solution, then a 13% solution, an 11% solution, and a 7% solution. Finally, 2 ml of a cell suspension containing 75 million cultured rodent kidney cells was loaded on top of the discontinuous gradient (a suspension generated as described in the 'renal cell culture' section above). Importantly, when the pump starts to deliver the gradient solution to the pipe, the fluid is carefully made to slowly flow down along the side of the pipe at a 45 ° angle to ensure that a suitable interface is formed between each layer of the gradient. Subsequently, the discontinuous gradient loaded with cells is centrifuged at 800xg for 20 minutes without a brake. After centrifugation, the pipe is carefully taken out so as not to disturb each interface. 5 different cell fractions (4 bands and precipitation) (B1-B4, + precipitation) (see Figure 1A, left conical tube) are produced. Sterile disposable ball pipette or 5ml pipette are used to collect each fraction, and the fraction (see the embodiment 10 of WO/2010/056328 et al., Presnell et al.) are characterized in phenotype and function. When the rodent nephrocyte suspension is immediately subjected to discontinuous gradient fractionation after separation, the fraction enriched in renal tubular cells (and comprising some cells from the collecting duct) is divided into a density of 1.062-1.088g/mL. In the present invention, the fraction of urinary tubular cell (and comprising some cells from collecting duct) is divided into the density of 1.051-1.062g/mL.Similarly, when the rodent nephrocyte suspension is immediately subjected to the discontinuous gradient fractionation after separation, the fraction (" B4 ") having been enriched in erythropoietin founder cell, glomerular podocyte and vascular cell is separated on the density of 1.025-1.035g/mL.In the present invention, the fraction of urinary tubular cell (and comprising some cells from collecting duct) is divided into the density of 1.051-1.062g/mL.Similarly, when the rodent nephrocyte suspension is immediately subjected to the discontinuous gradient fractionation after separation, the fraction (" B4 ") having been enriched in erythropoietin founder cell, glomerular podocyte and vascular cell is separated on the density of 1.025-1.035g/mL. Importantly, post-cultivation distribution of cells into "B2" and "B4" fractions was enhanced by exposing the cultures to a hypoxic culture environment (hypoxia is defined as oxygen levels less than 21% (atmospheric)) for periods ranging from approximately 1 hour to 24 hours, prior to harvest and step gradient analysis (additional details regarding the effects of hypoxia on banding distribution are provided in Example 3).

Each strip was washed by diluting with 3x volumes of KSFM, thoroughly mixed, and centrifuged at 300x g for 5 minutes. The pellet was resuspended in 2 ml of KSFM and viable cells were counted using trypan blue exclusion and a hemocytometer. One million cells were collected to extract RNA samples, washed in PBS, and then rapidly frozen in liquid nitrogen. Cells from B2 and B4 were used for transplantation studies in uremic and anemic female rats (generated by two-step 5/6 nephrectomy at Charles River Laboratory). Quantitative real-time PCR was used to confirm the characteristics of B4, including the expression of oxygen-regulated erythropoietin and vEGF, the expression of glomerular markers (phenylephrine, podocin), and the expression of vascular markers (PECAM). The phenotype of the 'B2' fraction was confirmed by the expression of E-cadherin, N-cadherin, and aquaporin-2. See Figures 49a and 49b of Presnell et al. WO/2010/056328.

Therefore, the use of the discontinuous gradient strategy not only allows the rare population (B4) of erythropoietin-producing cells to be enriched, but also is a method for generating a fraction (B2) of relatively enriched functional renal tubular cells (see Figures 50 and 51 of Presnell et al. WO/2010/056328). The discontinuous gradient strategy also allows EPO-producing cells and renal tubular cells to be separated from erythrocytes, cell debris, and other potential undesirable cell types such as large cell aggregates and certain types of immune cells.

The step gradient method may require adjustment in terms of the specific density used to provide good separation of cellular components. Preferred methods for adjusting the gradient include 1) running a continuous density gradient that progresses from a high density at the bottom of the gradient (e.g., 16-21% Optiprep) to a relatively low density at the top of the gradient (e.g., 5-10%). Continuous gradients can be prepared using any standard density gradient solution (Ficoll, Percoll, sucrose, iodixanol) according to standard methods (Axis Shield). The target cells are loaded onto the continuous gradient and centrifuged at 800xG for 20 minutes without a brake. Cells of similar size and granularity tend to separate together in the gradient, so the relative position in the gradient can be measured, and the specific gravity of the solution at that position can also be measured. Thus, a specific step gradient can then be derived that focuses on the separation of specific cell populations based on their ability to traverse the density gradient under specific conditions. Such optimization may be necessary when isolating cells from unhealthy versus healthy tissue, or when isolating specific cells from different species. For example, canine and human kidney cell cultures were optimized to ensure that specific B2 and B4 subpopulations identified in rats could be isolated from other species. The optimal gradient for isolating rodent B2 and B4 subpopulations consisted of 7%, 11%, 13%, and 16% (w/v) Optiprep. The optimal gradient for isolating canine B2 and B4 subpopulations consisted of 7%, 10%, 11%, and 16% (w/v) Optiprep. The optimal gradient for isolating human B2 and B4 subpopulations consisted of 7%, 9%, 11%, and 16% (w/v). Thus, the density ranges for localizing B2 and B4 from cultured rodent, canine, and human kidney cells are provided in Table 2.1.

Table 2.1. Species density ranges

Example 3 - Hypoxic incubation prior to gradient affects band distribution, composition, and gene expression

In order to measure the influence of oxygen condition on the distribution and composition of prototype B2 and B4, the new kidney cell preparation from different species is exposed to different oxygen conditions, and then gradient step is carried out.Use the standard method (as described above) that is used for rat cell separation and cultivation initialization to set up rodent neonatal kidney enhancement (neo-kidney augmentation) (NKA) cell preparation (RK069).All culture bottles were cultivated 2-3 days under 21% (atmosphere) oxygen condition.Change culture medium, subsequently half culture bottles are reset to the oxygen-controlled incubator that is arranged to 2% oxygen, and the remaining culture bottles are kept other 24 hours under 21% oxygen condition simultaneously.Use the standard enzymatic harvesting method described above to come from each group of condition harvesting cells subsequently.Prepare discontinuous gradient according to standard method, gather in the crops " normoxia " (21% oxygen) and " hypoxia " (2% oxygen) culture separately, it is used for identical discontinuous gradient (Fig. 2) in parallel. Although four bands and a precipitate were generated under both conditions, the distribution of cells across the gradient was different in the batches cultured at 21% and 2% oxygen (Table 1). Specifically, the yield of B2 increased due to hypoxia, accompanied by a decrease in B3. In addition, expression of B4-specific genes (e.g., erythropoietin) was enhanced in the resulting gradient generated from cells cultured under hypoxia (Figure 73 of Presnell et al. WO/2010/056328).

A canine NKA cell preparation (DK008) was established using standard methods for dog cell isolation and culture (similar to rodent isolation and culture methods), as described above. All culture flasks were cultured under 21% (hypoxia) oxygen conditions for 4 days, after which a subset of culture flasks was transferred to hypoxia (2%) for 24 hours, while a subset of culture flasks was maintained at 21%. Subsequently, each group of culture flasks was harvested and subjected to the same discontinuous gradient (Figure 3). Similar to the rat results (Example 1), hypoxic and atmospheric oxygen-cultured dog cells were differentially distributed throughout the gradient (Table 3.1). Again, the yield of B2 increased due to hypoxic exposure before the gradient was performed, accompanied by a decrease in distribution to B3.

Table 3.1.

The above data show that pre-gradient exposure to hypoxia increases the composition of B2 and the distribution of specific specialized cells (erythropoietin-producing cells, vascular cells, and glomerular cells) to B4. Therefore, as described above, hypoxic culture followed by density gradient separation is an effective method to generate 'B2' and 'B4' cell populations across species.

Example 4 - Isolation of tubular/glomerular cells from human kidney

Utilize the enzymatic separation method of detailed description to separate and breed renal tubules and glomerular cells from normal human kidney tissue.By the above-mentioned gradient method, after cultivation, in vitro enrichment of renal tubular cell fraction.As shown in Figure 68 of Presnell et al. WO/2010/056328 (incorporated herein in their entirety by reference), phenotypic attributes are maintained in separation and breeding.The renal tubular cell function assessed by the absorption of labeled albumin is also maintained after repeated passage and cryopreservation.Figure 69 of Presnell et al. WO/2010/056328 (incorporated herein in their entirety by reference) shows that when cultivating a colony of renal tubular cells and a colony of renal tubular cells consumed in 3D dynamic culture, the significant increase of the expression of renal tubular marker E-cadherin is shown in the colony of renal tubular cells enriched.This confirms that when cultivating cells in a 3D dynamic environment, the enrichment of renal tubular cells exceeding initial enrichment can be maintained.

Example 5 - Further separation of EPO-producing cells by flow cytometry

The same cultured population of kidney cells described above in Example 2 was subjected to flow cytometric analysis to examine forward and side scatter. A small, less granular EPO-producing cell population was discernible (8.15%) and could be isolated by positive selection of the small, less granular population using the sorting capabilities of flow cytometry (see Figure 70 of Presnell et al. WO/2010/056328, incorporated herein by reference in its entirety).

Example 6 - Unfractionated Mixture of Renal Cells Isolated from Autoimmune Glomerulonephritis Patient Samples Characterization

As mentioned above, the unfractionated mixture of nephrocytes is separated from autoimmune glomerulonephritis patient samples. In order to measure the unbiased phenotypic composition of the specific subpopulation of nephrocytes separated and amplified from renal tissue, quantitative real-time PCR (qrtpcr) analysis (Brunskill et al., the same, 2008) is used to identify differential cell type specificity and pathway specific gene expression patterns between cell subfractions. As shown in Table 6.1, HK20 is an autoimmune glomerulonephritis patient sample. Table 6.2 shows that the cells produced from HK20 lack glomerular cells, as measured by qRTPCR.

Example 7 - Therapeutically relevant renal bioactivities isolated from cases of focal segmental glomerulosclerosis Genetic profiling of cell populations

In order to determine the unbiased genotype composition of a specific subpopulation of renal cells isolated and amplified from renal tissue, quantitative real-time PCR (qrtpcr) analysis (Brunskill et al., supra, 2008) was used to identify differential cell type-specific and pathway-specific gene expression patterns between cell fractions. The presence of glomerular cells in the B4 fraction of human preparation HK023 (derived from a case of focal segmental glomerulosclerosis (FSGS) in which most glomeruli have been destroyed) was assessed at harvest. In brief, unfractionated (UNFX) cultures were generated individually (Aboushwareb et al., supra, 2008) and maintained from each of (4) core biopsies obtained from the kidney using a standard biopsy method. After UNFX was passaged in vitro (2), cells were harvested and subjected to a density gradient method (as in Example 8) to produce subfractions, including subfraction B4, which is known to be enriched in endocrine cells, vascular cells, and glomerular cells based on work performed in rodents, dogs, and other human samples.

Individually from each independent UNFX sample collection B4 fraction of HK023, described fraction shows as the unique cell band with the buoyant density of 1.063-1.091g/mL.From each sample separation RNA, its Podocin (glomerular cell marker) and PECAM (endothelial cell marker) expression was checked by quantitative real-time PCR.As expected from the sample generated from the biopsy of the case of serious FSGS, the existence of podocin (+) glomerular cell in the B4 fraction can not be detected inconsistent with podocin in 2/4 sample.On the contrary, PECAM+ vascular cells are always present in the B4 fraction of the culture started by the biopsy of 4/4.Therefore, can on 1.063-1.091g/mL density range, even from the people's kidney separation B4 fraction with severe disease state.

Table 7.1 Expression of Podocin and PECAM in glomerular and vascular cells isolated from FSGS cases using subfraction B4

Furthermore, as shown in Table 7.2, the human sample (HK018) showed no detectable Podocin (a glomerular marker) by qRTPCR after density gradient centrifugation.

Table 7.2. Characterization of gene expression after HK018 gradient in B2&B4'

Gene RQ(Unfx) RQ(B2) RQ(B4) B2/B4 Podocin 1 ND ND - VegF 1 1.43 1.62 0.9 Aqp1 1 1.7 1.2 1.4 Epo 1 0.9 0.5 1.8 Cubic protein 1 1.2 0.7 1.7 Cyp 1 1.2 1.4 0.85 Ecad 1 1.15 0.5 2.3 Ncad 1 1.02 0.72 1.4

Example 8 - Enrichment/depletion of viable kidney cell types using fluorescence activated cell sorting (FACS)

Fluorescence activated cell sorting (FACS) can be used to enrich for one or more isolated renal cells and/or deplete one or more specific renal cell types from isolated primary renal tissue.

Reagents: 70% ethanol; wash buffer (PBS); 50:50 renal cell culture medium (50% DMEM high glucose): 50% keratinocyte-SFM; trypan blue 0.4%; primary antibodies against target renal cell populations such as CD31 (for renal endothelial cells) and nephrine (for renal glomerular cells). Matched isotype-specific fluorescent secondary antibodies; staining buffer (0.05% BSA in PBS).

Methods: Following standard procedures in a clean biosafety kitchen (BSC), single-cell suspensions from primary isolated or cultured renal cells can be obtained from T500T/C-treated culture flasks, resuspended in renal cell culture medium, and placed on ice. Cells are then counted and viability determined using trypan blue exclusion. To enrich/deplete a heterogeneous population of renal cells, such as glomerular cells or endothelial cells, 10 to 50e6 viable cells with at least 70% viability are obtained. The heterogeneous population of renal cells is then stained with a primary antibody specific for the target cell type at a starting concentration of 1 μg/0.1 ml of staining buffer per 1 x 10 ⁶ cells (titered as needed). The target antibody can be conjugated (e.g., CD31PE (specific for renal endothelial cells)) or unconjugated (e.g., phenylephrine (specific for renal glomerular cells)).

The cells are then stained on ice or in the dark at 4°C for 30 minutes. After the 30-minute incubation, the cells are washed by centrifugation at 300 x g for 5 minutes. The pellet is then resuspended in PBS or staining buffer, depending on whether a conjugated isotype-specific secondary antibody is required. If the cells are labeled with a primary antibody conjugated to a fluorescent dye, the cells are resuspended in 2 ml of PBS per 10e7 cells and then proceed to a FACS aria or equivalent cell sorter. If the cells are not labeled with an antibody conjugated to a fluorescent dye, the cells are labeled with an isotype-specific secondary antibody conjugated to a fluorescent dye at a starting concentration of 1 ug/0.1 ml/1e6 cells.

The cells were then stained on ice or in the dark at 4°C for 30 minutes. After the 30-minute incubation, the cells were washed by centrifugation at 300 x g for 5 minutes. After centrifugation, the pellet was resuspended in PBS at a concentration of 5e6/ml and 4 ml was transferred to a sterile tube per 12 x 75 mm.

Prepare the FACs Aria for aseptic sorting of living cells according to the manufacturer's instructions (BD FACs Aria User Manual). Load the sample tube into the FACs Aria and adjust the PMT voltage after acquisition. Pull down the gate to use a specific wavelength and utilize fluorescence intensity to select kidney-specific cell types. Pull down another gate to select a negative colony. After the desired gate has been pulled down to enclose the positive target colony and the negative colony, use the manufacturer's instructions to sort the cells.

The positive target population was collected in one 15 ml conical tube, and the negative population was collected in another 15 ml conical tube containing 1 ml of renal cell culture medium. After collection, samples from each tube were analyzed by flow cytometry to determine purity. The collected cells were washed by centrifugation at 300 x g for 5 minutes, and the pellet was resuspended in renal cell culture medium for further analysis and experiments.

Example 9 - Enrichment/depletion of kidney cell types using magnetic cell sorting

Isolated primary renal tissue can be enriched for one or more isolated renal cells and/or depleted for one or more specific renal cell types.

Reagents: 70% ethanol; wash buffer (PBS); 50:50 renal cell culture medium (50% DMEM high glucose): 50% keratinocyte-SFM; trypan blue 0.4%; running buffer (PBS, 2 mM EDTA, 0.5% BSA); rinse buffer (PBS, 2 mM EDTA); cleaning solution (70% v/v ethanol); Miltenyi FCR blocking reagent; Miltenyi microbeads or secondary antibody specific for IgG isotype target antibodies such as CD31 (PECAM) or phenylephrine.

Methods: Following standard procedures in a clean biosafety kitchen (BSC), single-cell suspensions of renal cells from primary isolates or cultures were obtained and resuspended in renal cell culture medium. Cell counts and viability were determined using trypan blue exclusion.

For enrichment/depletion of renal cells, such as glomerular cells or endothelial cells, from a heterogeneous population, at least 10e6 up to 4e9 viable cells with a viability of at least 70% are obtained.

The optimal separation for the enrichment/depletion method is determined based on the target cells of interest. To enrich for targets with a frequency below 10%, such as glomerular cells (using phenylephrine), use the Miltenyi autoMACS, or equivalent, instrument program POSSELDS (double positive selection in sensitive mode). To deplete targets with a frequency greater than 10%, use the Miltenyi autoMACS, or equivalent, instrument program DEPLETES (depletion in sensitive mode).

Live cells were labeled with a target-specific primary antibody such as nephrine rb polyclonal antibody (for glomerular cells) by adding 1 μg/10e6 cells/0.1 ml of PBS containing 0.05% BSA in a 15 ml conical centrifuge tube and then incubating at 4°C for 15 minutes.

After labeling, wash the cells to remove unbound primary antibody by adding 1-2 ml of buffer per 10e7 cells and then centrifuge at 300 x g for 5 minutes. After washing, add isotype-specific secondary antibody such as chicken anti-rabbit PE at 1 ug/10e6/0.1 ml of PBS containing 0.05% BSA and incubate at 4°C for 15 minutes.

After incubation, the cells were washed to remove unbound secondary antibody by adding 1-2 ml of buffer per 10e7 cells and then centrifuging at 300 x g for 5 minutes. The supernatant was removed and the cell pellet was resuspended in 60 μl of buffer per 10e7 total cells, followed by adding 20 μl of FCR encapsulation reagent per 10e7 total cells, which were then mixed thoroughly. 20 μl of direct MACS beads (e.g., anti-PE beads) were added, mixed, and then incubated at 4°C for 15 minutes.

After incubation, cells were washed by adding 10-20x the labeled volume of buffer and then centrifuging the cell suspension at 300xg for 5 minutes and resuspending the cell pellet in 500 μl-2 ml of buffer per 10e8 cells.

Clean and prime the autoMACS system according to the manufacturer's instructions to prepare for magnetic cell separation using the autoMACS. Place a new sterile collection tube under the discharge port. Select the autoMACS cell separation program. For selection, use the POSSELDS program. For depletion, select the DEPLETES program.

The labeled cells are inserted into the uptake port and the procedure is started. After cell selection or depletion, the samples are collected and placed on ice until use. The purity of the depleted or selected samples is verified using flow cytometry.

Example 10 - Cells with therapeutic potential can be isolated and propagated from normal and chronically diseased kidney tissue

The purpose of this research is to measure the functional characteristics of human NKA cells by high content analysis (HCA). High-content imaging (HCI) provides the simultaneous imaging of multiple subcellular events using two or more fluorescent probes (multiplexing) across many samples. High-content analysis (HCA) provides quantitative measurement of multiple cell parameters captured in high-content imaging. In short, standard biopsy method is used to independently generate (Aboushwareb et al., the same, 2008) and maintain unfractionated (UNFX) culture from the core biopsy tissue obtained from 5 human kidneys with late-stage chronic kidney disease (CKD) and 3 non-CKD human kidneys. After UNFX is passed down to the next generation (2) in vitro, cells are harvested and subjected to density gradient method (as in Example 2) to generate subfractions, including subfractions B2, B3 and/or B4.

Obtain human kidney tissue from non-CKD and CKD human donors, as outlined in Table 10.1. Fig. 4 shows the histopathological characteristics of HK17 and HK19 samples. Set up in vitro culture from all non-CKD (3/3) and CKD (5/5) kidneys. Determine the high content analysis (HCA) of albumin transport in the people's NKA cells of target region (regions of interest) (ROI) and be shown in Fig. 5 (HCA of albumin transport in people's NKA cells). The quantitative comparison of albumin transport in the NKA cells deriving from non-CKD and CKD kidney is shown in Fig. 6. As shown in Figure 6, albumin transport is not weakened in the NKA cultures derived from CKD. The comparative analysis of marker expression between the B2 enriched renal tubular cells and the B4 subfraction consumed renal tubular cells is shown in Fig. 7 (CK8/18/19).

Comparative functional analysis of albumin transport between B2, which is enriched in tubular cells, and B4, which is depleted in tubular cells, is shown in Figure 8. Subfraction B2 is enriched in proximal tubular cells, thereby displaying enhanced albumin transport function.

Albumin uptake: The medium of cells grown to confluence in 24-well collagen IV plates (BD Biocoat ^™ ) was replaced with phenol red-free, serum-free, low-glucose DMEM containing 1X antimycotic/antibiotic and 2mM glutamine (pr-/s-/1 g DMEM) for 18-24 hours. Immediately prior to the assay, cells were washed with pr-/s-/1 g DMEM + 10mM HEPES, 2mM glutamine, 1.8mM CaCl2, and 1mM MgCl2 and incubated for 30 minutes. Cells were exposed to 25 μg/mL rhodamine-conjugated bovine albumin (Invitrogen) for 30 minutes, then washed with ice-cold PBS to stop endocytosis and immediately fixed with 2% paraformaldehyde containing 25 μg/mL Hoechst nuclear dye. For inhibition experiments, 1 μM receptor-associated protein (RAP) (Ray Biotech, Inc., Norcross, GA) was added 10 minutes prior to the addition of albumin. Microscopic imaging and analysis were performed using a BD Pathway ^™ 855 High-Content BioImager (Becton Dickinson) (see Kelley et al. Am J Physiol Renal Physiol. 2010 Nov;299(5):F1026-39. Epub 2010 Sep 8).

In summary, HCA produces cell level data and can show that can not be measured by other groups dynamics (populations dynamics) of gene or protein expression detection. The quantifiable isolated HCA assay (HCA-AT) for measuring albumin transport function can be used for characterizing the renal tubular cells of human kidney as the component of human NKA prototype. HCA-AT enables the comparative assessment of cell function, thereby showing that the cells with albumin transport capacity are retained in the NKA culture derived from human CKD kidney. It has also been shown that the specific subfractions B2 and B4 of NKA culture are significantly different from the B2 of the fraction enriched for renal tubular cells with enhanced albumin transport activity represented in phenotype and function. The B2 cell subpopulation from human CKD is similar to the rodent B2 cell showing effect (as shown above) in vivo in phenotype and function.

Example 11 - Marker Expression as Predictors of Kidney Regeneration

This study investigates the expression of stem and progenitor cell markers as predictors of renal regeneration in 5/6 nephrectomized rats treated with a subset of therapeutically bioactive primary renal cells. The mechanisms underlying the enhanced renal function achieved by NKA therapy will be characterized. Our investigations into the mechanism of action of NKA therapy involve intercellular signaling, engraftment, and fibrosis pathways. This work focuses on how NKA therapy may enhance the organ's inherent regenerative capacity—possibly by mobilizing renal stem cells. We hypothesize that the prolonged survival and enhanced renal function observed in NKA-treated 5/6 NX rats are associated with molecular expression of specific stem cell markers.

Using a rat 5/6 nephrectomy model of CKD, this study utilized molecular assays to evaluate the mobilization of resident stem and progenitor cells within the rat 5/6 nephrectomized kidney in response to direct injection of a population of primary renal cells with defined therapeutic bioactivity. This cell-based therapy was observed to be specifically associated with upregulation of key stem cell markers CD24, CD133, UTF1, SOX2, LEFTY1, and NODAL at both transcript and protein levels. Upregulation was detected by 1 week post-injection and peaked by 12 weeks post-injection. Activation of stem and progenitor cell markers was associated with increased survival and significant increases in serum biomarkers relative to untreated nephrectomized controls.

Materials and methods

Isolation of Primary Kidney Cell Populations from Rat Primary kidney cell populations were isolated from rat as previously described (Aboushwareb et al., supra, 2008; Presnell et al., 2009 FASEB J 23:LB143).

In situ research design and analysis. The separation of primary nephrocyte colony (Presnell et al. Tissue Eng Part C Methods. October 27, 2010 [prior to electronic publication of printing]) and the detailed description of the in vivo study (Kelley et al., the same, 2010) of assessing the biological activity of primary nephrocyte subpopulation in the rodent model of 5/6 nephrectomy of CDK. Announced elsewhere that the subpopulation of enriched renal tubular cells of primary nephrocytes has improved survival rate and enhanced renal function in the rodent model of chronic kidney disease. In this study, during autopsy, from the rat separation tissue processed with B2 (NKA#1) or B2+B4 mixture (NKA#2), it was compared with the rat (Nx) of nephrectomy and the rat (control) of non-nephrectomy of experience sham operation. In Figures 9 and 11 and Table 11.1, the data of the rat processed from NKA#1 and NKA#2 were gathered. By analyzing the blood samples extracted from the rats of research before weekly and autopsy, systemic data were obtained.

Table 11.1 shows survival data for sham-treated animals (control), n=3; nx controls (Nx), n+3; animals treated with B2 cells (NKA#1), n=7; and animals treated with B2+B4 cells (NKA#2), n=7. At the end of the study (weeks 23-24), no Nx animals remained. NKA-treated animals had superior survival compared to untreated Nx controls.

Table 11.1

Early mid-term At the end of the study Treatment group 1 week 12-13 weeks [23-24 weeks] comparison 2/3* 2/2 2/2 NX 2/3* 1/2 0/1 NKA#1 5/7** 3/7** 1/3** NKA#2 5/7** 3/7** 3/3

*One animal was sacrificed at the scheduled time points to obtain tissue

**Two animals were sacrificed at the scheduled time points to obtain tissues

RNA isolation, cDNA synthesis and qRT-PCR. RNA was isolated from tissue embedded in optimal cutting temperature (OCT) freezing medium as follows: the tissue blocks were placed at room temperature, excess OCT was removed, and the tissue was then placed in PBS to completely thaw and remove residual OCT. The tissue was washed three times in PBS, then roughly minced and aliquoted into microcentrifuge tubes. The aliquoted tissue was then ground into a powder using a pestle, and RNA was extracted using the RNeasy Plus Mini kit (Qiagen, Valencia CA). RNA integrity was determined by spectrophotometry, and cDNA was generated from one volume of RNA (equivalent to 1.4 μg) using the VILO ^™ cDNA synthesis kit (Invitrogen, Carlsbad CA). After cDNA synthesis, each sample was diluted 1:6 by adding 200 μl of diH ₂ O to a final volume of 240 μl. The expression levels of target transcripts were examined by quantitative real-time PCR (qRT-PCR) using cataloged primers and probes from ABI and ABI-Prism 7300 Real-Time PCR System (Applied Biosystems, Foster City CA). Amplification was performed using a gene expression premix (ABI, Cat# 4369016) with peptidyl prolyl isomerase B (PPIB) as an endogenous control. qRT-PCR reactions were performed using 10 μl premix (2X), 1 μl primers and probes (20X), 9 μl cDNA, and a total volume of 20 μl per reaction. Each reaction was set up using primers and probes as follows.

Western blot. Frozen whole kidney tissue embedded in OCT freezing medium was used for protein sample collection. OCT was removed as described above and all tissues were lysed in a buffer consisting of 50mM Tris (pH 8.0), 120mM NaCl, 0.5% NP40 and a protease inhibitor cocktail (Roche Applied Science, Indianapolis IN). Lysis was performed at room temperature for 15 minutes under shaking conditions, followed by centrifugation at 13,000RPM for 10 minutes. All supernatants were collected and protein concentration was determined using the Bradford assay. SDS PAGE gel electrophoresis was performed by adding 30μg of protein per sample to each well of a Novex 10% Bis-Tris gel (Invitrogen). The gel was electrophoresed at 200V in MES running buffer (Invitrogen) for 40 minutes. Proteins were then transferred to nitrocellulose filters using the I-Blot system (Invitrogen) and blocked for 2 hours at room temperature with 15 mL of 4% w/v low-fat milk dissolved in Tris-buffered saline (TBS-T) (Sigma, St. Louis, MO). The membranes were probed overnight at room temperature with the following antibodies (each diluted in 5 mL of TBS-T containing 2% w/v low-fat milk): anti-human Lefty-A long and short isoforms (R&D systems MAB7461); anti-human, mouse, and rat CD133 (Abcam AB19898); anti-human and mouse UTF1 (Millipore MAB4337); anti-human NODAL (Abcam AB55676); anti-human and rat CDH11 (OB-cadherin) (Thermo Scientific MA1-06306); and anti-rat CD24 (Becton Dickinson). The membrane was washed three times for 10 minutes each with TBS-T and then probed with the appropriate HRP-conjugated secondary antibody (Vector Labs PI-2000; PI-1000) diluted (1:60,000) in TBS-T containing 2% w/v low-fat milk for 1.5 hours at room temperature. The membrane was washed three times for 10 minutes each with TBS-T and then washed twice in diH ₂ O for 10 minutes each. The blot was developed using ECL Advance chemiluminescent reagent (GE Healthcare Life Sciences, Piscataway, NJ) and visualized using ChemiDoc ^™ XRS molecular imager Quantity software (BioRad, Hercules, CA).

Results. A molecular assay was developed to assess the mobilization of resident stem and progenitor cells in 5/6NX rats and used to study the temporal response of such markers to NKA treatment. NKA treatment was observed to be specifically associated with upregulation of key stem cell markers CD24, CD133, UTF-1, SOX-2, LEFTY, and NODAL at the mRNA transcript and protein levels. Upregulation was detected by 1 week after injection and peaked at 12 weeks after injection. Activation of markers of stem and progenitor cells was associated with increased survival and a significant increase in serum biomarkers (i.e., an increase in renal filtration) relative to untreated 5/6NX control animals.

Figure 9 shows the expression of SOX2 mRNA in host tissues after NKA treatment of 5/6 NX rats. Temporal analysis of SOX2 mRNA expression showed that by 12 weeks post-implantation, SOX2 mRNA in the NKA-treated group was 1.8 times that of the Nx control. By 24 weeks post-implantation, 2.7 times the expression of SOX2 mRNA in the NKA-treated group was observed compared to the Nx control. (1-week, n=3 for each of control (sham), Nx (control), and NKA-treated) (12 weeks, n=1 for each of control (sham) and Nx (control); n=4 for NKA-treated) (24 weeks, n=1 for each of control (sham) and Nx (control); n=4 for NKA-treated). * indicates p-value = 0.023 or < 0.05.

Figure 10 - Western blot showing the time course of expression of CD24, CD133, UTF1, SOX2, NODAL, and LEFTY in sham-operated controls (control), Nx controls (Nx), and rats treated with NKA#1 and NKA#2 at 1, 12, and 24 weeks after treatment. Whole kidney tissue (N=1 for each sample) embedded in OCT freezing medium was used for protein sample collection. Lanes were normalized using the total protein loaded. CD133, UTF1, NODAL, LEFTY, and SOX2 protein levels in NKA-treated tissues increased at all time points relative to control or Nx rats.

Figure 11 describes the time course of the regeneration response index (RRI). Densitometric analysis of individual protein expression (Figure 10) is used to generate a quantitative index or regeneration response index (RRI) of regeneration marker protein expression. Band intensity was calculated from each Western blot using Image J v1.4 software (NIH) and the value of each protein per unit area standardization. The average intensity of the sham, Nx and NKA treatment groups was determined by compiling the 5 markers used in the Western blot analysis at each time point. The graphic shows an XY scatter with a smooth line fit generated from 1, 12 and 24 week time points. The average intensity of each group was plotted over time to highlight the trend of stem cell marker protein expression in host tissue response. Assuming that each sample has equal variation, a standard two-tailed Student's t-test was used for statistical analysis. Statistical significance was determined using a 95% confidence interval (p-value < 0.05). (NKA treatment group n = 2; control (sham) n = 1; Nx (control) n = 1). In sham-operated control animals, the RRI showed only a slight decrease from 90.47 at 1 week post-treatment to 81.89 at 24 weeks post-treatment. In contrast, kidneys from 5/6 Nx controls showed essentially the opposite response, with the RRI increasing from 82.26 at 1 week post-treatment to 140.56 at 18 weeks post-treatment, at which point the animals died. In NKA-treated animals, the RRI increased dramatically from 62.89 at 1 week post-treatment to 135.61 at 12 weeks post-treatment, then decreased to 112.61 at 24 weeks post-treatment.

NKA treatment has been observed to be associated with upregulation of stem cell markers CD24, CD133, UTF-1, SOX-2, and NODAL at both transcript and protein levels in host tissues. Upregulation was detected by 1 week post-treatment and peaked by 12 weeks post-treatment. Global activation of markers of stem and progenitor cells in host tissues was associated with increased survival relative to untreated nephrectomized control animals (1) and increases in clinically relevant serum biomarkers.

Mobilization of resident stem and progenitor cell populations in response to NKA treatment can contribute to the restoration of renal function in 5/6NX animals by regenerating damaged renal tissue and organ structure. Therefore, the molecular assay used in this study provides a rapid, direct, and predictive assay for evaluating the regenerative outcomes of tissue engineering and regenerative medicine treatments for CKD.

Example 12 - Exosomes derived from primary kidney cells contain microRNAs

We sought to correlate specific exosome-derived miRNAs with functionally relevant outcomes in target cells in vitro to inform the design of in vivo studies to elucidate the mechanisms underlying regenerative outcomes.

Methods: Commercially available cells, including HK-2 (human proximal tubular cell line), primary human mesangial cells (HRMC), and human umbilical cord endothelial cells (HUVEC), were used to investigate the effects of conditioned media on signaling pathways associated with the regenerative healing response. PCR-based arrays designed to detect known miRNAs were used to screen the RNA content of exosomes from conditioned media from human and rat primary renal cell cultures (UNFX). Hypoxia has been reported to affect exosome excretion; therefore, one group of cultures was exposed to hypoxia (2% _O₂ ) for 24 hours, after which the media was collected. Exosomes were separated from cellular debris by FACS.

FIG12 provides a schematic diagram of the preparation and analysis of UNFX-conditioned medium.

Results: UNFX-conditioned medium was found to affect signaling pathways associated with regenerative healing responses; no such responses were observed in controls using unconditioned medium. Specifically, NFκB (immune response) and epithelial-to-mesenchymal transition (fibrotic response) were attenuated in HK-2 cells, PAI-1 (fibrotic response) was attenuated in HRMC cells, and angiogenesis was promoted in HUVECs. Preliminary data from PCR array screening of exosome contents from UNFX-conditioned medium showed that UNFX generates exosomes containing miRNA sequences consistent with the observed responses to UNFX-conditioned medium.

Figure 13A-C shows that conditioned medium from UNFX cultures affects multiple cellular processes potentially related to regenerative outcomes in vitro. NFkB signaling has been proposed as a key mediator of the inflammatory process in renal disease (Rangan et al., 2009. Front Biosci 12:3496-3522; Sanz et al., 2010. J Am Soc Nephrol 21:1254-1262) and can be activated by tumor necrosis factor (TNF). HK-2 cells were pre-incubated with unconditioned medium (left) or UNFX conditioned medium (right) at 37°C for 1 hour and then activated with or without 10 ng/ml TNFa.

Figure 13A shows that UNFX-conditioned medium attenuates TNF-a-mediated NF-kB activation. NFkB activation was measured by RelA/p65 immunofluorescence staining (green). Hoechst counterstaining of nuclei (blue) and phalloidin staining of filamentous actin (red) facilitated the assessment of RelA/p65 nuclear localization (white arrows).

Figure 13B shows that UNFX-conditioned medium enhances the pro-angiogenic behavior of HUVEC cell culture. HUVEC cells (100,000/well) are covered on a polymer matrix glue (Matrigel) in Media 200 plus 0.5% BSA. Unconditioned medium (left) or UNFX conditioned medium (right) are added, and the cell tissue response is visually monitored by image capture for 3-6 hours. Cell migration (white arrows), alignment (black arrows), tube formation (red arrows) and closed polygon formation (asterisks) of cell tissue are scored. UNFX conditioned medium induces more tubules and closed polygons compared to unconditioned culture, which indicates that pro-angiogenic factors are present in the culture medium.

Figure 13 C shows that UNFX-conditioned medium weakens the fibrosis pathway in epithelial cells.HK-2 cells lose epithelial features and obtain interstitial phenotype when exposed to transforming growth factor (TGF) in vitro, thereby replicating the epithelial to mesenchymal transition (EMT) (Zeisberg et al. 2003 Nat Med 9:964-968) associated with renal fibrosis progression.HK-2 cells are cultured in unconditioned medium (CTRL), unconditioned medium (TGFβ1) containing 10ng/ml TGFβ1 or UNFX conditioned medium (TGFβ1+CM) containing 10ng/ml TGFβ1 for 72 hours. CDH1 (epithelial marker), CNN1 (interstitial marker) and MYH11 (interstitial marker) of cell are determined by quantitative RT-PCR. Conditioned medium reduces the degree of EMT induced by TGFβ1-, as measured by CDH1, CNN1 and MYH11 gene expression. Error bars represent the standard deviation (SEM) of the mean value of 3 experimental repetitions.

FIG13D depicts a positive feedback loop established by TGFβ1 and plasminogen activator inhibitor-1 (PAI-1) that, when not inhibited, leads to the gradual accumulation of extracellular matrix proteins (Seo et al., 2009. Am J Nephrol 30:481-490).

Figure 14A-B shows the weakening of fibrosis pathway in mesangial cells.HRMC was cultured for 24 hours in UNFX conditioned medium (UNFX CM) with control (CTRL) or addition (+) or without addition (-) 5ng/ml TGFβ1. The Western blot analysis of PAI-1 showed that UNFX CM weakened the rising of TGFβ1-induced PAI-1 protein levels. b Actin was shown as the loading control. Human glomerular mesangial cells (HRMC) expressed elevated levels of PAI-1 in the presence (+) of 5ng/ml TGFb1. Co-culture of conditioned medium (CM) derived from human bioactive renal cells was used to weaken TGFb1-induced PAI-1 protein expression. CM did not change the expression of PAI-1 at the mRNA level (data not shown).

Figure 14B shows that CM from rat bioactive renal cells has similar effects on cultured HRMC induced (+) and not induced (-) with TGFb 1. CM supernatants collected after centrifugation (depleted rat CM) were less effective in attenuating PAI-1 expression, suggesting that the CM component responsible for the observed attenuation of PAI-1 protein may be associated with vesicles secreted by rat bioactive renal cells.

Figure 15 shows that the conditioned medium from UNFX contains secreted vesicles. Figure 145A describes secreted vesicles (including exosomes), which are double lipid structures (red) including internal components (green) of cytoplasmic origin. Phosphatidylserine (blue triangles) is a component of the membrane exposed to the extracellular space during vesicle biosynthesis (Thery et al., 2010. Nat Rev Immunol 9: 581-593).

PKH26 and CFSE label the lipid membrane and cytoplasm of secretory vesicles, respectively (Aliotta et al., 2010. Exp Hematol 38:233-245), whereas Annexin V binds phosphatidylserine.

Figure 15B-C shows FACS sorting. UNFX conditioned medium was labeled with PKH26, CFSE, and APC-conjugated annexin V and subsequently sorted using fluorescence-assisted cell sorting (FACS). Triple-positive particles representing secreted vesicles were collected and total RNA was extracted using TRIZol reagent. Commercially available RT-PCR-based arrays were used to screen for known sequences of microRNA contents.

Table 12.1 shows that secreted vesicles contain microRNAs with predicted therapeutic outcomes. UNFX cells excrete exosomes containing known miRNA sequences. UNFX-conditioned medium influences functionally relevant regenerative responses in human cell lines. The causal relationship between the detected miRNAs and the observed regenerative responses is under active investigation; however, the results obtained to date suggest that UNFX cells have the potential to generate therapeutically relevant paracrine effects through exosome-mediated transfer of miRNAs to target cells and tissues.

Table 12.1

miRNAs in exosomes gene target The role of prediction miR-146a TRAF6,IRAK1* Inhibition of NFkB miR-130a GAX,HOXA5** Promote angiogenesis miR-23b Smad 3/4/5*** Inhibits TGFβ signaling (anti-fibrosis)

*Taganov et al., 2006. Proc Natl Acad Sci USA 103:12481-12486.

**Chen and Gorski, 2008. Blood 111:1217-1226.

***Rogler et al., 2009. Hepatology 50:575-584.

The data support the conclusion that efflux vesicles from bioactive renal cell cultures contain components that attenuate PAI-1 induction by the TGFb1/PAI-1 feedback loop.

Microarray and RT-PCR analysis. Unfractionated (UNFX) bioactive renal cells from Lewis rats were cultured in basal medium (a 50:50 mixture of DMEM and KSFM without serum or additives) under hypoxic conditions (2% O2) for 24 hours. Conditioned medium was collected and ultracentrifuged at 100,000 x g for 2 hours at 4°C to precipitate secreted vesicles (e.g., microvesicles, exosomes). Total RNA was extracted from the resulting pellet and known microRNA species were determined using real-time RT-PCR (Rat MicroRNA Genome V2.0 PCR Array; Qiagen #MAR-100A). The following miRNAs were detectable.

Example 13 - Paracrine Factors Derived from Bioactive Renal Cells

In this study, we utilized a cell-based in vitro assay to investigate potential paracrine mechanisms by which bioactive renal cells may modulate fibrosis by regulating mediators such as lysosomal activator inhibitor-1 (PAI-1).

Materials and methods : Conditioned medium was collected from the culture of rat and human bioactive renal cells under serum-free and supplement-free conditions (Aboushwareb et al., World J Urol 26, 295, 2008; Presnell et al. 2010, supra) and used for in vitro assays. Commercially available rat and human-derived mesangial cells were used as a surrogate for host response tissue in in vitro assays because mesangial cells are the source of PAI-1 generation in damaged or diseased kidneys (Rerolle et al., Kidney Int 58, 1841, 2000.). PAI-1 gene and protein expression were measured by quantitative RT-PCR and Western blotting, respectively. Vesicle particles (e.g., exosomes) discharged from the cells to the culture medium were collected by high-speed centrifugation (Wang et al., Nuc Acids Res 2010, 1-12 doi: 10.1093/nar/gkq601, July 7, 2010), and total RNA was extracted from the precipitate using TRIzol reagent (Invitrogen). The RNA content of the vesicles was screened using a PCR-based array of known microRNA sequences (Qiagen).

Results : Conditioned medium from bioactive renal cell cultures attenuated the TGFβ1-induced increase in PAI-1 steady-state protein levels in mesangial cells, but did not affect steady-state mRNA levels; an observation consistent with a mechanism by which microRNAs regulate target genes. Based on the hypothesis that microRNAs can be transferred between cells via extracellular vesicle trafficking (Wang et al., supra, 2010), we analyzed the microRNA content of conditioned medium and confirmed the presence of microRNA 30b-5p (miR-30b-5p), a putative inhibitor of PAI-1.

The data shown here show that bioactive nephrocytes can directly regulate fibrosis through the intercellular transfer of miR-30b-5p to target mesangial cells through exosomes. As a result of mesangial cell absorption of miR-30b-5p, the increase in PAI-1 protein levels in the stable state of TGFβ1-induction is weakened (the response that can ultimately reduce the deposition of extracellular matrix in the glomerular space in kidney tissue). Current work is underway to confirm that PAI-1 is indeed a direct target of miR-30b-5p.

Figure 14A-B is shown in the control (CTRL) or to the culture medium to add (+) or not add (-) TGFβ1 biologically active renal cell conditioned medium (CM) cultured for 24 hours of human mesangial cells PAI-1 and α-actin protein expression of western blot.In CTRL culture, TGFβ1 increases the expression of PAI-1 protein.In CM culture, the response induced by TGFβ1 is weakened.

Secreted vesicles were analyzed for microRNAs that may be putative inhibitors of PAI-1. Secreted vesicles were collected from human and rat biocompetent renal cell CM by high-speed centrifugation, and the microRNA content of the vesicles was determined using a PCR-based array of known sequences. miR-449a, a putative regulator of PAI-1 (6), was identified. HRMC were transiently transfected with miR-449a or without miR-449a (CTRL). 24 hours after transfection, cells were exposed to 5 ng/ml TGFb1 (+) or without (-) for an additional 24 hours.

Figure 16 A shows that wherein prepare total protein and measure its PAI-1 and b-actin western blot.MiR-449a reduces steady state PAI-1 protein level (comparing lane 1 to lane 3) and the level of induced PAI-1 protein is also lower (comparing lane 2 to lane 4) in the culture transfected by miR-449a.The data support that the vesicle of efflux comprises miR-449a and the absorption of miR-449a in mesangial cells reduces the conclusion of PAI-1 expression.

Figure 16B depicts a microRNA, miR-30b-5p, which was also identified in the PCR-based array and is a putative regulator of PAI-1 based on prediction algorithms (http://mirbase.org - miRBase is hosted and maintained by the School of Life Sciences at the University of Manchester).

PAI-1 protein levels in glomeruli were examined in vivo after treatment of CKD induced by 5/6 nephrectomy using bioactive renal cells.

Figure 17A-C shows representative immunohistochemical images of PAI-1 (A-C) in Lewis rat kidneys that have undergone unilateral nephrectomy (A), 5/6 nephrectomy (B), or 5/6 nephrectomy and intrarenal delivery of bioactive renal cells (C). Due to 5/6 nephrectomy (B), the accumulation of PAI-1 in the glomeruli (arrows) was reduced as a result of treatment (C).

In a separate study, qRT-PCR was performed on kidney tissue harvested at necropsy, and relative gene expression values were plotted against the days of the study.

Figure 17D shows that 5/6 nephrectomized rats (red squares) exhibited more robust PAI-1 expression relative to rats treated with bioactive renal cells (blue circles) and sham-operated controls (green triangles).

Figure 17E shows representative Western blot analysis of kidney samples 3 and 6 months after treatment. Treated tissue (Nx+Tx) of 5/6 nephrectomized rats (Nx) has reduced accumulation of PAI-1 and fibronectin (FN) proteins (Kelley et al. 2010, supra).

The data support the conclusion that PAI-1 protein levels are reduced in vivo in glomerular disease following treatment of CKD induced by 5/6 nephrectomy using bioactive renal cells.

In summary, Examples 12-13 support the hypothesis that one mechanism by which intrarenal delivery of bioactive renal cells enhances renal function may be through the intercellular transfer of components that modulate the fibrotic pathway in resident renal cells.

Example 14 - Secreted Factors from Bioactive Renal Cells Attenuate the NFκB Signaling Pathway

In this study, we investigated the role of the NFκB pathway in NKA-mediated attenuation of disease progression in a 5/6 nephrectomy model and identified properties of bioactive renal cells that could contribute to regenerative outcomes by directly modulating NFκB activation. Figure 17G depicts exemplary activation of the NFκB pathway by TNFα.

Materials and methods: from wherein B2+B4 (NKA prototype) in PBS is processed before 6 weeks and carries out two-step 5/6 nephrectomy process Lewis rat harvest remnant kidney. NFκB activation of tissue of NKA-processing (TX) or unprocessing (UNTX) is analyzed by immunohistochemistry, RT-PCR, western blot analysis and electrophoretic mobility shift assay (EMSA). Conditioned medium (CM) collected from isolated NKA cell culture grown in serum-free and supplement-free culture medium is used for in vitro functional assay. Human proximal tubular cell line (HK-2) is used as the target cell type for the determination readout based on molecule and immunofluorescence. Vesicle particles (exosomes) discharged to culture medium by cells are collected by high-speed centrifugation. Total RNA isolated from exosomes is screened using PCR-based array (Qiagen) of known microRNA sequences.

Results: Nuclear localization of the NFκB subunit RelA/p65 was observed in the remnant kidneys of rats undergoing 5/6 nephrectomy, suggesting activation of the inflammatory pathway in UNTX tissues. Preliminary comparison with TX tissues by RT-PCR revealed a reduction in RelA gene expression, suggesting that NKA treatment may affect NFκB pathway activation by inhibiting RelA/p65 expression. This hypothesis is supported by the observation that CM attenuates TNFα-induced NFκB activation in vitro, as demonstrated by reduced nuclear localization of RelA/p65 in HK-2 cells exposed to CM relative to that seen in response to tumor necrosis factor-α (TNFα) (Figure 17F). RT-PCR analysis of NKA exosomal microRNAs was ongoing to investigate the presence of sequences known to influence the NFκB pathway.

Figure 17F shows that a 2-hour exposure to NKACM reduced the nuclear localization of NFκB p65 (green) in HK-2 cells in an immunofluorescence assay (compared to that observed in control cultures pretreated with TNFα). In HK-2, NFκB p65 (green) localized to the nucleus after 30 minutes of exposure to TNFα (control medium). However, pretreatment of HK-2 cells with NKA-conditioned medium for 2 hours before the addition of TNFα attenuated the nuclear localization response of NFκB p65. Nuclei were stained with DAPI (blue) and filamentous actin was stained with Alexa594-phalloidin (red) to help qualitatively assess the intensity of NFκB nuclear localization (note the slightly reduced phalloidin border in the TNFα-treated control cells in the merged image in the bottom row). In the merged image, the counterstain provides a reference for NFκB localization.

Immunohistochemistry of NFkB p65 subunit in renal tissue of Lewis rats shows more robust nuclear localization of NFkB p65 subunit, particularly in renal tubular epithelial cells (black arrows), in animals with progressive CKD induced by 5/6 nephrectomy (panel B) compared to control animals with non-progressive renal insufficiency induced by unilateral nephrectomy (panel A). Tissue was harvested 6 weeks after nephrectomy. Magnification 200X.

Panel C: Western blot analysis of NFkB p65 in cytoplasmic ('C') and nuclear ('N') protein extracts from kidney tissue of Lewis rats that had undergone 5/6 nephrectomy. Comparison of gtubulin levels (loading control) between weeks 1 and 13 shows a relatively constant level, while nuclear NFkB p65 increases over time, consistent with the immunohistochemical results.

Panel D: Electrophoretic mobility shift assay (EMSA) on nuclear extracts confirms that NFkB localized to the nucleus is activated for DNA binding after 5/6 nephrectomy. Lanes represent nuclear extracts prepared from two animals at each time point.

The NFkB pathway is progressively activated in the 5/6 nephrectomy model of chronic kidney disease. Immunohistochemistry was performed for the NFkB p65 subunit in renal tissue of Lewis rats.

Figures 18A-D show that animals with progressive CKD caused by 5/6 nephrectomy (panel B) have more robust nuclear localization of the NFkB p65 subunit, particularly in renal tubular epithelial cells (black arrows), compared to control animals with non-progressive renal insufficiency caused by unilateral nephrectomy (panel A). Tissues were harvested 6 weeks after nephrectomy. Magnification is 200X.

Figure 18C shows Western blot analysis of NFkB p65 in cytoplasmic ('C') and nuclear ('N') protein extracts from kidney tissue of Lewis rats that had undergone 5/6 nephrectomy. Comparing week 1 with week 13, where gtubulin levels (loading control) were relatively consistent, nuclear NFkB p65 increased over time, consistent with the immunohistochemistry results.

FIG18D shows an electrophoretic mobility shift assay (EMSA) on nuclear extracts and confirms that NFκB, which localizes to the nucleus, is activated for DNA binding after 5/6 nephrectomy. Lanes represent nuclear extracts prepared from two animals at each time point. 1 mg of nuclear protein was incubated with 5 ng of NFκB DNA binding site, which was electrophoresed on a 6% DNA retardation gel and subsequently stained with ethidium bromide.

Intrarenal delivery of NKA cells reduces nuclear localization of NFkB. Multiple defined renal cell subpopulations have been isolated and their bioactivity in promoting renal function has been determined in vivo in a 5/6 nephrectomy model of CKD (Presnell et al. 2010, supra). NKA cells showed bioactivity, whereas other subpopulations did not (Kelley et al. 2010, supra).

Figure 18E shows Lewis rats with established CKD that received intrarenal injections of NKA (A) or non-bioactive nephrocytes (B). Lewis rats with established CKD received intrarenal injections of NKA (A) or non-bioactive nephrocytes (B). Six months after treatment, tissues were harvested and assayed for NFkB p65 subunits by immunohistochemistry. Tissues from animals treated with NKA showed less nuclear localization of NFkB p65 (particularly in the proximal tubules) compared to tissues from animals treated with non-bioactive nephrocytes, suggesting that NKA treatment is involved in reducing NFkB pathway activity in vivo.

Analysis of the microRNA content of secretory vesicles isolated from human and rat NKA conditioned medium by high-speed centrifugation using a PCR-based array of known sequences identified several microRNA species that may influence immune responses through NFkB based on literature reports (Marquez RT et al. (2010) Am J Physiol Gastrointest Liver Physiol 298:G535; Taganov KD et al. (2006) Proc Natl Acad Sci USA 103:12481) or prediction algorithms (http://mirbase.org - miRBase is hosted and maintained by the School of Life Sciences at the University of Manchester).

In vivo and in vitro findings provide insights into how bioactive renal cells (NKA) can enhance renal function in chronically diseased kidneys by modulating immune response pathways, such as those affected by NFkB activation. Activated NFkB (p65 nuclear localization, particularly in proximal tubular cells) is associated with the establishment of chronic kidney disease in a 5/6 nephrectomy rodent model and is attenuated by NKA treatment. The in vitro response of proximal tubular cells (HK-2) to NKA-conditioned medium mimics the in vivo attenuation of NFkB nuclear localization in response to NKA treatment. Putative mediators of intercellular inhibition of NFkB activation (microRNAs) were identified in NKA-conditioned medium. Taken together, these data support the hypothesis that one mechanism by which intrarenal delivery of bioactive renal cells enhances renal function is through the intercellular transfer of components, such as RNA, that modulate the immune response of resident renal cells.

Example 15 - Functional evaluation of NKA constructs

Kidney cell colonies seeded on gelatin or HA-based hydrogels were able to survive and maintain a functional tubular epithelial phenotype during 3 days of in vitro maturation, as measured by transcriptomics, proteomics, secretomes, and confocal immunofluorescence assays. To investigate the potential mechanisms by which NKA constructs can affect disease states, the effects of conditioned medium on the TGF-β signaling pathway associated with tubulointerstitial fibrosis associated with CKD progression were assessed. Conditioned medium was observed to attenuate TGF-β-induced epithelial-mesenchymal transition (EMT) in vitro in a human proximal tubular cell line (HK2).

Materials and methods

Biomaterials. Biomaterials were prepared as beads (homogeneous spherical structures) or granules (heterogeneous populations with jagged edges). Gelatin pellets (Cultispher S and Cultispher GL) manufactured by Percell Biolytica (Sweden) were purchased from Sigma-Aldrich (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA), respectively. Cross-linked HA and HA/gelatin (HyStem ^™ and Extracel ^™ from Glycosan BioSystems, Salt Lake City, UT) particles were formed from lyophilized sponges produced according to the manufacturer's instructions. Gelatin (Sigma) particles were formed from cross-linked lyophilized sponges.

PCL was purchased from Sigma-Aldrich (St. Louis, MO). PLGA 50:50 was purchased from Durect Corp. (Pelham, AL). PCL and PLGA beads were prepared using a modified double emulsion (W/O/W) solvent extraction method. PLGA particles were prepared using a solvent casting porogen leaching technique. All beads and particles measured 65 to 355 microns when dry.

Cell isolation, preparation and culture. Human cadaver kidneys were obtained from the National Disease Research Institute (NDRI) in accordance with all NIH guidelines governing the use of human tissues for research purposes. Dog kidneys were obtained from a contract research organization (Integra). Rat kidneys (21-day-old Lewis rats) were obtained from Charles River Laboratories (MI). The preparation of primary renal cell colonies (UNFX) and a defined subpopulation (B2) from intact rats, dogs, and human kidneys has been previously described (Aboushwareb et al. World J Urol 26 (4): 295-300; 2008; Kelley et al., supra, 2010; Presnell et al. WO/2010/056328). Briefly, renal tissue was enzymatically dissociated in a buffer containing 4.0 units/mL dispase (Stem Cell Technologies, Inc., Vancouver BC, Canada) and 300 units/ml collagenase IV (Worthington Biochemical, Lakewood NJ), followed by centrifugation through 15% iodixanol (Axis Shield, Norton, MA) to generate UNFX. UNFX cells were seeded on tissue culture treated polystyrene plates (NUNC, Rochester NY) and then cultured in a 50:50 medium (a 1:1 mixture of high glucose DMEM: keratinocyte serum-free medium (KSFM) containing 5% FBS, 2.5 μg EGF, 25 mg BPE, 1X ITS (insulin/transferrin/sodium selenite medium supplement) and antibiotics/antimycotics (all from Invitrogen, Carlsbad CA)). B2 cells were isolated from UNFX cultures using centrifugation through a four-step iodixanol (OptiPrep; 60% w/v in unsupplemented KSFM) density gradient specifically layered for rodent (16%, 13%, 11%, and 7%), canine (16%, 11%, 10%, and 7%), or human (16%, 11%, 9%, and 7%) (Presnell et al. WO/2010/056328; Kelley et al., supra, 2010). The gradient was centrifuged at 800 x g for 20 minutes at room temperature (without brake). The band of interest was removed by pipetting and washed twice in sterile phosphate-buffered saline (PBS).

Cell/biomaterial complex (NKA construct). In order to perform in vitro analysis of cell function on biomaterials, a uniform layer of biomaterial (prepared as described above) was covered on a hole of a 6-well low-attachment plate (Costar#3471, Corning). UNFX or B2 cells (2.5x10 ⁵ / well) were directly seeded on the biomaterial. In order to study the adhesion of canine cells to biomaterials, 2.5x10 ⁶ UNFX cells were seeded with 50 μl of biomaterial in a non-adhesive 24-well plate (Costar#3473, Corning). After 4 hours on an oscillating platform, the canine NKA construct was matured overnight in a 5% CO ₂ incubator at 37 ° C. The next day, live cell/dead cell staining was performed using a live cell/dead cell staining assay kit (Invitrogen) according to the manufacturer's instructions. Rat NKA constructs were prepared in a 60cc syringe on a roller bottle device with a rotation speed of 1 RPM.

For the transcriptomic, secretomic, and proteomic analyses described below, NKA constructs were matured for 3 days. Cells were then harvested for transcriptomic or proteomic analysis, and conditioned media was collected for secretomic profiling.

Functional analysis of renal tubular cell-related enzyme activity. Canine NKA constructs (10 μl loose packed volume) were evaluated in 24-well plates using an assay for leucine aminopeptidase (LAP) activity modified from a previously published method (Tate et al. Methods Enzymol 113:400-419; 1985). Briefly, 0.5 ml of 0.3 mM L-leucine p-nitroanilide (Sigma) in PBS was added to the NKA constructs for 1 hour at room temperature. The wells were sampled in duplicate, and the absorbance at 405 nm was recorded as a measure of LAP activity. LLC-PK1 cell lysate (American Type Culture Collection or ATCC) was used as a positive control.

Transcriptome profile characterization. Polyadenylated RNA was extracted using the RNeasy Plus Mini kit (Qiagen, CA). Concentration and integrity were determined by UV spectrophotometry. cDNA was generated from 1.4 μg of isolated RNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). The expression levels of target transcripts were examined by quantitative real-time polymerase chain reaction (qRT-PCR) using commercially available primers and probes (Table 15.1) and an ABI-Prism 7300 real-time PCR system (Applied Biosystems, CA). Amplification was performed using TaqMan gene expression premix (ABI, Cat#4369016), and the TATA box binding protein gene (TBP) was used as an endogenous control. Each reaction consisted of 10 μl of premix (2X), 1 μl of primers and probes (20X), and 9 μl of cDNA. Samples were run in triplicate.

Table 15.1

Characterization of secretome profiles. Conditioned medium from human NKA constructs was collected and frozen at -80°C. The biomarker concentrations of the samples were assessed quantitatively. The results of a given marker concentration of the conditioned medium were normalized to the concentration of the same biomarker in the conditioned medium from the control culture (2D culture without biological material) and expressed as a unitless ratio.

Proteomic profiling. Proteins from three independent replicates were extracted from the cell/biomaterial complex and pooled for analysis by 2D gel electrophoresis. All reagents were from Invitrogen. Isoelectric focusing (IEF) was performed by adding 30 μg of protein resuspended in 200 μl of Zoom 2D Protein Stabilizer #1 (Cat#ZS10001), Zoom Carrier Ampholytes pH 4-7 (Cat#ZM0022), and 2MDTT (Cat#15508-013) to a pH 4-7 Zoom IEF strip (ZOOM IEF Strip) (Cat#ZM0012). After electrophoresis at 500 V for 18 hours, the IEF strips were loaded onto NuPAGE Novex 4-12% Bis-Tris ZOOM IPG well gels (Cat#NP0330BOX) for SDS-PAGE separation and electrophoresis was performed in MES buffer (Cat#NP0002) for 45 minutes at 200 V. Proteins were visualized using SYPRO Ruby protein gel stain (Cat#S-12000) according to the manufacturer's instructions.

Confocal microscopy. NKA constructs prepared from human or rat UNFX or B2 cells were matured for 3 days and then fixed in 2% paraformaldehyde for 30 minutes. The fixed NKA constructs were blocked and permeabilized by incubating in 10% goat serum (Invitrogen) + 0.2% Triton X-100 (Sigma) in D-PBS (Invitrogen) for 1 hour at room temperature (RT). For immunofluorescence, the NKA constructs were labeled overnight at RT with the primary antibody (Table 15.2) at a final concentration of 5 μg/ml. The labeled NKA constructs were washed twice with 2% goat serum/D-PBS + 0/2% Triton X-100 and then incubated with a secondary antibody of anti-rat IgG2A (Invitrogen) conjugated with goat or rabbit TRITC at a concentration of 5 μg/ml. For double labeling with DBA (Dolichos biflorus agglutinin), NKA construct candidates were further incubated with FTTC-conjugated DBA (Vector Labs) diluted to 2 mg/ml in 2% goat serum/D-PBS + 0.2% Triton X-100 for 2 hours at RT.

Table 15.2

Antibody source Manufacturer Catalog Number target IgG1 control mice BD 557273 Background control IgG control goat Invitrogen 026202 Background control IgG control rabbit Invitrogen 026102 Background control N-cadherin mice BD 610920 proximal renal tubules E-cadherin mice BD 610182 distal renal tubule Cubic protein (A-20) goat Santa Cruz Sc-20609 proximal renal tubules GGT-1 rabbit Santa Cruz Sc-20638 renal tubular epithelium Megaprotein rabbit Santa Cruz Sc-25470 proximal renal tubules

The samples were washed twice with D-PBS and optically sectioned using a Zeiss LSM510 laser scanning confocal microscope (Cellular Imaging Core, Wake Forest Baptist Medical Center) running LSM Image software (Zeiss) or using a Pathway 855 confocal microscope (BD Biosciences).

Analysis of TGF-β mediated EMT of HK2 cells.HK2 cells (ATCC) are cultured in 50:50 culture medium in fibronectin or collagen (IV) coated culture dishes (BD Biosciences). In order to perform EMT assay, HK2 cells are seeded in 24-well collagen (IV) coated plates with a confluence of 70-80% using 50:50 culture medium or conditioned medium collected from two-dimensional (2D) human UNFX cultures or NKA constructs prepared with human UNFX that mature for 3 days before culture medium collection. 10 ng/ml TGF-β is added to culture medium 3 days before RNA is isolated from cells for EMT assay to initiate TGF-β induction. Utilizing qRT-PCR, EMT is monitored by analyzing the relative expression of E-cadherin (epithelial marker) and calmodulin (mesenchymal marker) at the end of the 3-day incubation period. RNA is prepared from harvested HK2 cells for TaqMan qRT-PCR analysis, as described above. Statistical analysis was performed using a standard two-tailed Student's t-test, assuming equal variation for each sample. 95% (p-value < 0.05) and 99% (p-value < 0.01) confidence intervals were used to determine statistical significance.

In vivo implantation of acellular biomaterials and NKA constructs. Lewis rats (6 to 8 weeks old) were purchased from Charles River (Kalamazoo, MI). All experimental procedures were performed under the guidelines of PHS and IACUC at Carolina Medical Center. Under isoflurane anesthesia, female Lewis rats (approximately 2 to 3 months old) underwent a midline incision to expose the left kidney. 35 μl of filler biomaterial (acellular biomaterial or NKA construct) was introduced into the renal parenchyma by direct injection. Two injection trajectories were used: (i) from each pole toward the cortex (referred to as cortical injection), or (ii) from the midline of the kidney toward the pelvis (referred to as intramedullary injection). Rats were sacrificed 1, 4, or 8 weeks after injection. No early deaths occurred. The study design for the acellular implantation study is shown in Table 15.3 (ND=not performed).

Renal histology.Representative kidney samples were collected and placed in 10% buffered formalin for 24 hours. The sections were dehydrated in ascending grades of ethanol and then embedded in paraffin. Sections (5 μm) were cut and placed on a charged slide, and standard staining protocols (Prophet et al., Armed Forces Institute of Pathology:Laboratory methods in histotechnology.Washington, DC:American Registry of Pathology; 1992) were processed to carry out hematoxylin and eosin (H&E) staining, Masson's trichrome staining, and periodic acid-Schiff (PAS) staining. Digital micrographs were captured using a Nikon Eclipse 50i microscope equipped with a Digital Sight (DS-U1) camera at a total magnification of x40, x100, and x400. Renal morphological changes were assessed using the commonly used (Shackelford et al. Toxicol Pathol 30(1):93-96; 2002) severity grading scale (grades 1, 2, 3, 4), for which descriptive terms (minimal, mild, moderate, marked/severe) were used to describe the extent of observed glomerulosclerosis, tubular atrophy and dilatation, tubular casts, and interstitial fibrosis and inflammation.

Table 15.3

result

Mammalian kidney tissue is to the response of the injection of biomaterial into the renal parenchyma.By the direct injection analysis biomaterial in the possible use (table 15.3) of nephrocyte/biomaterial composite to healthy rat kidney.By measuring the degree of histopathological parameters (inflammation, fibrosis, necrosis, calcification/mineralization) and biocompatibility parameters (degradation, new blood vessel formation and new tissue formation of biomaterial) 1 week and 4 weeks after injection, tissue response is assessed.

Figures 19A-B show in vivo evaluation of the biomaterials at one week post-implantation. Trichrome X10 low-magnification image of a kidney cross section shows biomaterial aggregation. Trichrome X40: Close-up of biomaterial aggregation. H&E X400: High-magnification image of biomaterial aggregation to assess the extent of cell/tissue infiltration. Each kidney was injected at two locations as described in Materials and Methods. At one week post-implantation, the host tissue response elicited by each tested biomaterial was generally similar; however, the gelatin hydrogel appeared to elicit a less robust histopathological response and a more biocompatible response.

Figure 19 C shows the in vivo evaluation of biomaterials 4 weeks after implantation. At 4 weeks after implantation, the severity of the histopathological parameters of tissues injected with HA or gelatin particles was qualitatively reduced compared to 1 week after implantation. Gelatin particles were almost completely reabsorbed, and fewer giant cell reactions were observed than in tissues receiving HA particles. In most cases where biomaterials were injected via an intramedullary injection trajectory (e.g., deeper into the medulla/pelvis), undesirable results were observed, including obstruction leading to hydronephrosis, inflammatory reactions of greater severity, and renal arteriole and capillary microthrombosis leading to infarction (data not shown).

The functional phenotype of the upper relevant nephrocyte colony of assessment with biomaterial.After being directly injected into renal parenchyma, in the rodent model of chronic kidney disease, prolong survival and enhance renal function, the upper relevant nephrocyte colony (UNFX) of treatment has been characterized (Presnell et al. WO/2010/056328; Kelley et al., the same, 2010) and for its separation, characterization and amplification method has been developed and can be transplanted and used (Presnell et al., 2010, the same) between multiple species. In order to assess whether UNFX cells adhere to, maintain viability and retain mainly tubular epithelial phenotype when mixing NKA construct, transcriptomics, secretome, proteomics and confocal immunofluorescence microscopy analysis are carried out to the NKA construct generated from UNFX and various biomaterials.

Adhesion and viability. UNFX cells of canine origin were seeded on gelatin beads, PCL beads, PLGA beads, HA particles and HA/gelatin particles (3 NKA constructs/biomaterials) as described. The distribution and viability of cells were assessed one day after inoculation by live/dead cell staining.

Figures 20A-D show live/dead cell staining of NKA constructs seeded with canine UNFX cells (A = gelatin beads; B = PCL beads; C = HA/gelatin particles; D = HA particles). Green indicates live cells; red indicates dead cells. (A) Gelatin beads; (B) PCL beads; (C) HA/gelatin particles; and (D) HA particles. Live cells were observed on all hydrogel-based NKA constructs.

UNFX cells strongly adhered to natural-derived hydrogel-based biomaterials such as gelatin beads and HA/gelatin particles (black arrows in A, D), but showed minimal adhesion to synthetic PCL (B) or PLGA beads (not shown). Cells did not adhere to HA particles (C) but showed evidence of biological response (i.e., spheroid formation). The functional activity of UNFX cells seeded on the hydrogel-based NKA construct was confirmed by measuring leucine aminopeptidase, a proximal tubule-associated hydrolase (data not shown).

Transcriptomic Profile Characterization. Quantitative transcriptomic analysis was used to compare gene expression profiles of human UNFX cells in hydrogel-based NKA Constructs (3 NKA Constructs/biomaterial) and parallel 2D cultures of UNFX cells.

Figures 20E-G show transcriptomic profiling of NKA constructs. TC: Primary human UNFX cells cultured in 2D. Gelatin: NKA construct composed of human UNFX cells and gelatin hydrogel. HA-Gel: NKA construct composed of human UNFX cells and HA/gelatin particles. qRT-PCR data are presented in both graphical and table formats. Transcripts were examined and fell into four major categories: (i) tubular : aquaporin 2 (AQ2), E-cadherin (ECAD), erythropoietin (EPO), N-cadherin (NCAD), cytochrome P450, family 24, subfamily A, polypeptide 1-aka vitamin D 24-hydroxylase (CYP), cubosome, phenylephrine; (ii) interstitial : calmodulin (CNN1), smooth muscle myosin heavy chain (SMMHC); (iii) endothelial : vascular endothelial growth factor (VEGF), platelet endothelial cell adhesion molecule (PECAM); and (iv) glomerular : podocin. In summary, tubular marker expression was comparable between hydrogel-based NKA constructs and 2D UNFX cultures. Similarly, endothelial markers (VEGF and PECAM) were comparable. In contrast, the glomerular marker podocin showed significant variability between NKA constructs. Podocin levels in HA/gelatin-based NKA Constructs were most comparable to those observed in 2D UNFX cultures. Interestingly, expression of mesenchymal markers (CNN1 and SMMHC) was significantly downregulated in hydrogel-based NKA Constructs relative to 2D UNFX cultures (p < 0.05), suggesting that the fibroblast subpopulation of UNFX may not proliferate in hydrogel-based NKA Constructs in renal media formulations.

Secretome proteomic profiling characterization. NKA Constructs were generated using human UNFX and B2 cells and gelatin or HA/gelatin hydrogels (1 NKA Construct/biomaterial/cell type = 4 NKA Constructs total).

Figure 21A-B shows the secretome profile of NKA construct. Data are expressed as 3D:2D ratio. As described in Materials and Methods, NKA constructs were generated from human UNFX or B2 cells and gelatin (hydrogel 1) or HA/gelatin (hydrogel 2) hydrogels. The conditioned medium from the NKA constructs that had matured for 3 days was subjected to a secretome profile, and the secretome profile was compared with the parallel 2D cultures of human UNFX or B2 cells by calculating the ratio (3D:2D ratio) of the analyte expression of the NKA constructs (three-dimensional or 3D, cultures) to the 2D cultures. For each of the 3 NKA constructs inoculated with UNFX cells, the 3D:2D ratio was 1 or close to 1, which shows that the inoculation process carried out on this type of biomaterial and the 3-day maturation had almost no effect on the secretome profile of UNFX cells. Similar results were observed for NKA constructs seeded with B2 cells at 3D:2D ratios of 1 or close to 1, providing additional evidence that the seeding process and 3 days of maturation on this type of biomaterial have little impact on the secretome profile of therapeutically relevant kidney cells.

Proteomic profile characterization. Proteomic profiles of a given cell or tissue are generated by separating total cellular proteins using 2D gel electrophoresis and have been used to identify specific markers associated with kidney disease (Vidal et al. Clin Sci (Lond) 109(5):421-430; 2005).

Figure 22A-B shows the proteomic profile characterization of NKA constructs. Utilize people UNFX cells and biomaterials as shown to generate NKA constructs. As described in Materials and Methods, proteins in total protein extracts were separated by 2D gel electrophoresis. In this experiment, the proteomic profile characterization was used to compare the protein expression of people UNFX cells in NKA constructs (gelatin or based on HA/gelatin hydrogel, 3 NKA constructs/biomaterials) and 2D tissue culture. The proteomic profiles of the total proteins isolated from the NKA constructs or 2D cultures of UNFX cells are essentially the same, thereby providing additional evidence that the inoculation process carried out on such biomaterials and 3 days of maturation have little effect on the proteomics expressed by UNFX cells.

Confocal microscopy. The maintenance of the renal tubular epithelial phenotype of rat and human B2 cells (Presnell et al. 2010, supra) in NKA constructs was assessed by confocal imaging of established markers: Figures 23A-C show confocal microscopy of NKA constructs. Confocal microscopy of NKA constructs generated using human (A) or rat (B, C) B2 cells and gelatin hydrogels. (A) E-cadherin (red - solid white arrow), DBA (green - dotted green arrow), and gelatin hydrogel beads were visualized using DIC optics. (B) DNA visualized using DAPI staining (blue - solid white arrow) and each of the following markers shown in green (dashed white arrow): IgG control, N-cadherin, E-cadherin, cytokeratin 8/18/18, DBA. (C) Double-labeled images of markers, colors as shown. E-cadherin and DBA in human NKA constructs, and E-cadherin, DBA, N-cadherin, cytokeratin 8/18/19, gamma-glutamyl transpeptidase (GGT-1), and megaprotein in rat NKA constructs. Optical sectioning of confocal images also allows assessment of the extent of cell infiltration into the biomaterial after seeding and 3 days of maturation. B2 cells in human and rat NKA constructs showed expression of multiple renal tubular epithelial markers. Optical sectioning showed minimal cell infiltration of the hydrogel constructs, with cells generally confined to the surface of the biomaterial.

In vivo response to the implantation of NKA construct prototype. Based on the in vivo response to the injection of biomaterial into the renal parenchyma and the in vitro phenotype and functional characterization of UNFX and B2 cells in the above-mentioned NKA construct, gelatin hydrogel was selected to assess the in vivo response to the injection of NKA construct into the renal parenchyma of healthy Lewis rats. NKA construct was generated from isogenic B2 cells, implanted into two animals, and the animals were killed at 1, 4 and 8 weeks after implantation. All animals survived to autopsy at the scheduled time, and the sections of renal tissue were harvested, sliced, and then stained with Trichrome, hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS).

Figures 24A-B show in vivo evaluation of NKA Constructs at 1 and 4 weeks post-implantation. Trichrome X10 low-magnification image of a kidney cross section showing biomaterial aggregates. Trichrome X40: Close-up of biomaterial aggregates. H&E/PASX400: High-magnification image of biomaterial aggregates assessing the extent of cell/tissue infiltration. Each kidney was injected at two locations, as described in Materials and Methods.

Figure 24 A is presented at the in vivo assessment of NKA construct when implanting after 1 week.After injection 1 week, gelatin beads exist in the form of focus aggregates (left frame, circled area) of spherical porous material, and described spherical porous material is basophilic staining and is surrounded by multinucleated macrophage (phagocytic multi-nucleatedmacrophage) and giant cell of significant vascular tissue and phagocyte.Fibrovascular tissue is integrated into beads and shows the renal tubular epithelial component that represents newborn nephrocyte formation.In addition, renal tubules and vascular glomerulus (vasculoglomerular) structure are identified by morphology (PAS frame).

Figure 24B shows in vivo evaluation of the NKA Construct at 4 weeks post-implantation. By 4 weeks post-injection, the hydrogel was completely resorbed and the space was replaced by progressive renal regeneration and repair with minimal fibrosis (note the numerous functional tubules within the circled area of the 4-week Trichrome frame).

Figures 25A-D show in vivo evaluation of NKA Constructs at 8 weeks post-implantation. Trichrome X10 low-magnification image of a kidney cross section showing biomaterial aggregation. Trichrome X40: Close-up of biomaterial aggregation. H&E/PAS X400: High-magnification image of biomaterial aggregation assessing the extent of cellular/tissue infiltration. (A) Moderate chronic inflammation (macrophages, plasma cells, and lymphocytes), moderate number of hemosiderin-laden macrophages with a prominent fibrovascular response (stained blue by Masson’s Trichrome - black arrows) (chronic hemorrhage generated by injection); (B) Higher magnification of the framed area of (A) (trichrome stained, x400) showing the induction of a regenerative response consistent with the formation of new renal tissue (C) representative of the adjacent (normal) renal parenchyma showing typical cortical glomerular morphology (HE, x400); (D) HE-stained section, x400, comparing the new glomerular morphology observed in the treated area with that in Figure 154C.

Figure 25A-D shows the in vivo evaluation of NKA constructs at 8 weeks post-implantation. At 8 weeks post-implantation, evidence of new kidney-like tissue formation was observed, consistent with the induction of early events in nephrogenesis. Comparison of regeneration-induced areas (B, D) with adjacent cortical parenchyma (C) revealed the presence of multiple S-shaped bodies and newly formed glomeruli.

The effect of the conditioned medium from the NKA construct on the EMT induced by TGF-β of HK2 cells. The development of tubular-interstitial fibrosis in the progression of CKD is related to the EMT mediated by TGF-β of renal tubular epithelial cells (Zeisberg et al. Am J Pathol 160 (6): 2001-2008; 2002). Similarly, the weakening of TGF-β pathways was also observed in vivo in rodent models of progressive CKD, where survival was prolonged and renal function was enhanced by the treatment of UNFX and B2 cells (Presnell et al. WO/2010/056328). The human proximal tubular cell line HK2 has been well established as an in vitro model system for testing the stimulatory or inhibitory effects of small molecules or proteins on TGF-β-induced EMT (Dudas et al. Nephrol Dial Transplant 24(5):1406-1416; 2009; Hills et al. Am J Physiol Renal Physiol 296(3):F614-621; 2009). To investigate the potential mechanisms by which NKA constructs may affect renal tissue responses after implantation, conditioned medium collected from NKA constructs generated using UNFX cells and hydrogels was evaluated in the HK2 EMT assay system.

Figure 26 shows that the conditioned medium from the NKA construct weakens the TGF-β induced EMT of HK2 cells in vitro. EMT is monitored by the relative expression of quantitative ECAD (epithelium) and CNN1 (interstitial) markers. HK2 cells are cultured in 50:50 culture medium (control and TGFB control samples) or 2D culture from human UNFX cells (TC) or conditioned medium (CM) of the NKA construct generated from human UNFX cells and shown gelatin or HA/gelatin. In order to induce EMT, 10ng/ml TGF-β is added to each sample (except control) for 3 days and then measured. When HK2 cells are cultured in 50:50 culture medium (control), ECAD (epithelial marker) is expressed at a level higher than CNN1 (interstitial marker). When TGF-β is added to culture medium for 3 days (TGFB control), ECAD expression is significantly downregulated, while accompanied by the upregulation of CNN1, which is consistent with the induction of EMT events. Conditioned medium from 2D UNFX cell culture significantly (p < 0.05, for ECAD and CNN1) weakened HK2 cells to TGF-β EMT response (TC CM). Conditioned medium from NKA constructs (gelatin CM and HA/gelatin CM) also weakened the EMT response to TGF-β; however, the overall effect was less than that observed for the conditioned medium from 2D UNFX cell culture (for both NKA constructs, for ECAD, p < 0.05 was significant and trended towards control, although statistically not significant for CNN1). Additional interstitial markers were screened, and the markers generated similar results (data not shown). These data suggest that NKA constructs can potentially affect the TGF-β pathway associated with tubulointerstitial fibrosis in vivo in a manner similar to that observed for cell-based treatments (Presnell et al. WO/2010/056328). These data also suggest that in vitro EMT assays have potential application for screening/optimizing/monitoring the biotherapeutic efficacy of NKA constructs if the in vivo response can be shown to correlate statistically significantly with the in vitro EMT response, potentially reducing the need for time-consuming and expensive in vivo assays.

This study investigated the response of mammalian renal parenchyma to the implantation of synthetic and natural biomaterials (non-cellular and as bioactive renal cell/biomaterial complexes (i.e., NKA constructs)). The combination of in vitro functional assays and in vivo regeneration results was analyzed to functionally screen candidate biomaterials for potential incorporation into NKA construct prototypes. Implantation of hydrogel-based non-cellular biomaterials into the renal parenchyma (Figure 19) was generally associated with minimal fibrosis or chronic inflammation and no evidence of necrosis by 4 weeks after implantation. Appropriate cell/tissue ingrowth and neovascularization were observed with minimal residual biomaterial. Based on these in vivo data, hydrogel-based biomaterials were selected to generate NKA constructs to assess in vitro biofunctionality and in vivo regeneration potential. In vitro confirmation of material biocompatibility was provided by live cell/dead cell analysis of the NKA construct (Figure 20). Gelatin-containing hydrogels were associated with strong adhesion to primary renal cell populations. Phenotypic and functional analysis of NKA constructs generated from bioactive primary renal cell populations (UNFX or B2) and hydrogel biomaterials were consistent with the continuous maintenance of the renal tubular epithelial cell phenotype. Transcriptomic, secretome, proteomic, and confocal microscopy analyses of the NKA constructs confirmed no significant differences relative to primary renal cells seeded in 2D culture. Finally, implantation of the hydrogel-based NKA construct into the renal parenchyma of healthy adult rodents was associated with minimal inflammatory and fibrotic responses and regeneration of newly formed kidney-like tissue by 8 weeks post-implantation.

In summary, these data provide evidence that the regenerative response is induced in vivo by NKA constructs. These studies represent the first in vivo intrarenal studies of the biological response of mammalian kidneys to the implantation of therapeutically relevant primary renal cell/biomaterial complexes. The observed results indicate that NKA structures have the potential to promote the regeneration of newborn kidney tissue and weaken non-regenerative (e.g., reparative healing) responses.

Biological response of the mammalian kidney to implantation of polymeric materials. In another study, the host tissue response of the rodent kidney to intrarenal injection of natural and synthetic biomaterials was investigated to evaluate candidate biomaterials for forming cell/biomaterial complexes with bioactive renal cell populations (Presnell et al., supra, 2010). Methods: Natural biomaterials included gelatin and hyaluronic acid (HA). Synthetic biomaterials included polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA). Candidate biomaterials were evaluated in two different physical conformations: homogenous spherical beads or heterogeneous, non-uniform particles. PCL and PLGA beads were prepared using a modified double emulsion (water/oil/water) solvent extraction method. Gelatin beads were purchased (Sigma-Aldrich, St. Louis, MO). PLGA particles were prepared using a solvent casting particle leaching technique; gelatin and HA particles were prepared from cross-linked freeze-dried foams. Two 35 μl injections of the sparsely packed biomaterials were delivered to the left renal parenchyma of 3-month-old St. Louis rats. Histopathological evaluation of formalin-fixed renal tissue sections 1 and 4 weeks after injection was performed using a semiquantitative grading severity scale of inflammation, tissue/cell ingrowth, neovascularization, material breakdown, and fibro-cellular response from 0 (absent) to 4 (marked). The overall score was calculated as the ratio of % positive response to % negative response (the higher the overall score, the better the outcome).

Results. Representative 40X images of kidneys harvested 1 week after histopathological evaluation of biomaterial candidates, sections stained with Masson's Trichrome (data not shown). Materials composed of polymers of natural origin such as gelatin and HA are associated with weaker fibrous-cellular responses and chronic inflammation when compared with synthetic biomaterials such as PLGA and PCL (fibrous capsules of tissue), as well as more cells ingrowth, neovascularization, biomaterial decomposition, and the necessary inflammation required for tissue healing and integration. Overview of histopathological evaluation scores. The mean value (mean ± SD) of the scores was calculated according to the material composition. Synthetic materials (PLGA and PCL) scored the lowest, and gelatin materials were generally scored higher than HA materials. This trend was most evident at 4 weeks. This was attributed to factors unrelated to material injection, and not all samples tested in week 1 were available for analysis in week 4. The number of samples included in the gelatin, HA, and synthetic groups was 3, 4, 3, and 2, 3, 1, respectively, in week 1 and in week 4.

Four weeks after injection, naturally derived biomaterials (eg, gelatin or HA) delivered by injection to healthy renal parenchyma elicited tissue responses that were pathologically weaker than those of synthetically derived biomaterials, as measured by semiquantitative histopathological assessment.

Example 16 - Hypoxic exposure of cultured human kidney cells induces cell migration and attachment and mediators that contribute to renal Repair of tubular cell monolayers in vitro

Study oxygen tension is to have the effect in the separation and function of the selected colony of renal epithelial cells (B2) of the therapeutic function of confirmation in the model of chronic kidney disease (CKD).This study examines whether hypoxic exposure changes the composition and function of selected human renal cells (SRC) or bioactive renal cells (BRC) during processing.After being exposed to 2% oxygen, the following phenomenon is observed: the change of the distribution of cells across density gradient (referring to Presnell et al. WO 10/056328, incorporated herein by reference in its entirety), the raising of productive rate after overall gradient, the regulation of oxygen-regulated gene expression (previously reported in Kelley et al., the same, (2010)), the expression of increased erythropoietin, VEGF, HIF1-α and KDR (VEGFR2).In processing, the exposure to hypoxia enhances the ability of selected bioactive renal cells to repair/regenerate damaged renal tubules.

Figure 27 describes a method for exposing cells to hypoxia during treatment. Figure 28 shows that after exposure to 2% oxygen, the following phenomena were observed: altered cell distribution across the density gradient, improving overall post-gradient yield. Hypoxic exposure (<3%) increased the recovery of cultured human CKD-derived kidney cells from iodixanol-based density gradients (relative to atmospheric oxygen tension (21%)) (96% vs. 74%) and increased the relative distribution of selected cells (B2) to the high-density (>9% iodixanol) fraction (21.6% vs. 11.2%).

Competitive in vitro assays showed that B2 cells pre-exposed to hypoxia for 24 hours were more proficient at repairing damaged proximal tubular cell monolayers than B2 cells cultured under 21% oxygen tension, with 58.6% ± 3% of repair occurring within two hours of injury.

Figure 29A describes an assay developed to observe the repair of renal tubular cell monolayers in vitro. 1. Cells were labeled with fluorescent dye (2% oxygen, 21% oxygen, and HK2 renal tubular cells). 2. A renal tubular cell monolayer was established and the monolayer was damaged. 3. Labeled cells exposed to oxygen were added (cells exposed to 2% and 21% oxygen). They were seeded identically at 20,000/cm2. Cultured in serum-free medium at 5% O2 for 24 hours. 4. Cells that repaired the damage were quantified. Figure 29B - Quantitative imaging analysis (BD Pathway 855 BioImager) - Red circles = cells cultured at 2% O2, blue circles = 21% O2. Figure 29C - Cells induced by 2% oxygen were observed to attach faster (2 hours) and maintain a slight advantage for 24 hours. Cells induced by 2% oxygen are more proficient in repairing the renal tubular epithelial monolayer.

FIG30A depicts an assay developed to visualize the repair of renal tubular cell monolayers in vitro. 1. Cells were labeled with a fluorescent dye. 2. A renal tubular cell monolayer was established on the bottom of an 8 μm pore transwell insert and then damaged. 3. The insert was inverted and labeled cells exposed to oxygen (cells exposed to 2% and 21% oxygen) were added. These were seeded identically at 50,000/cm2. The cells were cultured in serum-free medium under 5% O2 for 24 hours. 4. Cells that repaired the damage were quantified.

Figure 30B shows that induction of cells with 2% oxygen enhanced migration and wound repair compared to uninduced (21% oxygen). Figure 30C plots the % of migrated cells versus migration time. The average number of cells and the average percentage of cells are provided in Table 16.1.

Hypoxia also induced the mRNA expression of CXCR4, MMP9, ICAM1, and dystroglycan, genes involved in cell migration and adhesion. Immunocytochemistry confirmed the local accumulation of MMP9 and the increase of connexin 43 aggregates on the cell membrane.

Figure 31A shows that osteopontin is secreted by renal tubular cells and is upregulated in response to damage (osteopontin immunohistochemistry: Hoechst nuclear stain (blue), osteopontin (red), 10x). Osteopontin is a secreted phosphorylated glycoprotein (Kelly et al. J Am Soc Soc Nephrol, 1999). Osteopontin is expressed in the renal tubules and is involved in adhesion and migration. Osteopontin is upregulated in established renal tubular cell monolayers due to damage, as shown by immunofluorescence (Figure 31A) and ELISA (Figure 31B).

Table 16.1

Quantitative image analysis using simple PCI

Figure 32A shows that the migratory response of the cells is mediated in part by osteopontin (green = migrated cells (5x)). Figure 32B shows that neutralizing antibodies (NAbs) to osteopontin reduce the migratory response of kidney cells by 50%.

Figure 33 shows that hypoxia induces the expression of tissue remodeling genes in cells. Caveolin-1 is a scaffold protein involved in the regulation of integrin signaling. MMP-9 is a metalloproteinase that facilitates migration by degrading the extracellular matrix. ICAM-1 is an intracellular adhesion molecule associated with epithelial cell motility. CXCR4 is a chemokine surface receptor that mediates cell migration.

FIG34 depicts a putative mechanism for hypoxic enhancement of cellular bioactivity leading to renal regeneration.

Taken together, these results indicate that hypoxic exposure facilitates the isolation of specific renal cell subpopulations that exhibit biological activity for in vitro repair of renal tubular damage, potentially enhancing the ability of such cells to migrate and engraft into diseased tissues after in vivo delivery. SRCs demonstrate the ability to stabilize renal function and enhance survival in rodent models of progressive CKD. Low oxygen levels (2% O2) provide the following: increased post-culture recovery of selected regenerative cells; enhanced cell adhesion and monolayer repair in response to tubular damage; and cell migration in response to stimulation of tubular damage. In addition, cell migration and adhesion are mediated in part by osteopontin in vitro, and hypoxia upregulates catenin, secreted proteins, and cell adhesion molecules that mediate tissue remodeling, migration, and intercellular communication.

Example 17 - Urine-derived microvesicles

Analysis of miRNA and protein in the luminal contents of kidney-derived microvesicles excreted into urine was performed to determine whether they could be used as biomarkers to assess regenerative outcomes. Because excess microvesicles are excreted into the extracellular space, some microvesicles fuse with adjacent cells, while others are excreted into urine (Zhou et al. 2008. Kidney Int. 74(5): 613-621). Such urinary microvesicles are now becoming excellent biomarkers for assay development (to better understand treatment outcomes).

Use the ZSF1 rodent model of metabolic disease with chronic progressive renal failure.B2+B4 cells are injected into the renal parenchyma of ZSF1 animals.Healthy animals and PBS vehicle are used as controls.As outlined below, urine-derived vesicles are analyzed at different time points.

1: ZSF1 animals - injected with PBS vehicle; urine collected 197 days after injection

2: ZSF1 animals - injected with PBS vesicles; urine collected 253 days after injection

3: ZSF1 animals - injected with fractions B2+B4; urine collected 197 days after injection

4: ZSF1 animals - injected with fractions B2+B4; urine collected 253 days after injection

5. ZSF1 animals - no injection; urine collected on study day 197

6. ZSF1 animals - no injection; urine collected on study day 253

7. Healthy animals - no injections; urine was collected on study day 197

8. Healthy animals - no injections; urine was collected on study day 253

Urine was collected from the test animals on the 197th and approximately 253rd day after treatment. Microvesicles were recovered from urine by standard methods known in the art (e.g., see Zhou et al. Kidney Int. 2008 September; 74 (5): 613-621). As shown by the standard Western blot in Figure 35, the microvesicles recovered from the urine of the treated animals (lanes 3-4) showed an increase in proteins (CD133 and WNT7A) associated with progenitor cells when compared with vehicle-treated (lanes 1-2) or untreated controls (lanes 5-8). In fact, microvesicles were recovered only from sick animals (lanes 1-6) but not from healthy controls (lanes 7-8), as shown by the expression of microvesicle-specific protein CD63 (Figure 35). Microvesicles containing CD133 appear to be prominosomes discharged from renal cells. Both CD133 and WNT7A are associated with regeneration and stem cell division (Romagnani P and Kalluri R. 2009. Fibrogenesis Tissue Repair. 2(1):3; Lie et al. 2005. Nature. 437(7063):1370-5; Willert et al. 2003. Nature. 423(6938):448-52; Li et al. 2009. Am J Physiol Renal Physiol. 297(6):F1526-33). Taken together, this supports the use of target proteins expressed in microvesicles as biomarkers for the development of assays designed to monitor regeneration.

miRNA Microarray and RT-PCR. Microarray and RT-PCR analysis of miRNA from urine-derived vesicles was performed using standard methods known in the art (e.g., see Wang et al., supra, 2010). In addition to proteins, miRNAs were also found in the contents of isolated microvesicles. Table 17.1 provides examples of miRNAs found to be increased by treatment.

Table 17.1

miRNA RQ value miRNA RQ value miRNA RQ value miR-15b 6.5206 miR-21 6.4755 miR-30a 6.0002 miR-30a* 2.4666 miR-30b-5p 9.8833 miR-30c 6.1688 miR-30d 5.9176 miR-30d* 4.1482 miR-30e 8.0836 miR-30e* 2.1622 miR-141 5.1515 miR-146a 2.3054 miR-151 3.4462 miR-200a 9.3340 miR-200c 8.0278 miR-429 9.7136

ZSF1 animals treated with B2+B4 were analyzed for changes in miRNAs over time (days 197 and 253). Fold changes were observed for the following miRNAs:

The miRNA levels of ZSF1 animals treated with B2+B4 (day 253) were analyzed and compared to the miRNA levels of ZSF1 animals treated with PBS vehicle (day 253). The fold changes of the following miRNAs were observed:

The miRNA levels of ZSF1 animals treated with B2+B4 (day 197) were analyzed and compared to the miRNA levels of ZSF1 animals treated with PBS vehicle (day 197). The fold changes of the following miRNAs were observed:

The miRNAs listed in Table 17.1 provide examples of miRNAs that have been implicated in processes related to tissue regeneration. MiR-15b has been implicated in regulating apoptosis through BCL-2 and caspase regulation (Guo et al. 2009. J Hepatol. 50(4):766-78) and in regulating cell cycle progression through regulation of cyclins (Xia et al. 2009. Biochem Biophys Res Commun. 380(2):205-10). MiR-21 has been shown to inhibit apoptosis by regulating the survival pathway MAPK/ERK. The miR-30 family of miRNAs is crucial for podocyte structure and function, suggesting that an increase may be necessary for glomerular genisis. MiR-141, 200a, 200c, and 429 are all involved in regulating epithelial to mesenchymal transition (EMT) in response to TGF-β signaling, which may reduce fibrosis (Saal et al. 2009. Curr. Opin. Nephrol. Hypertens. 18:317-323). MiR-146a and 151 are implicated in NFκB regulation, thus potentially reducing inflammatory responses in vivo (Taganov et al. 2006. Proc Natl Acad Sci U S A. 103(33):12481-6; Griffiths-Jones et al. 2006. NAR. 34 Database Issue:D140-D144). Taken together, these miRNAs regulate processes associated with successful regenerative outcomes; thus, making them candidate biomarkers for assay development. Taken together, these data support the concept that, in addition to providing physicians with a noninvasive method for monitoring therapy, urinary microvesicles and/or their luminal contents may be viable targets for regenerative assays because they contain proteins and miRNAs that regulate multiple pathways, including TGFβ-1, NFκB, apoptosis, cell division, and pluripotency.

Claims (13)

1. A composition comprising isolated human secretory vesicles produced by an enriched population of renal cells, the composition being used in a method for reducing renal fibrosis in a human subject with nephropathy, the method being carried out by administering the composition to the subject in vivo. The vesicles described herein include miRNAs that inhibit TGFβ1 or PAI-1, and The vesicles thereon have been isolated from the enriched population of kidney cells. 2. The composition of claim 1, wherein the vesicle comprises microvesicles and/or exogenous bodies. 3. The composition of claim 1 or 2, wherein renal fibrosis is reduced by inhibiting epithelial-mesenchymal cell transformation. 4. The composition of claim 3, wherein epithelial-mesenchymal transition is inhibited by inhibiting TGFβ1 signaling or inhibiting plasminogen activator inhibitor-1 (PAI-1) signaling. 5. The composition of claim 3 or 4, wherein the epithelial-mesenchymal transition and/or plasminogen activator inhibitor-1 (PAI-1) is induced by TGFβ1. 6. The composition of any one of claims 3 to 5, wherein the vesicle comprises a miRNA that inhibits TGFβ1. 7. The composition of claim 1, wherein the enriched population of renal cells generating the vesicles comprises: (a) A first cell population, wherein the first cell population is enriched for renal tubular cells and also includes epithelial cells from the collecting duct system; or (b) A combination of a first cell population and a second cell population enriched with renal tubular cells and also including epithelial cells from the collecting duct system, wherein the second cell population includes one or more of glomerular cells and vascular cells. 8. The composition of any one of claims 1-7, wherein the vesicles comprise miRNA. 9. The composition of claim 8, wherein the miRNA is selected from the group consisting of miR-30b-5p, miR-449a, miR-146a, miR-130a, miR-23b, miR-21, miR-124, and miR151. 10. The composition of claim 8, wherein the miRNA is selected from two or more of miR-21, miR-23a, miR-30c, miR-1224, miR-23b, miR-92a, miR-100, miR-125b-5p, miR-195, and miR-10a-5p. 11. The composition of claim 1, wherein the enriched population of kidney cells is non-autologous to the autologous kidney of the human subject. 12. The composition of claim 1, wherein the enriched population of kidney cells is autologous to the human subject's own kidney. 13. Use of the composition of any one of claims 1-12 in the preparation of a pharmaceutical composition for reducing renal fibrosis in the treatment of a human subject with nephropathy.