WO2015020267A1 - Method for manufacturing cell implant including early-endothelial progenitor cells and islet cells - Google Patents

Abstract

The present invention relates to a method for manufacturing a cell implant, the method comprising the steps of: (a) culturing early-endothelial progenitor cells from a mammal; (b) culturing islet cells from a mammal; and (c) mixing the cultured early-endothelial progenitor cells and islet cells to manufacture a cell implant. According to the present invention, the cell implant of the present invention provides excellent ability to reach normoglycemia and ability to grow blood vessels.

Description

 【Specification】

 [Name of invention]

 Manufacturing method of cell transplants including early-endothelial progenitor cells and pancreatic islet cells

 The present invention has been made by the task number 20110023257 under the support of the Ministry of Education, Science and Technology, the research management professional organization of the task is the Korea Research Foundation, the research project name is "general researcher support project" research title is "nanoparticle and magnetic resonance image Validation of vascular endothelial cell-pancreatic islet joint transplantation ", the host institution is Samsung Seoul Hospital, and the research period is Sept. 01, 2014 ~ Aug. 31, 2011.

 The present invention has been made by the overweight number 2012R1A2A2A01013810 under the support of the Ministry of Education, Science and Technology, the research management specialized agency of the project, the Korea Research Foundation research project name is "medium-level researcher support project", the research title is "in the liver context of pancreatic islet allograft Blocking PEGylat ion and IL-1 receptor ", the lead institution is Samsung Seoul Hospital, and the research period is 2012. 05. 01-2015. 04. 30.

 This patent application claims priority to Korean Patent Application No. 10-2013 # 0092735 filed with the Korean Patent Office on August 5, 2013, the disclosure of which is incorporated herein by reference.

 The present invention relates to a method for producing a cell transplant comprising early-endothelial progenitor cells and pancreatic islet cells. [Background technology]

With the establishment of the i slet transplantat ion protocol in Edmonton, Canada, clinical islet cell transplantation has emerged as a possible treatment for the treatment of patients with type 1 diabetes. However, low engraftment rate of transplanted pancreatic islets is a major cause of long-term glycemic control failure. Pancreatic islets should be transplanted successfully through new blood vessel regeneration and blood flow control within a few days after transplantation. However, transplanted somatic cells are exposed to lower blood vessel density, oxygen partial pressure, and nutrients than endogenous islets. Cells Difficulty in engraftment of normal pancreatic islets such as the death process and problems in the regulation of insulin secretion occurs. Completion of the new vascular network takes place approximately 10-14 days after transplantation (3, 4).

 Bone Marrow-derived Stem Cells have been recognized as important therapeutics for clinical cell therapy. Bone marrow-derived pleasant cells include a variety of cells, including Hematopoietic Stem Cells (HSCs), Mesenchymal Stem Cells (MSCs), and endothelial progenitor cells (EPCs). Bone marrow-derived EPCs play an important role in the regeneration of damaged ischemic organs and contribute to the induction of new neovascularization in patients with ischemic disease (5-7).

EPC reports that at least two types exist during the incubation period in laboratory conditions. Early-EPC, or endothelial cell colony-forming units (CFU—FCs) and late-end endothelial colony forming cells (ECFCs) (8-10). ). The two types of EPCs differ in appearance, culture duration, cell proliferation capacity, and expression protein, but have endothelial cell functional characteristics and improved performance through angiogenesis in animal models (8). Late-vascular endothelial progenitor cells show the ability to repair vascular damage, but previous clinical results show that after myocardial infarction, the effective timing of bone marrow-derived cell delivery is 4-10 days. Because of the need for proliferation of late-vascular endothelial progenitor cells, the clinical practice lacks practical access for autologous or allogeneic cell therapy (11). In addition, the origin of late-vascular endothelial progenitor cells has not yet been defined for clinical application, and clinical trials have not yet been made. This has the disadvantage that there are risks of unexpected cancer in long-term laboratory culture conditions, such as mesenchymal enjoyment cells (MSCs) (12,13). Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are The level of the technical field to which the present invention pertains and the content of the present invention are more clearly described by reference to the present specification as a whole.

[Content of invention]

 【Problem to solve】 ᅳ

 The present inventors have sought to ameliorate problems such as low engraftment rate of transplanted islet cells of conventional Islet Cells transplantation. As a result, by culturing the early-Endothelial Progenitor Cells and pancreatic islet cells, the present invention was found to exhibit excellent normal blood glucose uptake and angiogenesis ability in the case of a cell transplant prepared by mixing immediately before transplantation. Completed this year.

 Accordingly, it is an object of the present invention to provide a method for producing a cell transplant bull.

 Another object of the present invention is to provide a cell transplant.

 Another object of the present invention to provide a pharmaceutical composition for improving, preventing or treating diabetes. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

[Measures of problem]

 According to one aspect of the present invention, the present invention provides a method for weeding a cell transplant for pancreatic islet cell transplantation comprising the following steps:

 (a) culturing mammalian early-Endothelial Progenitor Cells;

(b) culturing mammalian islet cells; And ^'

(c) mixing the cultured early-endothelial progenitor cells and pancreatic islet cells to prepare a cell transplant. The present inventors have tried to ameliorate problems such as low engraftment of transplanted islet cells of conventional Islet Cells transplantation. That As a result, it was confirmed that the cell transplants prepared by culturing the early-Endothelial Progenitor Cells and the islet cells, respectively, exhibited excellent engraftment rate and angiogenic ability.

 To prepare a cell transplant for pancreatic islet cell transplantation of the present invention, mammalian early-endothelial progenitor cells are cultured. According to one embodiment of the invention, the early-endothelial progenitor cells used in the present invention are isolated early-endothelial progenitor cells. According to one embodiment, the early-endothelial progenitor cells used in the present invention are bone marrow-derived early-endothelial progenitor cells.

 The culture of early endothelial progenitor cells can be cultured in various media known in the art, such as EGM-2 SingleQuots and EBM-2 (endothelial basal medium-2) supplemented with fetal bovine serum (FBS).

 The mammal of the present invention is human, cow, horse pig, goat, dog, cat, chicken, mouse, rat, rabbit or guinea pig. According to one embodiment of the invention, the mammal is a human or a pig.

 The early stage endothelial progenitor cells for producing a cell transplant of the present invention are progenitor cells prior to differentiation into endothelial cells, and mean cells that are likely to differentiate into endothelial cells under specific conditions. According to one embodiment of the present invention, the early-endothelial progenitor cells separate the bone marrow from the femur and tibia of the mammal to remove erythrocytes and the cells obtained by culturing mononuclear cells in endothelial cell differentiation medium for 5-12 days. it means.

 The early-endothelial progenitor cells of the present invention have the form of fusiform (3 ^ 1 16 ᅳ 3113 6 € 1). According to one embodiment of the invention, the early-endothelial progenitor cells exhibit a spindle-like form when cultured in endothelial cell differentiation culture medium for 7-10 days.

 Early-endothelial progenitor cells of the present invention have the characteristics of endothelial progenitor cells, monocytes and hematopoietic stem cells. According to one embodiment of the invention, the early-endothelial progenitor cells of the present invention are CD106, CD31, Flk-1, eNOS, vWF and VE-cadherin, markers of endothelial progenitor cells, CDllb and CD45, markers of monocytes, and It expresses CD34 and Sca-1, markers of hematopoietic stem cells.

As used herein, the term 'expression' refers to gene expression analysis methods commonly used in the art, such as real-time RT-PCR methods (see Sambrook, J. et. al. , Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Western Blot ("Imaging Systems for Westerns: Chemi luminescence vs. Infrared Detect ion, 2009, Methods in Molecular Biology, Protein Blotting and Detect ion, vol. 536". Humana Press. Retrieved 2010) , FACS (Flow cytometry or Fluorescence-act ivated cell sorting, Loken MR (1990) .Immunofluorescence Techniques in Flow Cytometry and Sorting (2nd ed.). Wieley.pp. 341-53) I) unocytochemistry methods and protocols In case of analyzing gene expression or protein expression according to molecular biology experiments of Edited by Lorette C. Javois, 2nd edition, 1999.Human Press, etc., gene expression and / or than control group without transplantation of the cell transplant of the present invention. It means the case in which the protein expression is analyzed as much.

 The premature endothelial progenitor cells of the present invention are Di 1 -ac-LDL (Acetylated Low Density Lipoprotein labeled with 1,1 '-di oct adecy 1 -3, 3,3', 3 '-t et r amethy 1 i ndo- carbocyanine perchlorate) and BS-1 (from Bandeiraea simpl icifol ia) lectin binding capacity. The Di l-ac-LDL absorption capacity is a marker for identifying endothelial cells. When the cells are in contact with Di I-ac-LDL, the lipoprotein is degraded by lysosomal enzymes and Di l, a fluorescent dye, is used for intracellular membranes. Accumulate in the and can be seen visually. The BS-1 lectin binding capacity is a marker capable of identifying endothelial cells, and when the BS-1 lectin is reacted to the endothelial cells, the endothelial cells show binding capacity with BS 1.

 One of the main features of the present invention is that, after preparation of the early-endothelial progenitor cells and pancreatic islet cells, respectively, in order to prepare a cell transplant for islet cell transplantation, they are mixed.

 According to one embodiment of the present invention, the early endothelial progenitor cells of the present invention are 5-

Incubate for 12 days. According to another embodiment of the present invention, the early stage endothelial progenitor cells of the present invention are cultured for 7-10 days.

 The day before the preparation of the cell transplant for transplantation of pancreatic islets, the culture of the pancreatic islets is disclosed.

According to one embodiment of the invention, the islet cells are cultured for 16-24 hours. Another of the main features of the present invention is that the cell transplant mixes premature endothelial progenitor and pancreatic islet cells just prior to transplantation.

 According to one embodiment of the present invention, the cell transplant has a cell number mixing ratio of 1000: 1 to 10000: 1. According to another embodiment of the present invention, the cell transplant has a cell number mixing ratio of 2500: 1 to 7500: 1.

 The cell transplant of the present invention has excellent normal blood sugar reaching ability.

 According to one embodiment of the present invention, the cell transplant has a normal blood sugar reaching ability of 1.5 to 3.0 times as compared to the pancreatic islet cell transplant. As used herein, the term "normal blood glucose reachability" refers to the efficacy of a cell transplant to reach a blood glucose level of 11.1 mmol / L when a cell transplant is transplanted based on a mouse and injected with 1 g / kg of glucose. Means to come. More specifically, if cumulative diabetic reversal curves were created for the cell transplant, 11. Normal blood glucose reachability is indicated by the percentage of receptors that reach blood glucose levels below 1 mmol / L (see FIG. 2G). According to another embodiment of the present invention, when transplanting the cell graft of the present invention, it showed a normal blood sugar reaching ability of about 82%, the pancreatic islet cell transplant showed a normal blood sugar reaching ability of about 38¾. .

 The cell transplant of the present invention has excellent blood vessel generating ability.

 According to one embodiment of the present invention, the cell transplant has two to three times the capacity to produce blood vessels as compared to the islet cell transplant.

 As used herein, the term "angiogenic ability" refers to angiogenic ability by cell transplantation, and specifically, to confirm the degree of angiogenesis by cell transplantation, immunocytochemistry using CD31 antibody (based on mouse scatter) The number of blood vessels generated by performing the method is confirmed (see FIGS. 4E and 4F).

 According to another embodiment of the present invention, when transplanting the cell graft of the present invention, the number of angiogenesis is increased by 2 to 3 times as compared to the islet cell alone cell transplant.

According to another aspect of the present invention, the present invention provides a cell transplant for pancreatic islet cell transplantation prepared by the above method. According to another aspect of the present invention, there is provided a pharmaceutical composition for improving, preventing or treating diabetes, including a cell transplant prepared by the method of the present invention. When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in the formulation, lactose, textose, sucrose, sorbbi, manny, starch, acacia rubber, phosphate, alginate, gelatin, calcium silicate, microcrystalline selreul Ross, ^polyvinyl, a pyrrolidone, selreul Ross, water, syrup, methyl selreul Ross, methylhydroxy heuksi benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils such as It includes, but is not limited to. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

 The pharmaceutical composition of the present invention can be administered parenterally, and in the case of parenteral administration, it can be administered by topical transplantation or the like.

 Appropriate dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as formulation method, mode of administration, age of patient, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction response. Can be. Preferably, the dosage of the pharmaceutical composition of the present invention is 1- on an adult basis.

10000 cell count / kg body weight.

The pharmaceutical composition of the present invention is readily available to those of ordinary skill in the art. Depending on the methods that can be implemented, it can be prepared in unit dose form or formulated using a pharmaceutically acceptable carrier and / or excipient or incorporated into a multi-dose container. The formulation may be in the form of a solution, suspension, syrup or emulsion of an oil or aqueous medium, or may be in the form of extracts, powders, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer. Since the cell grafts and compositions for improving, preventing or treating diabetes of the present invention are prepared by the method for producing cell grafts, the common contents between the two are omitted in order to avoid excessive complexity of the present specification. 【Effects of the Invention】

 In summary, features and advantages of the present invention are as follows:

 (a) The present invention provides a method for producing a cell transplant for pancreatic islet cell transplantation, a cell transplant and diabetic improvement, prevention or treatment composition.

 (b) The cell transplant of the present invention provides excellent normal blood glucose reaching ability and blood vessel producing ability.

 (c) The cell transplant of the present invention can overcome the low engraftment rate of conventional pancreatic islet cell transplantation.

[Brief Description of Drawings]

 La to le are early marrow bone marrow-derived early -EPCXear ly-Endothel ial

Progeni tor Cel ls). La shows microscopic images of early -EPC cultured for 7 days in endothelial cell culture medium. Scale bar represents 100 ms. FIG. Lb shows the results of analysis of CD31, Flk-1, CD106, CD34, CD117, Sea-1, CDllb and CD45 expressed on the surface of early -EPC by flow cytometry. Figure lc shows the results confirming the Di I-ac-LDL (red) absorption capacity and BS-1 lectin (green) binding capacity of the early EPC on day 7, culture. Scale bars represent 50 μπι. ID is CD31 of early EPC. Flk-1, vWF and eNOS expression is confirmed by RT-PCR. FIG. Le shows the results of confirming the endothelial cell markers CD31, VE-cadher in and Flk-1 by immunochemical staining. 2A-2F show non-fasting blood glucose levels after transplantation. Degree

2a to 2c show the ratio of reaching normal blood glucose according to the number of transplanted islets. FIG. 2A is a group transplanted with 100 pancreatic islets (0/7), FIG. 2B is a group transplanted with 200 pancreatic islets (6/12), and FIG. 2C is a group transplanted with 300 pancreatic islets (3/3) 2D to 2F show changes in blood glucose when islets alone (n = 13), islets + non-EPC (n = 14) and islets + BM-EPC transplantation (η = Γ7). Cumulative rate of diabetic recovery to normal blood glucose over time The graph (cumul at ive di abetes reversal curves, percentage of mice reaching normal blood glucose with a blood glucose concentration of less than 11 mmo 1/2) (* <0.05, vs. -EPC).

3A-3C show insulin measurements from serum at glucose load test and fasted state on day 28 after transplantation. FIG. 3A shows glucose load test for pancreatic islet + EPCs co-transplant group (white square) and pancreatic islet cell transplant group (black circle). Figure 3b shows the AUC _giu (area under the glucose curve) for glucose load test. Figure 3c shows the measurement of serum insulin levels at 0 and 30 minutes after glucose injection in fasted state 0: p <0.05, **: p <0.01).

 4A to 4F show that neovascularization was improved in the area where pancreatic islets were implanted when HF cells were transplanted with islet cells and early -EPC. 4A and 4B show that pancreatic islet cells isolated from a genetically engineered mouse (GFP-Tg) are consistent with a portion stained with insulin antibody. The right panel of FIG. Bars represent 25 i, and FIG. 4B shows the comparison of the area where pancreatic islet cells have settled between the islets alone and the co-transplants of islets + early -EPC. 4C and 4D show the shape and configuration of the settled islet cells and quantitatively show the number of glucagon. The dotted white squares are enlarged to show the composition of glucagon of each group, and the right panel of FIG. 4C shows the distribution of glucagon in one islet cell of the mouse (bar size of 50 um). 4E and 4F show the degree of blood vessel formation in the anchored area, and staining of blood vessels quantitatively shows the number of blood vessels using CD31 antibody (red) (FIG. 4F). Transplanted pancreatic islet cells (isolated from GFP-Tg) are shown in green, with dashed white lines indicating the extent of engraftment. * Part indicates kidney cortex The data are expressed as mean standard error (*: p <0.05, **: p <0.01). .

5A-5C show whether angiogenesis at the site of implantation is due to donor black or recipient origin. FIG. 5A is an outline of an experiment for evaluating the contribution of recipient blood vessels. For this experiment, GFP-Tg mice were used as recipients, and pancreatic islets and early -EPCs were used as donor mice for transplantation. It was. CD31 antibody staining is transplanted in FIG. 5B Total blood vessels at the site are shown and GFP antibody staining represents blood vessels derived from the recipient. The white dotted line shows the implanted area, and the * part shows the kidney cortex. The bar bar size is 100 mm 3. 5C shows quantitatively formed blood vessels, black bars represent totally formed blood vessels, and white bars represent blood vessels derived from recipients with CD31 + / GFP + (**: p <0.01, †: p <0.05 vs Islets cells alone).

 6A-6D show the role of early EPC co-grafted with islet cells. 6A is an experimental summary for evaluating the contribution of transplanted early -EPC. Early-EPC derived from GFP Tg was used to track the role of early -EPC, and normal mice were used for pancreatic islets and recipient mice. Figure 6b is a representative picture showing the insertion of GFP-Tg-derived EPC transplanted into blood vessels stained with CD31. The scale bar is 50. White dashed lines indicate implanted sites. 6C quantitatively shows the number of co-located GFP-positive cells and CD31-positive cells in the islet cell + EPCs group, but not in the islet cell alone transplant group. 6D shows that GFP-Tg-derived EPCs transplanted with pancreatic islets are present at the site of transplantation (left panel), and the remaining EPCs show VEGF expression (middle panel). The scale bar is 100. [Specific contents to carry out invention]

 Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . Example

Experimental Materials and Methods

 Laboratory animals

Green fluorescent transgenic mice (GFP-Tg) derived from C57BL / 6J were purchased from the Jackson Laboratory. An overview of early -EPC and pancreatic islet cell co-transplantation is shown in Figures 5a and As shown in 6a, 10-12-week-old GFP-Tg and normal mice were used as donors and recipients according to experimental conditions. GFP-Tg mice were used to evaluate the degree of contribution of blood vessels from recipients. Intraperitoneal injection of 180 nig / kg of streptozotocin (streptozotocin) was used to induce diabetes in the mice, and mice maintained with sustained hyperglycemia (≥20 μL / l) were used for transplantation. Blood glucose measurement was performed using a blood glucose meter after collecting blood through the tail vein. Animal management and testing procedures were conducted in accordance with the regulations of the Animal Experiment Ethics Committee of Samsung Medical Center. Myeloid-derived vascular endothelial progenitor cells (BM-EPC) isolation and culture

Mouse derived bone marrow was obtained from the femur and tibia. Muscles and connective tissue were removed from the bones, and then bone marrow in the bones was obtained using a 30-gauge follower. Red blood cells were removed by adding ACK lysis buffer to the obtained bone marrow. The obtained bone marrow mononuclear cells (mononuclear eel I s) of EBM-2 (Endothel ial Basal Medi) containing EGM-2 Sin leQuots (EGM-2 medium, Lonza Inc, USA) and 5% Fetal Bovine Serum (FBS) After suspension in 에 -2), 6-well plates coated with 2% gelatin were inoculated with 1 × 10 ⁷ cells / well. After 3-4 days of incubation, the non-stick cells were removed by washing with PBS and replaced with fresh culture. BM-EPC cultured for 7-10 days confirmed the characteristics of endothelial progenitor cells by FACS and immunofluorescence staining. The identified BM-EPCs were transplanted with pancreatic islets. GFP-Tg-derived EPC (GFP EPC) and pancreatic islet cells were co-transplanted to evaluate the effect on angiogenesis. Isolation of Pancreatic Islets

Pancreatic islet cells were isolated from GFP-Tg and normal mice. The pancreatic islets were isolated by injecting 0.8 mg / kg collagenase PCcol lagenase P, Roche, Germany into the cochlear biduct, and only the islet cells were purified using Fi coU density difference. . Pancreatic islets are 10% FBS and 1% Cultured in M199 medium containing penicillin / streptomycin. One day later, pancreatic islet cells were selected for transplantation. Flow cytometry

 Mouse Early—Surface markers of EPCs were analyzed by FACSO CS Calibur).

5 10 ⁵ cells were reacted with primary or isotype control antibody diluted in PBS 100 ^ containing 0.5% BSA for 20 minutes at 4 ^° C and washed three times with PBS. The antibodies used were PEXphycoerythr in) -conjugated anti-mouse CD31, PE-anti-mouse CD117, PE-antimouse Flk-1 (VEGFR2), PE anti-mouse Sea-1 (BE Pharmingen, USA), PE -CD106 (AbD Serotec, UK)-fluoresce in isothiocynate (FITC) -conjugated term—mouse

CD34 (eBioscience, USA), FITC-CDllb, and FITC-CD45 (BD Pharmingen). After washing and fixation a minimum of 10,000 cells were analyzed with Cell Quest Pro software.

RT-PCR Analysis

 Total RNA was extracted from mononuclear cells isolated from bone marrow and early -EPC with TRIzol reagent (Invitrogen) and RT-PCR was performed according to the manual of 1 // g RA Superscript Π Reverse TranscriptaseC Invitrogen (USA) after 7 days of incubation. It was. RT-PCR was performed using AccuPower PCR premix and amplified each cDNA. The primer sequences and PCR product sizes used are as follows:

CD31 forward 5′-TGCAGGAGTCCTTCTCCACT-3 ′ and reverse 5 ′ ACGGriTGATTCCACTTTGC-3 ′, product size: 245 bp); Flk-1 (forward 5′-GGCGGTGGTGACAGTATCTT-3 ′ and reverse 5′-GTCACTGACAGAGGCGATGA-3 ′, product size: 162 bp); vWF (forward 5′-CAGCATCTCTGTGGTCCTGA— 3 ′ and reverse 5′-GATGTTGTTGTGGCAAGTGG-3 ′, product size: 217 bp); eNOS (forward 5'- GACCCTCACCGCTACAACAT-3 ¹ and reverse 5 '-CT (CCTTCTGCTCATTTTC-3', product size: 209 bp); and β-actin (forward -TGTTACCAACTGGGACGACA-S ¹ and reverse 5 ¹ -GGGGTG GAAGGTCTCAAA-3 ' , Product size: 165 bp). Immunochemical staining

Holding the cells at room temperature for 20 minutes in 4% paraformaldehyde and washed three times with PBS H ^■ were ^'. Blocking for 45 min with PBS containing 5¾ normal serum and 0.1% Triton X-100. After blocking, the primary antibody was reacted overnight at 4 ^° C. Primary antibodies are rat anti-CD31 (1: 100, BD Phamingen), rat anti-VE-cadherin (CD144, 1: 100, BD Phamingen) and rabbit anti-Flk-1 (l: 100, Cell Signaling Technology) . After washing three times with PBS, cells were treated with Alexa-568-conjugated goat anti-rat and Alexa-568-conjugated goat anti-rabbit (1: 200, Molecular Probes) for 1 hour at room temperature. Primary antibody was reacted. Cell nuclei were visualized using DAPI (1: 2500, Molecular Probes).

Di I -ac-LDL uptake and BS-1 lectin binding capacity

Early -EPC were incubated in gelatin-coated 8-well chamber slides (Lab-Tek, Nunc, Germany) 에 10 Λι Dil-ac-LDL (l, l'- dioctadecyl-3,3,3 ', 3 'ᅳ tetramethy 1 i ndocarbocyan ine ᅳ labeled acetyl at ed low density lipoprotein, Molecular Probes, USA) was incubated at 37 ^° C for 1 hour, and then fixed with 1% paraformaldehyde. After washing the cells three times with PBS, 37 ^{° with} 10 g / n Fluorescein isothiocyanate (FITC) -conjugated mouse endothelial cell-specific BS ᅳ 1 lectin (Bandeiraea simpl ici fol ia lectin 1, Vector Laboratories, USA) Stain for 1 hour at C. Fluorescence was visualized by confocal microscopy (Carl Zeiss Inc., USA) and fluorescence microscope (Elympus, Japan). Early -EPC and Pancreatic Islet Co-Transplantation

Early -EPC and pancreatic islet cells were collected in tubes (Eppendorf, USA) and centrifuged. Early -EPC and pancreatic islets, each suspension were mixed by pipetting and transferred to a polyethylene (-50) tube using a Hamilton syringe and transplanted under the mouse kidney membrane. Diabetic-induced mice were tested as outlined in the experiments of FIGS. 4, 5 and 6 (Fig. 4-① 200 fluorescent pancreatic islets, ② 200 fluorescent pancreatic islets + 1 10 ⁶ Normal early -EPC (normal mouse used as recipient) Figure 5-① 200 normal islet cells, ② 200 normal islet cells + IX 10 ⁶ Normal early -EPC (used as GFP-Tg recipient); Figure 6- ① 200 normal islets, ② 200 normal islets cells + 1 X 10 ⁶ GFP- early -EPC (normal mice used as recipient)). After transplantation, body weight and blood glucose were measured twice every two weeks. Islet Cell Function Test after Transplantation into Diabetic Mouse

 Intraper i toneal glucose tolerance tests (IPGTT) were performed 14 and 28 days after transplantation. After fasting for 10 hours, 1 g / kg glucose was injected intraperitoneally and blood glucose was measured hourly (0, 15, 30, 45, 60, 90 and 120 minutes). Blood was obtained through retro-Orbi tal plexus at 0 and 30 minutes, and the isolated serum insulin was measured using a rat / mouse inulin i ISA unosorbent assay kit. It was. The percentage of mice that reached normal blood glucose and the time of arrival (day) were calculated for each group, and it was judged to be effective when the blood glucose level fell below 11.1 画 ol / l for two consecutive days. Renal excision with transplanted pancreatic islet cells was performed to show the transplant dependence on whether the diabetic mouse recovered from transplantation. Immunostaining and Morphological Analysis

After transplantation, kidneys were extracted at 14 and 28 days, fixed with 4¾ paraformaldehyde, washed with PBS, and then immersed in 30% sucrose to prepare a frozen block. The tissue was cut into 10 mm 3 and stained. Tissue staining may include anti-insulin antibodies for pancreatic islet cell staining, anti-CD31 antibodies for new blood vessel staining, anti-VEGF antibody, a growth factor involved in angiogenesis, anti-GFP antibodies for staining GFP-derived cells, and Anti-glucagon antibodies were used for alpha cell staining. Tissue was blocked with 10% normal goat serum and then rat anti-mouse CD31 antibody, rat polyclonal anti-GFP, rabbit anti-VEGF antibody, guinea pig anti-insulin antibody and rabbit anti-glucagon antibody. Was used as the primary antibody. In addition, 568-conjugated goth anti—rat, 488 or 568-conjugated goth anti-rabbit antibody and Cy3-conjugated anti-guinea pig Antibodies were used as secondary antibodies. DAPI (4 ′, 6-diamidino-2-phenyi indole) was used for nuclear staining. Morphological measurements and analysis were performed using Image-Pro Plus software version 5.1. Statistical analysis

 Data are expressed as mean value and standard error of measurement. The differences between the groups were analyzed by two-tai led unpai red Student's ί-test and log-rank test. Was considered to be statistically significant when <0.05. Experiment result

Early Mouse -EPC Characterization

Mouse premature -EPC was incubated in endothelial cell differentiation medium (EGM-2 Bul letKi t) for 7 days, and the microscopic observation confirmed that the cells were spindle-shaped. Early -EPC was expressed in macrophages as well as in the expression of endothelial marker proteins CD31, CD106 and Flk-1 _. And CDllb and CD45, which are known as markers of mononuclear leukocytes, and CD34 and Sca-1, which are markers of blood stem cells, were expressed by FACS. In addition, these cells showed the ability to absorb Di I-ac-LDL and BS-1 lectin, which are characteristic of EPC, and expressed endothelial cell marker proteins CD31, Flk-1, vWF, eNOS and VE-cadherin. PCR was confirmed by immunofluorescence staining (FIGS. La to le). Improvement of Diabetes Reversal Rate by Joint Transplantation

We examined whether the number of pancreatic islet cells showing diabetic cure rate at 50% after transplantation improved with early -EPC. In the group transplanted with 100 pancreatic islets alone, there was no blood glucose reduction effect (0/7), but the group with 300 pancreatic islets transplanted alone showed 100% (3/3) blood glucose reduction. When 200 pancreatic islets were transplanted alone, 5OT (6/12) showed a blood glucose reduction effect, and 200 pancreatic islets were treated during co-transplantation (FIGS. 2A to 2C). In the 200 islet cell transplant group alone, 13 of the 13 patients had diabetes recovery, while 14 of the 17 cohorts with early -EPC were normal. Blood glucose was maintained (FIGS. 2D and 2F). The mean blood glucose level of the islet and EPCs co-transplant group was significantly lower than that of the islet cell alone group (10.4 cm 0.7 mmol / L vs. 20.6 mm 2.9 mmol / L, p <0.001). Insulin immunofluorescence staining was performed on the pancreas taken from the transplanted groups. This shows that the remaining beta cells of the pancreas did not contribute to the recovery of diabetes due to regeneration, indicating that diabetes was blessed by the transplanted pancreatic islets. There was a significant difference in the time to reach normal blood glucose (day) between the pancreatic islets alone and the islets and early -EPC co-transplant groups (17.6 土 3.1 days vs. 9.8 sul 1.4 days, <0.05). In addition, the cumulative percentage of normal blood glucose attained over time was higher in the co-transplant group of pancreatic islets and early -EPC (FIG., P <0.05). After transplantation, the body weight decreased in all groups, but the time of co-transplantation of pancreatic islets and early -EPC was significantly increased compared to that of pancreatic islets alone. EPC alone and non-transplanted diabetic groups were unable to maintain blood glucose and eventually were euthanized due to excessive weight loss. When bone marrow-derived non-EPC (non-endothelial progenitor cells) were co-transplanted with islet cells, we examined whether they could improve glycemic control. The results were shown (FIG. 2E). Enhanced function over islet cell transplantation alone

Intraperitoneal glucose tolerance tests (IPGTT) were performed to investigate the function of transplanted islet cells in vivo. On the 14th and 28th day after transplantation, the co-transplant group of pancreatic islets and early -EPC showed improved blood glucose change after glucose loading compared to the islet cell transplant group, and showed significant difference in all time intervals (Fig. 3a). In addition, the value of the area under the glucose curve (AUG _glu ) was significantly lower in the islet and early-EPC co-transplant groups than in the islet-only group. <0.01, Figure 3b). The improved blood glucose control in the islet and early -EPC co-transplant groups was confirmed to be due to increased insulin production from the transplanted islet cells. The pancreatic islet and early -EPC co-transplant groups had significantly higher levels of fasting serum insulin in comparison with the pancreatic islet cells alone (0 min, ^ c0.05; 30 minutes / Ο .ΟΙ). These results show that co-transplantation of pancreatic islets and early -EPC can provide improved function than islets alone (FIG. 3C). Normal morphology and angiogenesis of the islets

To assess the degree of angiogenesis, the transplanted kidney was extracted on day 28. First, the established endocrine (endocrine) areas and non-endocr ine (non-insulin area) areas were evaluated. Since the transplanted GFP-islet cells corresponded to the insulin stained portion, the GFP portion was evaluated as the endocrine region. Co-transplantation groups of pancreatic islets and early -EPCs were significantly wider in the endocrine and non-endocrine areas than in the islets alone group. It was observed that the group of cavities was divided into several masses, and that the pancreatic islet cell transplantation group consisted of loose lumps (<0.05) (FIGS. 4A and 4B). In addition, glucagon-positive cells in co-alpha transplant group is and the distribution around most endocrine area, which was similar to that seen ^"par cells in pancreatic cells of the pancreas is located (Fig. 4c). On the other hand, alpha cells were irregularly distributed in the islet cell transplantation group. And the number of alpha cells between the two groups showed a significant difference (O <0.01) (Fig. 4d). To compare the angiogenic density, the tissues used CD31 antibody. Vascular density was significantly higher in the co-graft group than in the pancreatic islet cell alone group, and blood vessels were observed in and around the endocrine region (p <0.01) (FIGS. 4E and 4F). In addition, AUG _glu by glucose _loading test was correlated with insul in—positive area and vessel densi ty. These data suggest that pancreatic islet and early -EPC co-transplantation can improve the shape preservation of established endocrine cells and the formation of angiogenesis and the establishment of islet cells after transplantation. Enhancement of Angiogenesis

Co-transplantation of islets and early -EPCs has been shown to improve angiogenesis from settled islets. But blood vessels from the recipient I did not know how much it contributed. Therefore, in order to easily identify the recipient-derived angiogenesis, a GFP ᅳ Tg mouse was used as the recipient. Tissues were stained with anti-CD31 antibody and anti-GFP antibody at 14 and 28 days after transplantation. In staining for CD31 (staining blood vessels throughout the anchored area), the blood vessel density was significantly higher in the islet and early -EPC co-transplant groups than in the islet-only transplant group? <0.01), the density of recipient-derived blood vessels (CD31 + / GFP +) was also increased in the islet cell + EPC group? <0.05) (FIGS. 5B and 5C). However, the recipient-derived vascular density made up less than 40% of all CD31 positive vessels. These results indicate that angiogenesis mainly accounts for donor-derived angiogenesis rather than donor-derived angiogenesis. Promotion of neovascularization

 We investigated whether co-implanted early-EPCs persist in the transplanted area and if so, what are their functions. By using GFP-Tg-derived early -EPC for this experiment, it was easy to confirm the presence at the transplanted site. Transplanted GFP-early -EPC was found to be distributed in or around the endocrine region, and EPC in the blood vessels was also observed (4.2 * 0.79 GFP + / CD31 +) (FIGS. 6B and 6C). Occasionally, GFP-early-EPC was observed in the kidney cortex. The transplanted early -EPC was not insulin stained, indicating no differentiation into insulin secreting cells. Early -EPC remaining after transplantation was shown to express VEGF (FIG. 6D). These results suggest that early co-transplantation—EPC can enhance angiogenesis at the site of implantation by both the paracrine effect and the direct incorporat ion mechanism. references

 Shapiro AM, Lakey JR, Ryan EA et al. Islet transplantat ion in seven pat ients wi th type 1 di abetes mel 1 i tus using a glucocort ico id- free immunosuppressive regimen. N Engl J Med 2000; 343: 230-238.

2. Ryan EA, Paty BW, Senior PA et al. Five-year fol low 一 up after cl inical islet transplantat ion. Diabetes 2005; 54: 2060-2069. Jansson L, Car lsson P0. Graft vascular function after transplantation of pancreatic islets. Diabetologia 2002; 45: 749-763.

 4. Vajkoczy P, Olof sson AM, Lehr HA et al. Histogenesis and ul trastructure of pancreatic islet graft microvasculature. Evidence for graft revascularization by endothelial eel Is of host origin. Am J Pathol 1995; 146: 1397-1405.

 5. Takahashi T, Kalka C, Masuda H et al. Ischemia- and cytokine ᅳ induced mobilization of bone mar row 一 derived endothelial progenitor eel Is for neovascularization. Nat Med 1999; 5: 434-438.

 6. Kawamoto A, Gwon HC, Iwaguro H et al. Therapeutic potential of ex vivo expanded endothelial progenitor eel Is for myocardial ischemia. Circulation 2001; 103: 634-637.

 7. Raf i i S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 2003; 9: 702-712.

 8. Hur J, Yoon CH, Kim HS et al. Characterization of two types of endothelial progenitor eel Is and their different contributions to neovasculogenesis. Arterioscler Thromb Vase Biol 2004; 24: 288-293.

 9. Yoder MC, Mead LE, Prater D et al. Redefining endothelial progenitor eel Is via clonal analysis and hematopoietic stem / progeni tor cell principals. Blood 2007; 109: 1801-1809.

 10.Ingram DA, Cap 1 ice NM, Yoder MC. Unresolved questions, changing definitions, and novel paradigms for defining endothelial progenitor cells. Blood 2005; 106: 1525-1531.

 ll. Sekiguchi H, I i M, Losordo DW. The relative potency and safey of endothelial progenitor eel Is and unselected mononuclear eel Is for recovery from myocardial infarct ion and ischemia. J Cell Physiol 2009 219: 235-242.

12. Scolding N, Marks D, Rice C. Autologous mesenchymal bone marrow stem cells: practical consider at ion. J Neurol Sci 2008 2265: 111-115. 13.Siatskas C, Payne NL, Short MA, Bernard CC. A consensus statement addressing mesenchymal stem cell transplant at ion for multiple sclerosis: it's time !. Stem Cell Rev 20066: 500-506. Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

[Patent Claims] [Claim 1]  A method for preparing a cell transplant for islet cell transplantation, which comprises the following steps: (a) Early-Endothelial Progenitor in Mammalian Culturing the cells;  (b) culturing mammalian islet cells; And  (c) mixing the cultured early—endothelial progenitor cells and pancreatic islet cells to produce a cell transplant. [Claim 2]  The method of claim 1, wherein the mammal is a human, cow, horse, pig, goat, dog, cat, chicken, mouse, rat, rabbit or guinea pig. [Claim 3]  The method of claim 1, wherein the early endothelial progenitor cells of step (a) are derived from bone marrow. [Claim 4] The method of claim 1, wherein the early-endothelial progenitor cells of step ( _a ) are spindle-shaped: [Claim 5]  The method of claim 1, wherein the early-endothelial progenitor cells of step (a) express CD106, CD31, Flk-1, eNOS, vWF and VE-cadherin. [Claim 6] The method of claim 1, wherein the early-endothelial progenitor cells of step (a) express CDllb, CD45, CD34 and Sca 1. [Claim 7]  The method of claim 1, wherein the early-endothelial progenitor cells of step (a) have Di l-ac LDL uptake and BS-1 lectin binding capacity. [Claim 8]  The method of claim 1, wherein the early endothelial progenitor cells of step (a) are cultured for 5-12 days. [Claim 9]  The method of claim 1, wherein the islets of step (b) are cultured for 16-24 hours. [Claim 10]  The method of claim 1, wherein the cell transplant of step (c) is characterized in that the cell number mixing ratio of 1000: 1 to 1000 (3: 1). [Claim 11]  The method of claim 1, wherein the mixing of step (c) is performed immediately before transplantation. [Claim 12]  The method of claim 1, wherein the cell transplant has a 1.5 to 3.0-fold normal blood glucose uptake as compared to islet cell transplantation. [Claim 13] The method of claim 1, wherein the cell transplant is characterized in that it has two to three times the capacity to produce blood vessels compared to the islet cells alone. [Claim 14]  A therapeutic cell transplant for pancreatic islet cell transplantation by the method of any one of claims 1 to 13. [Claim 15]  14. A pharmaceutical composition for improving, preventing or treating diabetes, comprising a cell transplant weed by the method of any one of claims 1 to 13.