HK1217179B - Repair and/or reconstitution of invertebral discs - Google Patents

Description

Intervertebral disc repair and/or reconstruction

Field of the Invention

The present invention relates to methods of intervertebral disc repair and reconstruction in an individual. The methods of the present invention are useful in treating spinal conditions characterized by intervertebral disc degeneration.

Background of the Invention

The intervertebral disc (IVD) is the largest major avascular, aneural and lymphatic structure in the human body. The intervertebral disc is essential for the normal function of the spine because it provides flexibility and mechanical stability in axial compression, bending and extension. The IVD is composed of several specific connective tissues: (i) hyaline cartilage of cartilaginous endplates (CEPs), which cover the surface of the vertebral bones (body) above and below the intervertebral disc; (ii) the fibrocartilaginous annulus fibrosus (AF) that encapsulates the nucleus pulposus (NP); and (iii) the central gelatinous nucleus pulposus (NP), which is not hyaline cartilage, although it contains chondrocytes. A transition zone (TZ) has been identified, which, as its name suggests, is located between the AF and the NP. The fibrocartilaginous AF is composed of concentric collagen layers (bone plates) that are connected to the bony edges of the vertebral body.

Proteoglycans (PGs) and collagens of type I, II, III, V, VI, IX, X, and XI are the major matrix components of all these intervertebral disc tissues, but their relative abundance and distribution depend on their anatomical location. PGs, which have a high affinity for water molecules, are most abundant in the NPs of "healthy intervertebral discs." The water absorbed by the PGs generates hydrostatic pressure within the NPs that "expands" the encapsulated fibrocartilage AF. The combination of these specific connective tissues and their respective physiochemical properties contributes to the hydrodynamic and viscoelastic properties of the IVD, which are essential for the normal biomechanical function of the spine.

The IVD undergoes significant matrix changes during aging and degeneration. Studies of human cadaveric and spinal disc specimens obtained during spinal surgery have shown that discs from individuals in the middle-aged to elderly age group often have extensive lesions (1, 2).

Three major types of disc lesions have been identified in these specimens: (i) rim lesions, which are cross-sectional defects proximal to the bone connection of the AF to the vertebral margin; (ii) concentric (circumferential) tears, in which the annular bony plates separate from one another; and (iii) radial tears, which arise from the extension of a tear originating within the NP (1, 2, 3). Rim lesions are of particular interest because they more commonly appear during adolescence and early adulthood within the anterior portion of the AF, proximal to its insertion into the bone at the vertebral margin, suggesting that they may be mechanistically mediated. The presence of rim lesions suggests early failure of the AF and is the primary cause of disc degeneration, but studies of cadaveric specimens have also shown that other pathological features (concentric tears, cystic annular degeneration, NP dehydration, ligamentous osteophytes at the vertebral margins, and osteoarthritis of the posterior intervertebral joints) are also consistently present to some extent (1, 2). Although the temporal history of these respective disc lesions remains a subject of debate, it is generally accepted that PGs and their associated water loss from the NP are early etiological determinants of disc degeneration (4).

As already discussed, the disc functions as a flexible hydroelastic cushion, which is largely regulated by the uptake of water molecules in the NP. A reduction in the NP water content and hence the oncotic pressure would result in supraphysiological mechanical stress imposed on the AF, leading to local failure.

90% of the population will experience medical problems related to back and neck pain due to intervertebral disc degeneration at some time in their lives (5, 6). In humans, the incidence of back or neck pain severe enough to require medical intervention increases in the 30s and 40s, peaks in the 50s, and declines thereafter (5).

In the United States, back pain is the second most common reason for doctor visits, and medical conditions related to back and neck pain result in more hospitalizations than any other musculoskeletal condition. Back pain is the leading cause of lost work time. For example, in the UK, it is estimated that more than 11 million work days are lost each year due to this condition. Furthermore, as longevity increases in the population over the next few decades, back and neck pain problems are expected to increase accordingly.

Despite the high incidence and economic burden of neck and back pain in today's society, the causes are still poorly understood. However, there is a consensus that degeneration and/or failure of the IVD is the primary cause of pain, either directly from the nerves present in the external AF or from adjacent spinal structures that become mechanically compromised due to loss of the hydroelastic function of the disc (7, 8, 9, 10). Disc disease is the cause of 23%-40% of all low back pain cases (11, 12). The external AF is innervated and nerve fibers can extend to its inner third of the depth, so any pathological changes in the external AF may cause pain (13, 14, 15).

The existing paradigm for treating back or neck pain of disc origin is empirical, focusing on lifestyle changes or the use of anti-inflammatory/analgesics to alleviate symptoms or surgical intervention that may require resection of degenerative tissue or spinal arthrodesis for motion restriction. Although spinal fusion is widely used to relieve neck or lower back pain, it is known that it is not a harmless procedure because the mechanical compression imposed on the adjacent disc by the introduction of a rigid segment across the disc space accelerates degenerative changes in the adjacent disc, which may become symptomatic at a later stage (16). Alternative methods of treatment are clearly needed.

It has been reported that intradiscal administration of the protein osteogenic protein-1 (OP-1) (bone morphogenetic protein-7) can stimulate disc matrix repair after experimental degeneration. Disc degeneration was induced in rabbits by pre-injecting the depolymerase chondroitinase ABC into the disc NP, a procedure known as chemonucleolysis (17). After chemonucleolysis, NP and AF cells were found to be more efficient in reconstructing a functional matrix. Disc cells embedded in a normal density extracellular matrix were found to be largely unresponsive to the stimulatory effect of OP-1 on PG synthesis (17).

Using autologous chondrocytes, the research of detecting potential cell therapy to realize the repair of degenerative canine and human IVD has been reported (18,19). The cells used are chondrocytes harvested from healthy NP of the same species and then re-implanted into the intervertebral disc of the defect. The shortcoming of the method is that the cells for this purpose need to be harvested from adjacent healthy intervertebral discs or from other donors of the same species. The violation of AF requires obtaining these cells and the process not only damages the AF structure but also removes living cells from NP and accelerates the degenerative changes in the tissue. Obviously, this method has limited human application.

SUMMARY OF THE INVENTION

This application describes for the first time the in vivo use of STRO-1 ⁺ multipotent cells to promote reconstruction of the nucleus pulposus and annulus fibrosus of degenerative intervertebral discs. STRO-1 ⁺ multipotent cells were derived from an allogeneic source and were well tolerated in the animal model used in this study. This suggests that STRO-1 ⁺ multipotent cells from donors can be grown in large quantities and developed as an "off-the-shelf" product for the treatment of degenerative intervertebral discs.

Thus, the present invention provides a method of repairing and/or reconstructing an intervertebral disc in an individual, the method comprising administering mesenchymal precursor cells (STRO-1 ⁺ multipotent cells) and/or progeny cells thereof to the intervertebral disc.

In an embodiment of the present invention, STRO-1 ⁺ multipotent cells and/or progeny cells thereof are administered to the nucleus pulposus of an intervertebral disc.

Preferably, the STRO-1 ⁺ multipotent cells are also TNAP ⁺ , VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ , CD45 ⁺ , CD146 ⁺ , 3G5 ⁺ , or any combination thereof.

STRO-1 ⁺ multipotent cells and/or their progeny cells can be derived from autologous, allogeneic or xenogeneic sources. In one embodiment, the cells are derived from an allogeneic source.

The methods of the present invention also include administering glycosaminoglycans (GAGs) to the intervertebral disc, such as hyaluronic acid (hyaluronan) (HA), chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, or heparin sulfate. The GAGs can be administered in the same or different composition as the STRO-1 ⁺ multipotent cells and/or their progeny cells.

It should be understood that the methods of the present invention can be practiced on any vertebrate. For example, the subject can be a mammal, such as a human, dog, cat, horse, cow, or sheep.

The methods of the present invention may be used to treat or prevent spinal conditions characterized by disc degeneration, such as low back pain, age-related disc changes, or spondylolisthesis.

Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Hereinafter, the present invention is described by means of the following non-limiting examples and with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic representation of lumbar spinal segments treated with STRO-1 cells in all sheep groups.

Figure 2. Radiographic Disc Height Index (DHI). Mean ± SE of X-ray-derived DHI in sheep receiving low (A) and high (B) doses of STRO-1 cells at baseline and 3 months after chondroitinase ABC-induced degeneration (3 months, pre-STRO-1 cells).

Figure 3. Total disc degeneration scores measured by MRI in discs injected with chondroitinase ABC. Mean ± standard error of total disc degeneration scores measured by MRI before treatment with low-dose STRO-1 cells + HA or HA alone for 3 months (A) or 6 months (B) after chondroitinase ABC injection.

Figure 4. Total disc degeneration scores measured by MRI in discs injected with chondroitinase ABC. Mean ± standard error of total disc degeneration scores measured by MRI before treatment with low-dose STRO-1 cells + HA or HA alone for 3 months (A) or 6 months (B) after chondroitinase ABC injection.

Figure 5. Total histopathological disc degeneration scores. Mean total histopathological disc degeneration scores at 3 months (A) and 6 months (B) for low-dose STRO-1 cells. # indicates a significant difference from the control (p < 0.001), and Ω indicates a significant difference from STRO-1 ⁺ cells (p < 0.01).

Figure 6. Total histopathological disc degeneration scores. Mean total histopathological disc degeneration scores at 3 months (A) and 6 months (B) for high-dose STRO-1 cells. # indicates a significant difference from the control (p < 0.001), and Ω indicates a significant difference from STRO-1 ⁺ cells (p < 0.01).

Figure 7. Total MRI disc degeneration scores. Total MRI disc degeneration scores at 3 months (A) and 6 months (B) for low-dose STRO-1 cells. # indicates a significant difference from the control (p < 0.05), and Ω indicates a significant difference from STRO-1 ⁺ cells (p < 0.05).

Figure 8. Total MRI disc degeneration scores. Total MRI disc degeneration scores at 3 months (A) and 6 months (B) for high-dose STRO-1 cells. # indicates a significant difference from the control (p < 0.05), and Ω indicates a significant difference from STRO-1 ⁺ cells (p < 0.05).

Figure 9. Nucleus pulposus (NP) histopathological degeneration scores. Mean values of NP histopathological degeneration scores at 3 months (A) and 6 months (B) for low-dose STRO-1 cells.

Figure 10. Nucleus pulposus (NP) histopathological degeneration scores. Mean values of NP histopathological degeneration scores at 3 months (A) and 6 months (B) for high-dose STRO-1 cells.

Figure 11. Glycosaminoglycan (GAG) content in the nucleus pulposus as determined by biochemical analysis. Mean ± SD values for glycosaminoglycan (GAG) content in the nucleus pulposus of intervertebral discs injected with low- or high-dose STRO-1 cells for 3 months (A) or 6 months (B). # indicates significant difference from control (p < 0.05).

Figure 12. Radiographically measured disc height index (DHI). A: Mean ± standard error of the X-ray DHI of chondroitinase ABC-induced degenerative discs at 3 and 6 months after injection of HA or HA + low-dose STRO-1 cells. B: Mean ± standard error of the X-ray DHI of chondroitinase ABC-induced degenerative discs at 3 and 6 months after injection of HA or HA + high-dose STRO-1 cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

General techniques and chosen definitions

Unless otherwise defined, all technical and scientific terms used herein should be understood to have the same meanings as commonly understood by those skilled in the art (e.g., in cell culture, stem cell biology, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures well known to those skilled in the art. These techniques are described and explained in the literature in, for example, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

As used herein, the terms "treating,""treat," or "treatment" include administering a therapeutically effective amount of STRO-1 ⁺ multipotent cells and/or progeny cells thereof sufficient to reduce or eliminate at least one symptom of the specified disease state.

As used herein, the terms "preventing,""prevent," or "prevention" include administering a therapeutically effective amount of STRO-1 ⁺ multipotent cells and/or progeny cells thereof sufficient to halt or impede the development of at least one symptom of the specified disease condition.

STRO-1 ⁺ multipotent cells or progeny cells

As used herein, the phrase "STRO-1 ⁺ multipotent cells" should be taken to mean STRO-1 ⁺ and/or TNAP ⁺ progenitor cells that are capable of forming multipotent cell clones.

STRO-1 ⁺ multipotent cells are cells found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestines, lungs, lymph nodes, thymus, bones, ligaments, tendons, skeletal muscle, dermis and periosteum; and can differentiate into germ lines, such as mesoderm and/or endoderm and/or ectoderm. Therefore, STRO-1 ⁺ multipotent cells can differentiate into a large number of cell types, including, but not limited to, adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue and fibrous connective tissue. The specific lineage commitment and differentiation pathways that these cells enter depend on various influences from mechanical influences and/or endogenous bioactive factors such as growth factors, cytokines and/or local microenvironmental conditions established by the host tissue. In one embodiment, STRO-1 ⁺ multipotent cells are non-hematopoietic progenitor cells that divide to produce progeny cells, which are stem cells or precursor cells that will eventually irreversibly differentiate to produce phenotypic cells.

In another embodiment, STRO-1 ⁺ multipotent cells are enriched in a sample obtained from an individual, such as an individual to be treated or a related individual or an unrelated individual (whether the same species or a different species). As used herein, the terms "enriched,""enrichment," or variants thereof, describe a cell population in which the proportion of a particular cell type or the proportion of multiple specific cell types is increased compared to an untreated population.

In another embodiment, the cells used in the present invention express one or more markers selected individually or collectively from TNAP ⁺ , VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ , CD45 ⁺ , CD146 ⁺ , 3G5 ⁺ or any combination thereof.

"Individually" means that the invention individually encompasses the recited marker or marker groups, and although the individual markers or marker groups may not be individually listed herein, the appended claims may define these markers or marker groups individually and separably from each other.

"Collectively" means that the invention includes any number or combination of the listed markers or peptide groups, and although these numbers or combinations of markers or marker groups may not be specifically listed herein, the appended claims may define these combinations or subcombinations individually and separably from any other combinations of markers or marker groups.

Preferably, the STRO-1 ⁺ cells are STRO-1 ^bright (STRO-1 ^bright ) (same as STRO-1 ^bri ). Preferably, the STRO-1 ^bright cells are additionally one or more of TNAP ⁺ , VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ and/or CD146 ⁺ .

In one embodiment, the STRO-1 ⁺ multipotent cells are peripheral blood mesenchymal precursor cells as defined in WO 2004/85630.

A cell is said to be "positive" for a given marker if it expresses a low (lo or dim) or high (bright, bri) level of that marker, depending on the extent of the marker's presence on the cell surface, where the term relates to the intensity of the fluorescent or other marker used in the cell sorting process. The distinction between lo (or dim or dull) and bri should be understood as being in the context of the marker used on the particular cell population being sorted. A cell is said to be "negative" for a given marker if it is not necessarily completely absent from the cell. The term indicates that the cell expresses the marker at a relatively very low level and, when detectably labeled, produces a very low signal or is undetectable above background levels.

As used herein, the term "bright" refers to a marker that produces a relatively high signal on the surface of a cell when the marker is detectably labeled. While not wishing to be bound by theory, it is proposed that "bright" cells express more of the target marker protein (e.g., an antigen recognized by STRO-1) than other cells in the sample. For example, when labeled with a FITC-conjugated STRO-1 antibody, STRO-1 ^bri cells produce a stronger fluorescent signal than non-bright cells (STRO-1 ^{dull /} dim) as determined by fluorescence activated cell sorting (FACS) analysis. Preferably, the "bright" cells constitute at least about 0.1% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In other embodiments, the "bright" cells constitute at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In a preferred embodiment, STRO-1 ^bright cells have a 2 log magnitude higher expression of STRO-1 surface expression relative to "background," i.e., STRO-1 ^negative cells. By comparison, STRO-1 ^dim and/or STRO-1 ^{intermediate cells} have less than 2 log magnitude higher expression of STRO-1 surface expression than "background," typically about 1 log or less than "background."

As used herein, the term "TNAP" is intended to include all isoforms of tissue-nonspecific alkaline phosphatase. For example, the term includes the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In preferred embodiments, TNAP is BAP. In particularly preferred embodiments, TNAP, as used herein, refers to a molecule that binds to a STRO-3 antibody produced by a hybridoma cell line deposited with the ATCC on December 19, 2005, under the terms of the Budapest Treaty, under deposit accession number PTA-7282.

In one embodiment, the STRO-1 ⁺ multipotent cells are capable of giving rise to clonogenic CFU-F.

It is preferred that a substantial proportion of STRO-1 ⁺ multipotent cells be able to differentiate into at least two different germ lineages. Non-limiting examples of lineages to which multipotent cells can be committed include bone progenitor cells; hepatocyte progenitor cells, which are multipotent for bile duct epithelial cells and hepatocytes; neural-restricted cells, which can give rise to glial progenitor cells that develop into oligodendrocytes and astrocytes; neuronal progenitor cells that develop into neurons; myocardium and cardiomyocytes, precursors of the glucose-responsive, insulin-secreting pancreatic β cell lineage. Other lineages include, but are not limited to, odontoblasts, tooth-producing cells, and chondrocytes, and precursor cells of the following: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal tubular epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, myocardium, smooth muscle, skeletal muscle, pericytes, vessels, epithelia, glia, neurons, astrocytes, and oligodendrocytes.

In another embodiment, the STRO-1 ⁺ multipotent cells are incapable of becoming hematopoietic cells in culture.

In one embodiment, cells are taken from the individual to be treated, cultured in vitro using standard techniques, and used to obtain supernatant or soluble factors or expanded cells for administration to the individual as an autologous or allogeneic composition. In an alternative embodiment, cells from one or more established human cell lines are used. In another useful embodiment of the invention, non-human animal cells (or from another species if the patient is not human) are used.

The present invention also contemplates the use of supernatants or soluble factors obtained from or derived from STRO-1 ⁺ multipotent cells and/or progeny cells (the latter also referred to as expanded cells) produced by in vitro culture thereof. The cells expanded by the present invention may have a variety of phenotypes depending on culture conditions (including the quantity and/or type of stimulating factors in the culture medium), number of passages, etc. In certain embodiments, the progeny cells are obtained after about 2 generations, about 3 generations, about 4 generations, about 5 generations, about 6 generations, about 7 generations, about 8 generations, about 9 generations or about 10 generations of passage from the parent population. However, progeny cells can be obtained after any number of passages from the parent population.

Progeny cells can be obtained by culturing in any suitable culture medium. The term "culture medium" used in connection with cell culture includes the components of the environment surrounding the cells. Culture medium can be a solid, liquid, gaseous, or a mixture of phases and materials. Culture medium includes liquid growth medium and liquid culture medium that does not support cell growth. Culture medium also includes colloidal culture medium, such as agar, agarose, gelatin, and collagen matrix. Exemplary gaseous culture medium includes the gas phase to which cells grown on a petri dish or other solid or semisolid carrier are exposed. The term "culture medium" also refers to materials intended for cell culture, even if they are not in contact with cells. In other words, a nutrient-rich liquid prepared for bacterial culture is a culture medium. When a powder mixture becomes suitable for culturing cells when mixed with water or other liquids, the powder mixture can be referred to as a "powder culture medium."

In one embodiment, TNAP ⁺ STRO-1 ⁺ multipotent cells are isolated from the bone marrow using magnetic bead-labeled STRO-3 antibodies and then cultured and expanded to obtain progeny cells useful for the methods of the present invention. (For examples of suitable culture conditions, see Gronthos et al. Blood 85:929-940, 1995).

In one embodiment, such expanded cells (progeny) (preferably, after at least 5 generations) may be TNAP- ^, CC9 ⁺ , HLA class I ⁺ , HLA class ^II- , ^CD14- , ^CD19- , CD3-, CD11a ^{- c-} , ^CD31- , ^CD86- , ^{CD34- ,} and/or ^CD80- . However, it is possible that the expression of different markers may vary under culture conditions different from those described herein. Furthermore, while cells of these phenotypes may predominate in the expanded cell population, it does not mean that a minority of cells do not have this phenotype (e.g., a small percentage of the expanded cells may be ^CC9- ). In a preferred embodiment, the expanded cells still have the ability to differentiate into different cell types.

In one embodiment, the expanded cell population used to obtain supernatant or soluble factors or the cells themselves comprises cells wherein at least 25%, more preferably at least 50%, of the cells are CC9 ⁺ .

In another embodiment, the expanded cell population used to obtain supernatant or soluble factors or the cells themselves comprises cells wherein at least 40%, more preferably at least 45%, of the cells are STRO-1 ⁺ .

In another embodiment, the expanded cells may express one or more markers collectively or individually selected from LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5, CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD90, CD29, CD18, CD61, integrin beta 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R, leptin-R (STRO-2=leptin-R), RANKL, STRO-1 ^beta and CD146, or any combination of these markers.

In one embodiment, the progeny cells are specialized amplified STRO-1 ⁺ specialized cell progeny (MEMPs) as defined and/or described in WO 2006/032092. Methods for preparing an enriched population of STRO-1 ⁺ specialized cells are described in WO 01/04268 and WO 2004/085630, and the progeny can be derived from the STRO-1 ⁺ specialized cells. In an in vitro context, STRO-1 ⁺ specialized cells rarely exist as absolutely pure preparations and are usually present together with other cells, which are tissue-specific committed cells (TSCCs). WO 01/04268 relates to harvesting such cells from the bone marrow at a purity level of about 0.1% to 90%. The colony of STRO-1 ⁺ specialized cells from which the progeny are derived can be harvested directly from a tissue source, or alternatively, it can be a colony that has been amplified in vitro.

^For example, progeny can be obtained from a population of harvested, unexpanded, substantially purified STRO-1 ⁺ multipotent cells that constitute at least about 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 95% of the total cells of the population in which they are present. This level can be achieved, for example, by selecting cells that are positive for at least one marker selected individually or collectively from the group consisting of TNAP, STRO-1 ^bright , 3G5 ⁺ , VCAM-1, THY-1, CD146, and STRO-2.

The difference between MEMPs and freshly harvested STRO-1 ⁺ multipotent cells is that MEMPs are positive for the marker STRO-1 ^bri and negative for the marker alkaline phosphatase (ALP). In contrast, freshly isolated STRO-1 ⁺ multipotent cells are positive for both STRO-1 ^bri and ALP. In a preferred embodiment of the present invention, at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the administered cells have the phenotype STRO-1 ^bri , ALP ^- . In another preferred embodiment, MEMPs are positive for one or more of the markers Ki67, CD44 and/or CD49c/CD29, VLA-3, α3β1. In still another preferred embodiment, MEMPs do not exhibit TERT activity and/or are negative for the marker CD18.

The initial population of STRO-1 ⁺ multipotent cells may be derived from any one or more of the tissue types set out in WO 01/04268 or WO 2004/085630, i.e. bone marrow, dental pulp cells, adipose tissue and skin; or may be derived more broadly from adipose tissue, teeth, dental pulp, skin, liver, kidney, heart, retina, brain, hair follicles, intestine, lung, spleen, lymph nodes, thymus, pancreas, bone, ligament, bone marrow, tendon and skeletal muscle.

It will be appreciated that in practicing the present invention, the separation of cells carrying any given cell surface marker may be achieved by many different methods, however, preferred methods rely on binding of a binding agent (e.g., an antibody or antigen binding fragment thereof) to the marker of interest, followed by separation of those that exhibit binding, either high level binding, low level binding, or no binding. The most convenient binding agents are antibodies or antibody-based molecules, preferably monoclonal antibodies or monoclonal antibody-based molecules due to the specificity of these latter reagents. Antibodies may be used in both steps, however other reagents may also be used, such that ligands for these markers may also be used to enrich for cells carrying the marker or lacking the marker.

Can antibody or ligand be connected to solid support to allow crude separation.Preferably, separation technology maximizes the vitality of the component to be collected.Can use various technologies with different efficiencies to obtain relatively crude separation.The specific technology used depends on separation efficiency, relevant cytotoxicity, the simplicity and speed of implementation and the needs for advanced equipment and/or technical skills.Separation operation can include, but is not limited to, magnetic separation, affinity chromatography and " panning " with the antibody-coated magnetic beads of antibody.Technology that provides accurate separation includes, but is not limited to FACS.The method for implementing FACS is well known to those skilled in the art.

Antibodies to each of the markers described herein are commercially available (eg, monoclonal antibodies to STRO-1 can be purchased from R&D Systems, USA), available from the ATCC or other depositories, and/or can be prepared using art-recognized techniques.

Preferably, the method for isolating STRO-1 ⁺ multipotent cells, for example, comprises a first step of a solid phase sorting step using, for example, magnetic activated cell sorting (MACS) that identifies high expression of STRO-1. If desired, a second sorting step is then performed to produce higher levels of precursor cell expression as described in patent specification WO 01/14268. This second sorting step may involve the use of two or more markers.

The method for obtaining STRO-1 ⁺ multipotent cells may also include harvesting the cell source using known techniques prior to the first enrichment step. Thus, the tissue is surgically removed. Cells comprising the source tissue are then isolated into a so-called single-cell suspension. This isolation can be achieved by physical and/or enzymatic means.

Once a suitable population of STRO-1 ⁺ multipotent cells is obtained, they can be cultured or expanded by any suitable method to obtain MEMPs.

In one embodiment, cells are taken from the individual to be treated, cultured in vitro using standard techniques and used to obtain supernatant or soluble factors or expanded cells for administration to the individual as an autologous or allogeneic composition. In an optional embodiment, cells of one or more established human cell lines are used to obtain supernatant or soluble factors. In another useful embodiment of the present invention, non-human animal cells (or if the patient is not human, from another species) are used to obtain supernatant or soluble factors.

The present invention can be practiced using cells from any non-human animal species, including, but not limited to, non-human primate, ungulate, canine, feline, lagomorph, rodent, avian, and fish cells. Primate cells that can be used to practice the present invention include, but are not limited to, orangutans, baboons, cynomolgus macaques, and any other New World or Old World monkeys. Ungulates that can be used to practice the present invention include, but are not limited to, bovine, porcine, ovine, goat, horse, buffalo, and bison cells. Rodent cells that can be used to practice the present invention include, but are not limited to, mouse, rat, guinea pig, hamster, and gerbil cells. Examples of lagomorph species that can be used to practice the present invention include rabbits, jackrabbits, hares, cottontail rabbits, snow hares, and pikas. Chickens (Gallus gallus) are an example of avian species that can be used to practice the present invention.

Cells useful for the methods of the present invention may be preserved prior to use or obtaining supernatant or soluble factors. Methods and procedures for preserving or storing eukaryotic cells, and in particular mammalian cells, are known in the art (see, for example, Pollard, J.W. and Walker, J.M. (1997) Basic Cell Culture Protocols, Second Edition, Humana Press, Totowa, N.J.; Freshney, R.I. (2000) Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.). Any method for maintaining the biological activity of isolated stem cells, such as mesenchymal stem cells/progenitor cells, or their progeny, may be used in conjunction with the present invention. In a preferred embodiment, the cells are maintained and stored by cryopreservation.

Administration and composition

The dose of STRO-1 ⁺ multipotent cells or their progeny administered can vary depending on factors such as the disease state, age, sex and weight of the individual. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be reduced or increased accordingly depending on the urgency of the therapeutic situation. For ease of administration and uniformity of dosage, it is advantageous to formulate parenteral compositions in dosage unit form. "Dosage unit form" as used herein refers to physically separate units suitable as unit doses for the individual to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier.

In one example, the dose of STRO-1 ⁺ multipotent cells administered ranges from 0.1×10 ⁶ to 4.0×10 ⁶ cells. For example, the dose can be 0.5×10 ⁶ cells.

It should be understood that STRO-1 ⁺ multipotent cells and/or their progeny can be processed into various forms (eg, solution suspension, solid, porous, woven, non-woven, particles, gel, paste, etc.) before being added to the intervertebral disc space.

It is contemplated that many biomaterials and synthetic materials may be used to co-inject with STRO-1 ⁺ specialized cells and/or their offspring into the nucleus pulposus to repair normal mechanical and/or physiological properties for damaged intervertebral discs. For example, one or more natural or synthetic glycosaminoglycans (GAGs) or mucopolysaccharides, such as hyaluronic acid (HA), chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparin sulfate, galactosamine glucuronide (galactosaminoglycuronglycan sulfate) (GGGS), see previous changes and others, including their physiological salts, may be directly injected into the nucleus pulposus. It has been suggested that HA plays a role in stimulating the synthesis of endogenous HA in synoviocytes and the synthesis of proteoglycans in chondrocytes, inhibiting the release of cartilage-degrading enzymes and acting as a scavenger of oxygen free radicals, which play a role in cartilage degradation. Chondroitin sulfate and glucosamine injections similarly show the progression of blocking articular cartilage degeneration. Arguably, other GAGs can provide similar protective or repair properties with therapeutic value, which makes them ideal candidates for injection into the intervertebral disc experiencing degenerative disc disease. Another valuable property of GAGs is their strong ability to attract and retain water. Therefore, it is suitable to mix GAGs with water or other aqueous materials to form a viscous gel, which can then be injected into the gap created by the suction of the nucleus pulposus, or alternatively, added to the existing nucleus pulposus as a supplement. Thereby forming a natural "hydrogel", which can three-dimensionally fill the gap and act similarly to a packing material that resists extrusion and enables the intervertebral disc to absorb the associated vibrations of the activity.

Synthetic hyaluronic acid gels such as HYALURONIC® (Ferring Pharmaceuticals) or HYALURONIC® (Q-MedAktiebolag Co., Sweden) are suitable for use in the present invention.

Examples of other injectable synthetic materials that can be used for co-administration include medical-grade silicone, (solid silicone particles suspended in a polyvinylpyrrolidone carrier; Uroplasty BV, Netherlands), (polymethyl methacrylate (PMMA) microspheres suspended in a gelatin carrier; Artcs Medical, USA), (smooth PMMA spheres suspended in a bovine cartilage carrier; Artepharma Pharmazeu Tische, GMBH Co., Germany). In addition, synthetic hydrogel compositions can be used as filler materials to restore the normal shape of the intervertebral disc, thereby restoring normal biomechanical function.

Antioxidants with known cartilage protection ability are also candidates for injection into the nucleus pulposus. Examples of these antioxidants include tocopherol (vitamin E), superoxide dismutase (SOD), ascorbic acid (vitamin C), catalase and other. In addition, the amphoteric derivatives of sodium alginate for injection are also contemplated herein. In addition, recombinant osteoprotein-1 (OP-1) is a good candidate for injection because it promotes the formation of a proteoglycan-enriched matrix in the nucleus pulposus and annulus fibrosus cells.

The use of synthetic injectables is also contemplated. These synthetic injectables are particularly suitable when the primary goal is to restore the biomechanical function of the intervertebral disc.

Hyaluronic acid, alone or in combination with other glycosaminoglycans, can be used as a carrier to deliver bioactive materials. In a preferred embodiment, hyaluronic acid and/or other GAGs are used as carriers for STRO-1 ⁺ multipotent cells or their progeny cells. The concentration and viscosity of the hyaluronic acid/GAG composition are routinely adjusted to be suitable for a given use.

In another example, a mixture of STRO-1 ⁺ multipotent cells or their progeny cells and fibrin glue can be delivered. The term "fibrin glue" as used herein refers to an insoluble matrix formed by cross-linking of fibrin polymers in the presence of calcium ions. Fibrin glue can be formed from the following: fibrinogen or its derivatives or metabolites, fibrin (soluble monomers or polymers) and/or its complex derived from biological tissue or liquid forming a fibrin matrix. Alternatively, fibrin glue can be formed from the following: fibrinogen or its derivatives or metabolites or fibrin produced by recombinant DNA technology.

The interaction of the catalyst (for example thrombin and/or factor XIII) formed by fibrinogen and fibrin glue can also form fibrin glue.It will be appreciated by those skilled in the art that in the presence of catalyst (for example thrombin), fibrinogen is cut by proteolysis and is converted into fibrin monomer.The fibrin monomer can form polymer subsequently, and this polymer can cross-link to form fibrin glue matrix.The existence of catalyst such as factor XIII can strengthen the cross-linking of fibrin polymer.The catalyst that fibrin glue forms can be from blood plasma, condensation precipitate or other blood plasma components containing fibrinogen or thrombin.Selectively, catalyst can be produced by recombinant DNA technology.

The rate of clot formation depends on the concentration of thrombin mixed with fibrinogen. As an enzyme-dependent reaction, the higher the temperature (up to 37°C), the faster the clot formation rate. The tensile strength of the clot depends on the concentration of fibrinogen used.

When fibrin clot is produced in the presence of hyaluronic acid, it undergoes interaction and becomes interlaced with a cross-linked matrix. It is known that the matrix plays a major role in tissue regeneration and performs cell regulation functions in tissue repair [Weigel PH, Fuller GM, LeBoeuf RD. (1986) A model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing (hyaluronic acid and fibrin in the early events of inflammatory response and wound healing) .J Theor Biol. 119: 219–34]. In the HA-fibrin matrix, the dissolution rate of hyaluronic acid can also be extended, which is beneficial in extending the therapeutic effect of the GAG (Wadstrom J and Tengblad A (1993) Fibrin glue reduces the dissolution rate of sodium hyaluronate (fibrin glue reduces the dissolution rate of sodium hyaluronate), Upsala J Med Sci. 98: 159–167).

Several publications describe the use of fibrin glue for the delivery of therapeutic agents. For example, U.S. Patent No. 4,983,393 discloses a composition for use as an intravaginal insert comprising agarose, agar, saline glycosaminoglycans, collagen, fibrin, and an enzyme. In addition, U.S. Patent No. 3,089,815 discloses an injectable pharmaceutical formulation consisting of fibrinogen and thrombin, and U.S. Patent No. 6,468,527 discloses a fibrin glue that promotes the delivery of various biological and non-biological agents to specific locations in the body. However, the use of fibrin + hyaluronic acid + to promote chondrogenic differentiation of STRO-1 ⁺ allogeneic cells has not yet been described.

The composition comprising STRO-1 ⁺ multipotent cells and/or their progeny cells is "surgically added" to the intervertebral disc space. That is, the material is added by intervention of medical personnel, as opposed to "addition" through the body's natural growth or regenerative processes. The surgical procedure preferably comprises injection via a hypodermic needle, but other surgical methods of introducing the collagen-based material into the intervertebral disc may also be used. For example, the material may be introduced into the intervertebral disc by extrusion through an enlarged annular opening, infusion through a catheter, insertion through an opening created by trauma or surgical incision, or by other means of invasively or less invasively placing the material into the intervertebral disc space.

In some embodiments of the present invention, it may not be necessary or desirable to suppress the patient's immune response before initiating treatment with the cell composition. Indeed, the results presented herein show that transplantation of allogeneic STRO-1 ⁺ multipotent cells in sheep is well tolerated in the absence of immunosuppression.

In other cases, however, it is desirable or suitable to suppress the patient's immune response with drugs before starting cell therapy. This can be achieved by using systemic or local immunosuppressants, or this can be achieved by cells delivered in a packaged device. Cells can be encapsulated into capsules that are permeable to nutrients and oxygen required for the cells and therapeutic factors, and cells are impermeable to immune humoral factors and cells. Preferably, the sealant is hypoallergenic, easily and stably located in the target tissue and provides more protection to the implant structure. These means and other means for reducing or eliminating the immune response for transplanted cells are known in the art. Alternatively, cells can be genetically modified to reduce their immunogenicity.

It should be understood that STRO-1 ⁺ multipotent cells or their progeny can be administered together with other beneficial drugs or biomolecules (growth factors, trophic factors). When administered together with other agents, they can be co-administered in a single pharmaceutical composition, or administered simultaneously or sequentially with the other agents in separate pharmaceutical compositions (before or after the other agents are administered). Biologically active factors that can be co-administered include anti-apoptotic agents (e.g., EPO, EPO mimetics, TPO, IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-β inhibitors, statins, IL-6 and IL-1 inhibitors, phenobarbital, tranilast, remicade, sirolimus, and NSAIDs (non-steroidal anti-inflammatory drugs, e.g., tepoxalin, tolmetin, suprofen); immunosuppressive/immunomodulatory agents (e.g., calcineurin inhibitors, such as cyclosporine, tacrolimus); mTOR inhibitors (e.g., sirolimus, everolimus); anti-proliferative agents (e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g., prednisolone, hydrocortisone); Antibodies, such as monoclonal anti-IL-2Rα receptor antibodies (e.g., basiliximab, daclizumab), polyclonal anti-T cell antibodies (e.g., antithymocyte globulin (ATG); antilymphoglobulin (ALG); monoclonal anti-T cell antibody OKT3)); antithrombotic agents (e.g., heparin, heparin derivatives, urokinase, PPack (D-phenylalanine proline arginine chloromethyl ketone), antithrombin compounds, platelet receptor antagonists, antithrombin antibodies, antiplatelet receptor antibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandin inhibitors and platelet inhibitors); and antioxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine) and local anesthetics.

Genetically modified cells

In one embodiment, the STRO-1 ⁺ multipotent cells and/or progeny cells thereof are genetically modified, e.g., to express and/or secrete a protein of interest, e.g., a protein that provides therapeutic and/or prophylactic benefit, such as insulin, glucagon, somatostatin, trypsinogen, chymotrypsinogen, elastase, carboxypeptidase, pancreatic lipase, or amylase, or a polypeptide associated with or causative for enhanced angiogenesis, or a polypeptide associated with cell differentiation into pancreatic cells or vascular cells.

The method for genetically modifying cells will be apparent to those skilled in the art. For example, nucleic acid to be expressed in the cell is operably connected to a promoter for inducing expression in the cell. For example, nucleic acid is connected to an operable promoter in a variety of individual cells, such as a viral promoter, for example a CMV promoter (for example a CMV-IE promoter) or a SV-40 promoter. Other suitable promoters are known in the art and should be applied in embodiments of the present invention with necessary modifications.

Preferably, nucleic acid is provided in the form of expression construct.Term used herein " expression construct " refers to the nucleic acid with the ability of expression of nucleic acid (for example, reporter gene and/or opposite selection reporter gene) that gives cell, and this nucleic acid is operably connected to the described nucleic acid that is given expression.In background of the present invention, should be understood that expression construct can comprise or can be plasmid, phage, phagemid, clay, viral subgenome or genomic fragment or can keep and/or copy other nucleic acid of heterologous DNA in expressible mode.

Methods for constructing suitable expression constructs for practicing the present invention will be readily apparent to those skilled in the art and are described, for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) or Sambrooke et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 3rd edition 2001). For example, each component of the expression construct is amplified from a suitable template nucleic acid using, for example, PCR and subsequently cloned into a suitable expression construct, such as a plasmid or phagemid.

Suitable vectors for the expression construct are known in the art and/or described herein. For example, expression vectors suitable for the methods of the present invention in mammalian cells are, for example, vectors of the pcDNA vector set provided by Invitrogen, vectors of the pCI vector set (Promega), vectors of the pCMV vector set (Clontech), pM vectors (Clontech), pSI vectors (Promega), VP16 vectors (Clontech), or vectors of the pcDNA vector set (Invitrogen).

Those skilled in the art will be aware of other vectors and sources of such vectors, for example, Invitrogen Corporation, Clontech, or Promega.

The nucleic acid molecule of separation or the gene construct comprising it are introduced into the cell for the means of expression are known to those skilled in the art.The technology for given biology depends on known successful technology.The means by which recombinant DNA is introduced into the cell include microinjection, DEAE-dextran-mediated transfection, such as by using liposome-mediated transfection, PEG-mediated DNA uptake, electroporation and such as by using the tungsten particles or gold particles (Agracetus Inc., WI, USA) coated with DNA.

Alternatively, the expression construct of the present invention is a viral vector. Suitable viral vectors are known in the art and can be purchased commercially. Conventional viral-based systems for delivering nucleic acids and integrating the nucleic acids into the host cell genome include, for example, retroviral vectors, lentiviral vectors or adeno-associated viral vectors. Alternatively, adenoviral vectors are useful for introducing free nucleic acids into host cells. Viral vectors are effective and versatile methods for gene transfer in target cells and tissues. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.

For example, retroviral vectors typically contain cis-acting long terminal repeats (LTRs) with a packaging capacity of up to 6-10 kb of foreign sequences. Minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate the expression construct into target cells to provide long-term expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SrV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J Virol. 56:2731-2739 (1992); Johann et al, J. Virol. 65:1635-1640 (1992); Sommerfelt et al, Virol. 76:58-59 (1990); Wilson et al, J. Virol. 63:274-2318 (1989); Miller et al, J. Virol. 65:2220-2224 (1991); PCT/US94/05700; Miller and Rosman BioTechniques 7:980-990, 1989; Miller, A.D. Human Gene Therapy 7:5-14, 1990; Scarpa et al Virology 75:849-852, 1991; Burns et al. Proc. Natl. Acad. Sci USA 90:8033-8037, 1993).

Various adeno-associated virus (AAV) vector systems have been developed for nucleic acid delivery. AAV vectors can be readily constructed using techniques known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. Molec. Cell. Biol. 5:3988-3996, 1988; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter Current Opinion in Biotechnology 5:533-539, 1992; Muzyczka. Current Topics in Microbiol, and Immunol. 158:97-129, 1992; Kotin, Human Gene Therapy 5:793-801, 1994; Shelling and Smith Gene Therapy 7:165-169, 1994; and Zhou et al. J. Exp. Med. 179:1867-1875,1994.

Other viral vectors useful for delivering the expression constructs of the invention include, for example, those derived from the pox family or alphavirus genus of viruses such as vaccinia virus and fowlpox virus, or conjugative virus vectors (e.g., described in Fisher-Hochet et al., Proc. Natl Acad. Sci. USA 56:317-321, 1989).

Example

Example 1: Experimental Design

24 sheep received an injection of 1.0 IU chondroitinase ABC (cABC) (Seikagaku Corporation, Japan) into three adjacent lumbar intervertebral discs (designated L3-L4, L4-L5, and L5-L6) to initiate progressive disc degeneration. The remaining lumbar intervertebral discs (designated L1-L2 and L2-L3) were not injected with cABC and served as normal controls. After 15 weeks (± 3 weeks) of cABC administration, injections of high or low doses (4×10 ⁶ or 0.5×10 ⁶ cells, respectively) of STRO-1 ⁺ multipotent cells or ProFreeze ^™ NOA freezing medium (Lonza Walkersville Md.) mixed with an equal volume of hyaluronic acid (Ferring Pharmaceuticals) were directly administered into the nucleus pulposus of the intervertebral disc (Table 1). Animals were autopsied 3 or 6 months after injection. Figure 1 is a schematic diagram of the spinal segments and treatment regimens used for the study.

Table 1: Summary of study design

Example 2: Expansion of immune-selected STRO-1 ⁺ multipotent cells

The STRO-1 ⁺ multipotent cells used in these experiments were derived from French sheep and were prepared by Lonza (USA).Bone marrow (BM) aspirates were obtained from sheep and BM mononuclear cells (BMMNCs) were prepared essentially as described previously (US 2005-0158289).

STRO-1 ⁺ multipotent cells were subsequently isolated by magnetic activated cell sorting as previously described (WO 2006/108229) using STRO-3 antibodies.

Example 3: Radiology

Under induced anesthesia, lateral radiographs of the lumbar spine were taken at the following time points: day 0 (injection of cABC), the day of test article administration (15 ± 3 weeks after induction of lumbar disc degeneration), and 3 and 6 months after test article implantation. Radiographs were evaluated by an observer blinded to the experimental design using the intervertebral height index (DHI), which was calculated as previously described (24) by averaging the measurements from the anterior, middle, and posterior portions of the IVD and dividing them by the mean of the adjacent intervertebral body heights.

Example 4: Magnetic Resonance Imaging (MRI)

Lumbar spine MRI was performed on all sheep under induced anesthesia approximately 15 weeks after induction of disc degeneration by injection of chondroitinase ABC and before treatment with intradiscal injection of HA or high and low doses of HA + STRO-1 ⁺ cells (Tx). Spine MRI was performed on all sheep again 3 and 6 months after treatment and immediately before necropsy (Nx).

Imaging was performed on one of two MRI scanners. The MRI performed before administering STRO-1 ⁺ cells was performed using a 1.5T Siemens VISION MRI scanner equipped with Numaris33G software. The MRI performed after administering STRO-1 ⁺ cells was performed using a 1.5 Tesla Siemens AVANTRO MRI scanner equipped with syngo B13 software. The localization scan was followed by sagittal imaging of the lumbar and sacral spine in T1, T1 gradient echo (T1_Flash), T2 and STIR weighted, followed by T2-weighted axial imaging of the 6 intervertebral disc segments L6/S1, L5/L6, L4/L5, L3/L4, L2/L3, L1/L2. The image sequence is provided on a CD that displays each scan as a series of 12 sagittal images. Two qualified observers who were blinded to the experimental design reviewed the digital images obtained for the 12 sagittal MRI acquisition levels using the grading criteria of Pferrmann et al (25) for the disc degeneration scoring system summarized in Table 2. The final score corresponded to the mean of the scores from the two assessors who were blinded to the experimental design.

Table 2. MRI grading of ovine disc degeneration using the Pferrmann grading system (25)

Example 5: Histopathological analysis

Each intervertebral disc unit for histological and biochemical analysis was isolated by cutting through the adjacent cranial and caudal vertebral bodies proximal to the growth plate with a bone saw. These spinal segments were fixed whole in 5% neutral buffered formalin for 56 hours and decalcified in 10% formic acid in 5% neutral buffered formalin with several changes of solution for 2 weeks under constant agitation until complete decalcification was confirmed using a Faxitron HP43855A X-ray cabinet (Hewlett Packard, McMinnville, USA).

Decalcified specimens were processed for paraffin embedding and sectioning by standard histological methods. Paraffin sections, 4 μm thick, were mounted on Superfrost Plus glass microscope slides (Menzel-Glaser), dried at 85°C for 30 minutes, and then at 55°C overnight. Sections were deparaffinized in xylene (2 minutes each, 4 changes) and rehydrated through graded ethanol washes (100-70% v/v) in tap water. One section from each block prepared from sagittal sections was stained with hematoxylin and eosin (H&E). H&E-stained tissue sections were numbered and examined by an independent histopathologist blinded to the laboratory design using a published (26) four-point semi-quantitative grading system (see Table 3) to assess the degree of degeneration. Other stains, including Alcian Blue counterstained with neutral red, were also used to identify the distribution and assembly of matrix components in the disc sections.

Table 3: Grading system for histological changes in the lower lumbar intervertebral disc (BEP is the bony endplate, CEP is the cartilage endplate)

Example 6: Biochemical Analysis of Fixed Intervertebral Disc Tissue

After removing midsagittal sections for histological analysis, the annulus fibrosus (AF) and nucleus pulposus (NP) were carefully dissected from the processed decalcified disc tissue. This process was more difficult for severely degenerated discs, in which the demarcation between the NP and AF was lost. Aliquots of finely dissected NP and AF tissue were lyophilized to constant weight, and triplicate portions (1-2 mg) of the dried tissue were hydrolyzed in 6 M HCl at 110°C for 16 hours. Aliquots of the neutralized digest were assayed for hydroxyproline as a standard for tissue collagen content as previously described (4). Triplicate portions of lyophilized tissue (~2 mg) were also digested with papain, and aliquots were assayed for sulfated glycosaminoglycans (GAGs) using the metachromatic dye 1,9-dimethylmethylene blue as previously described (27).

Example 7: Data Statistical Analysis

Statistical comparisons of DHI and biochemical data were performed for all 3-month and 6-month treatment groups using Student's unpaired t-test, with p < 0.05 considered significant. For histological and MRI total degeneration and NP scores, comparisons between different disc treatments were performed using the Kruskal-Wallis or Friedman test (nonparametric repeated measures ANOVA) with Dunn's multiple comparison post hoc test. p < 0.05 was considered statistically significant between groups.

Example 8: Results of sheep intervertebral disc regeneration studies using immunoselected STRO-1 ⁺ multipotent cells

All animals in the high-dose (4.0×10 ⁶ STRO-1 ⁺ cells) (Groups 2 and 4) and low-dose (0.5×10 ⁶ STRO-1 ⁺ cells) (Groups 1 and 3) injection groups maintained normal body weight and showed no evidence of adverse side effects during the experimental period.

As shown in Figure 2, injection of the chemical nucleus pulposus dissolving agent chondroitinase ABC into the NP of the target intervertebral disc resulted in a decrease of approximately 50% in the disc high index (DHI) over a 3-month period; this confirmed the significant loss of PG and water from the extracellular matrix of the intervertebral disc. The total MRI disc degeneration score supported the DHI pre-treatment data. As shown in Figures 3 and 4, for all groups, the control intervertebral discs that were not injected with chondroitinase ABC gave an MRI degeneration score of approximately 50% of the score of the intervertebral discs injected with chondroitinase ABC, which confirmed the effectiveness of the model.

The collection of average histopathological grading scores measured for the intervertebral discs of animals sacrificed 3 months after injection of low-dose STRO-1 ⁺ cells showed a decrease in scores, which was not significantly different from the scores of the uninjected control discs and the discs injected with cABC or cABC + HA. However, the scores of the latter two were significantly higher (p < 0.001) than the control disc scores (Figure 5A). For the low-dose STRO-1 ⁺ cell group that was sacrificed 6 months after treatment, the disc scores were significantly lower (p < 0.01) than the discs injected with cABC and cABC + HA, but were equivalent to the scores of the uninjected control discs (Figure 5B). High-dose STRO-1 ⁺ cell discs maintained this pattern at 3 months (p < 0.05) (Figure 6A), but not at 6 months after injection (Figure 6B).

Analyzed the microphotographs of tissue sections of intervertebral discs injected with cABC, cABC+HA and cABC+STRO-1 ⁺ cells of 3-month low-dose sheep and 6-month low-dose sheep and their respective histopathological scores. This analysis highlighted the significant structural and cellular changes in different intervertebral discs treated after cABC administration. As shown by the normalization of intervertebral disc structural integrity and new extracellular matrix deposition 6 months after low-dose cell administration, the beneficial effect of STRO-1 ⁺ cell mediation was confirmed. Although these tissue sections were selected based on their different histopathological scores, they were consistent with the overall average total scores obtained from the 3-month and 6-month low-dose groups summarized in Figure 5.

The disc histopathology scores complemented the disc degeneration scores assessed by MRI. In addition, in all groups, the MRI scores followed the same pattern: regardless of the dose used, the scores of the discs injected with cABC alone and cABC+HA were higher than those of the cABC+STRO-1 ⁺ cells and control discs. Nevertheless, for the MRI degeneration scores, significant differences were observed in the scores of the discs injected with low-dose STRO-1 ⁺ cells at 3 and 6 months relative to the cABC+HA scores (p<0.05) (Figures 7A and 7B). At 3 months, the MRI scores of the high-dose STRO-1 ⁺ cells injected discs were not significantly different from the control, cABC alone or cABC+HA values, but were lower than cABC alone at 6 months (Figures 8A and 8B).

Since the experimental model of disc degeneration used in these experiments was induced by direct injection of the PG depolymerase chondroitinase-ABC into the NP, the histopathological scores for this site are shown separately. In addition, the biochemical changes that occurred in the NP tissue in response to the different treatments were also shown. As demonstrated in Figures 9 and 10, the average NP histopathological scores of the discs injected with cABC+STRO-1 ⁺ cells followed the same pattern as the overall disc histopathological scores, i.e., the discs injected with cABC and cABC+HA showed higher degeneration scores. Nevertheless, due to the inter-animal sample variation within the small group size (N=6) used, the differences were not statistically significant.

The radiographically measured disc height index (DHI) is a validated indicator of disc degeneration (24). As already discussed, the DHI decreased by approximately 50% at 3 months after intradiscal injection of the PG-degrading enzyme cABC.

Biochemical determination of the glycosaminoglycan (GAG) content of the disc NP tissue (a marker of PG) in all treated discs showed a significant loss of this component relative to uninjected control discs (Figure 11). Significant differences in GAG levels were observed in cABC and cABC+HA tissue relative to control NP tissue for all groups (p<0.05), while discs injected with high-dose STRO-1 ⁺ cells at 3 months and low-dose STRO-1 ⁺ cells at 6 months showed no statistical difference from controls (Figure 11).

As shown in Figure 12, all injected discs, regardless of treatment, showed partial recovery of their DHI at 3 months post-treatment. Nevertheless, the discs injected with low-dose STRO-1 ⁺ cells showed the greatest improvement in DHI, with the improvement being confirmed to be statistically significant (p < 0.02). In addition, the DHI of the discs injected with low-dose STRO-1 ⁺ cells at 6 months was not significantly different from the mean DHI of the uninjected control discs (Figure 12A).

discuss

The results of this study indicate that intradiscal administration of low-dose STRO-1 ⁺ cells + HA to degenerated discs resulted in a greater degree of structural recovery than HA-injected discs. This conclusion was supported by data generated from three independent studies evaluating disc integrity and matrix recovery from a baseline degenerative state equivalent to 50% of the normal disc height index.

The deposition of remodeled extracellular matrix in the degenerated disc could explain the increased DHI observed at 6 months in discs injected with low doses of STRO-1 ⁺ cells. In the healthy spine, the presence of high concentrations of PG and its bound water molecules in the nucleus pulposus and inner annulus maintains disc height (DHI) (2,4). These entities confer a high turgor pressure on the disc that not only maintains disc height but also allows the disc to recover from deformation after axial compression (2,4). Indeed, the use of chondroitinase-ABC to induce disc degeneration in this animal model relies on the ability of this enzyme to selectively degrade and remove most of the PG from the extracellular matrix of the NP and inner AF (28). Histochemical studies using Alcian blue dye, which binds to negatively charged PG, showed that sections obtained from discs injected with low doses of STRO-1 ⁺ cells had stronger staining than sections from discs injected with cABC alone or cABC + HA, confirming the presence of high concentrations of PG in these tissues. Examination of these stained sections by both white light and fluorescence microscopy also confirmed the normalization of the AF collagen fibril-assembled lamellae structure and the hyaline cartilage endplate (CEP) morphology. The CEP is the major pathway for nutrient diffusion into the avascular NP, and physical disruption of its structure reduces the survival of resident cells.

The concentration of PG in the NPs, as determined biochemically for GAG content, only partially supported the histopathological findings. The GAG content of the high- and low-dose STRO-1 ⁺ cell-injected NPs was not found to be statistically different from that of the control NPs at 3 and 6 months, whereas the other treated discs were statistically different from the control NPs. However, no statistical difference was demonstrated between the GAG levels in the NPs of the cABC + STRO-1 ⁺ cell-injected discs and the cABC or cABC + HA discs, suggesting similar levels of GAGs in the tissues of these groups. Because the disc tissue used for biochemical analyses had been fixed in buffered formalin for several months prior to biochemical analysis, variable amounts of PG leached from the matrix during this time period is possible. Furthermore, the covalent cross-links of collagen and proteins formed during formalin fixation may have affected adequate macroscopic distinction and exfoliation of the NP and AF sites of the disc from each other. Exfoliation is particularly difficult in degenerative discs, where the boundary between the NP and AF has been lost. Intervertebral disc AF has a much lower GAG content than NP, and therefore sampling error can have a significant impact on analytical data (1–4).

In this study, ovine STRO-1 ⁺ multipotent cells were co-administered with a suitable carrier, such as high molecular weight hyaluronic acid (HA), to the nucleus pulposus of experimentally degenerated IVDs, which has been shown to accelerate the regeneration of the disc extracellular matrix, as assessed radiologically for restoration of disc height. This interpretation is based on the hypothesis that in loaded spinal columns, the presence of high concentrations of matrix proteoglycans in the NP and the inner annulus maintains disc height, which together with their bound water molecules impart a high turgor pressure to the structure. Indeed, at the outset of these experiments, the induction of disc degeneration using chondroitinase-ABC relied on the ability of this enzyme to degrade and remove most of the proteoglycans from the NP extracellular matrix.

The present results indicate that the recovery of the disc from the degenerative state induced by early cABC injection is more sustained with a lower dose ^of STRO-1 ⁺ cells (0.5×10 ⁶ ) than with a higher dose (4.0×10 ⁶ ). A possible explanation for this observation could be related to the poor nutritional supply to the NPs of the disc, which rely on the exchange of O ₂ /CO ₂ and metabolites between the blood vessels under the CEP (1, 2). As already mentioned, even a mild disruption of the NP-CEP interface and the subchondral blood supply of the vertebrae, as observed in degenerative discs, would be expected to affect the nutritional supply to the NPs. Insufficient nutritional status could impose an upper limit on the number of injected STRO-1 ⁺ cells that can survive in an oxygen-poor environment, leading to a loss of their viability and, therefore, a loss of therapeutic benefit.

The therapeutic mechanisms responsible for the restoration of disc integrity following administration of low doses of STRO-1 ⁺ multipotent cells in this animal model remain to be resolved. However, it is possible that anti-inflammatory cytokines and growth factors released by STRO-1 ⁺ multipotent cells within the degenerative disc space can modulate the pro-catabolic and anti-anabolic effects mediated by cytokines and other toxic factors released by resident disc cells in response to biochemical and biomechanical insults. These STRO-1 ⁺ multipotent cell-derived paracrine factors could also support the normal physiological anabolic response of resident disc cells to PG depletion from their extracellular environment, as occasionally observed in some discs not injected with STRO-1 ⁺ cells. On the other hand, it is possible that some STRO-1 ⁺ multipotent cells can migrate into the degenerated matrix and undergo differentiation into NP chondrocytes or other disc cells.

It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit and scope of the present invention as broadly described. Therefore, the present embodiments are to be considered in all aspects as illustrative and non-restrictive.

All publications discussed and/or referred to herein are incorporated herein in their entirety.

Any discussion of documents, activities, materials, devices, articles and the like included in the specification of the present invention is solely for the purpose of providing a background to the present invention and is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

References

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Claims (12)

1. Use of a population of mesenchymal precursor cells enriched to co-express STRO-1 and TNAP and ^bright for STRO-1, or a population of cells expanded from such cells by culture, in the preparation of a pharmaceutical composition for the treatment or prevention of lower back pain characterized by intervertebral disc degeneration in human individuals, wherein the mesenchymal precursor cells are capable of forming specialized cell clones. 2. The use as described in claim 1, wherein the cell population is derived from autologous, allogeneic, or xenogeneic sources. 3. The use as described in claim 2, wherein the cell population is derived from an allogeneic source. 4. The use as described in claim 1, wherein the cell population is genetically modified to express and/or secrete proteins. 5. The use as described in claim 4, wherein the protein is selected from insulin, glucagon, somatostatin, trypsinogen, chymotrypsinogen, elastase, carboxypeptidase, pancreatic lipase, amylase, polypeptides associated with or causing enhanced angiogenesis, and polypeptides associated with cell differentiation into pancreatic cells or vascular cells. 6. The use as claimed in claim 1, wherein the pharmaceutical composition further comprises glycosaminoglycans. 7. The use as described in claim 6, wherein the glycosaminoglycan is selected from hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate, and galactosamine glucuronide sulfate. 8. The use as described in claim 1, wherein the pharmaceutical composition further comprises an antioxidant. 9. The use as claimed in claim 8, wherein the antioxidant is selected from tocopherol, superoxide dismutase, ascorbic acid, and catalase. 10. The use as claimed in claim 1, wherein the pharmaceutical composition further comprises an amphoteric derivative of sodium alginate. 11. The use as claimed in claim 1, wherein the pharmaceutical composition further comprises recombinant osteoprotein-1. 12. The use as claimed in claim 1, wherein the pharmaceutical composition further comprises fibrin glue.