Intervertebral disc repair and / or reconstruction

Abstract

To provide a method for repair and reconstitution of invertebral discs.SOLUTION: The problem of the invention is solved by using STRO-1multipotent cells. Use of the STRO-1multipotent cells makes it possible to treat spinal conditions characterized by degeneration of the invertebral disc.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to methods for the repair and reconstruction of an invertebral disc in a subject. The methods of the present invention are useful in the treatment of spinal conditions characterized by degeneration of the intervertebral disc. [Background technology]

[0002] The intervertebral disc (IVD) is the largest primarily avascular, aneural, and aneural structure in the human body. The disc is crucial for normal function of the spine, as it provides flexibility and mechanical stability during axial compression, flexion, and extension. The IVD is composed of several specialized connective tissues: (i) the hyaline cartilage of the cartilaginous endplates (CEPs) that cover the surfaces of the vertebrae (bodies) located at the top and bottom of the disc, (ii) the fibrocartilaginous annulus fibrosus (AF) that encapsulates the nucleus pulposus (NP), and (iii) the central gelatinous nucleus pulposus (NP), which contains cartilage-like cells but is not hyaline cartilage. As the name suggests, a transition zone (TZ) located between the AF and NP has also been identified. The fibrocartilaginous AF is composed of concentric collagen layers (lamellae) that connect to the bony periphery of the vertebral body.

[0003] Proteoglycans (PGs) and collagen types I, II, III, V, VI, IX, X, and XI are the major matrix components of all these disc tissues, but their relative amounts and distribution depend on their anatomical location. PGs, which have a high affinity for water molecules, are most abundant in the NP of "healthy discs." Water absorbed by the PGs generates hydrostatic pressure inside the NP that "swells" the encapsulating fibrocartilage AF. It is the combination of these specialized connective tissues and their individual physiochemical properties that contribute to the hydraulic and viscoelastic properties of the IVD that are essential for the normal biomechanical function of the spine.

[0004] IVDs undergo profound matrix changes during aging and degeneration. Studies of human cadaveric specimens and intervertebral disc specimens obtained during spinal surgery have revealed that discs from individuals in the middle-aged to elderly age groups generally harbor extensive pathology (1, 2).

[0005] From these specimens, three major types of disc pathology have been identified: (i) peripheral lesions, i.e., transverse defects close to the attachment of the AF to the bone at the perivertebral body, (ii) concentric (circumferential) tears where the annular lamellae separate from one another, and (iii) radial tears resulting from the growth of a cleft that begins within the NP (1, 2, 3). Peripheral lesions are of particular interest because they appear more commonly within the anterior region of the AF close to its insertion into the bone at the perivertebral body during adolescence and early adulthood, suggesting that peripheral lesions may be mechanically mediated. The presence of peripheral lesions suggests early failure of the AF and is the primary cause of disc degeneration, although studies in cadaveric specimens have also shown that other pathological features (concentric tears, cystic annular degeneration, dehydration of the NP, osteophytes of the perivertebral ligaments, and osteoarthritis of the posterior facet joints) are always present to some degree (1, 2). Although the time course of each of these disc pathologies remains a subject of debate, it is generally agreed that the loss of PG and its associated water from the NP is an early etiological determinant of disc degeneration ( 4 ).

[0006] As discussed previously, the intervertebral disc functions as a flexible, hydroelastic cushion, mediated primarily by the absorption of water molecules into the NP. A decrease in the water content, and therefore the swelling pressure of the NP, would result in supraphysiological mechanical stresses on the AF, leading to focal failure.

[0007] Medical problems associated with back and neck pain resulting from disc degeneration are experienced by 90% of the population at some time in their lives (5, 6). In men, back or neck pain severe enough to justify medical intervention increases in incidence during the third and fourth decades of life, peaking in the fifth decade and then declining thereafter (5).

[0008] In the United States, back pain is the second most common reason for doctor's visits, and medical conditions related to back and neck pain account for more hospitalizations than any other musculoskeletal disorder. Back pain is a leading cause of lost work time; in the UK, for example, it is estimated that more than 11 million work days are lost each year from this condition. Furthermore, as the life expectancy of the population increases over the coming decades, back and neck pain problems are expected to increase accordingly.

[0009] Despite the high incidence and economic burden of back and neck pain in modern society, its causes remain poorly understood. However, there is general agreement that degeneration and / or failure of the IVD is the primary cause of pain, whether directly from nerves present in the lateral AF or from adjacent spinal structures that are mechanically compromised by loss of disc fluid-elastic function (7, 8, 9, 10). Disc disease is responsible for 23–40% of all cases of low back pain (11, 12). The lateral AF is irritated and nerve fibers can extend up to one-third of the depth within the AF, therefore any pathological changes to the lateral AF can cause pain (13, 14, 15).

[0010] Existing paradigms for treating back or neck pain of discogenic origin are empirical and tend towards lifestyle changes or the use of anti-inflammatory / analgesic drugs to minimize symptomatology, or towards surgical interventions that may require the removal of degenerative tissue or spinal arthrodesis to restrict movement. Although spinal fusion has been used extensively to relieve neck or lower back pain, it is known to be a non-benign procedure because the introduction of a rigid segment into the disc space places mechanical stress on the adjacent discs, accelerating degenerative changes in the adjacent discs that may become symptomatic at a later stage (16). Clearly, alternative treatments are needed.

[0011] Intradiscal administration of the protein osteogenic protein-1 (OP-1) (bone morphogenetic protein-7) has been reported to stimulate disc matrix repair following experimentally induced degeneration. Disc degeneration was induced in rabbits by preinjection of the depolymerase chondroitinase ABC into disc NPs, a procedure known as chemonucleolysis (17). Both NP and AF cells were found to be much more efficient at reconstituting a functional matrix after chemonucleolysis. Disc cells embedded in normal density extracellular matrix were found to be largely unresponsive to the stimulatory effects of OP-1 on PG synthesis (17).

[0012] Studies have been reported investigating a potential cell therapy to achieve repair of degenerated canine and human IVDs using autologous chondrocytes (18, 19). The cells used were chondrocytes harvested from allogeneic healthy NPs and subsequently reimplanted into the defective disc. A drawback of this approach is that the cells used for this purpose would need to be harvested from adjacent healthy discs or from other donors of the same species. Obtaining such cells would require breaching the AF, a process that would not only damage the AF structure, but would also accelerate the degenerative changes of this tissue if viable cells were removed from the NP. Clearly, this approach has limited its applicability in humans. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] International Publication No. 2006 / 032092 [Patent Document 2] WO 01 / 04268 [Patent Document 3] International Publication No. 2004 / 085630 [Patent Document 4] U.S. Patent No. 4,983,393 [Patent Document 5] U.S. Patent No. 3,089,815

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Summary of the Invention

Problems to be Solved by the Invention

[0015] The present application describes a method for the preparation of STRO-1 for promoting the remodeling of the nucleus pulposus and annulus fibrosus of a degenerative intervertebral disc. + This is the first description of the in vivo use of pluripotent cells. + The multipotent cells were derived from an allogeneic source and were well tolerated in the animal model used in this study. This allowed the donor-derived STRO-1 + It is suggested that the multipotent cells can be expanded in large numbers and developed as a "non-autologous" product for the treatment of degenerative discs. [Means for solving the problem]

[0016] Accordingly, the present invention provides a method for reconstructing and / or repairing an intervertebral disc in a subject, comprising administering to a subject mesenchymal progenitor cells (STRO-1 + The method includes administering to the intervertebral disc a multipotent cell (multipotent cell) and / or its progeny.

[0017] In an embodiment of the present invention, STRO-1 + The multipotent cells and / or their progeny are administered into the nucleus pulposus of the intervertebral disc.

[0018] Preferably, STRO-1 + Multipotent cells are TNAP + , VCAM-1 + , THY-1 + , STRO-2 + , CD45 + , CD146 + , 3G5 + Or any combination thereof.

[0019] STRO-1 + The pluripotent cells and / or their progeny may be derived from an autonomous, allogeneic or xenogeneic source. In one embodiment, the cells are derived from an allogeneic source.

[0020] The method of the invention may also include administering to the intervertebral disc glycosaminoglycans (GAGs), such as, for example, hyaluronic acid (hyaluronan) (HA), chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, and heparin sulfate. GAGs are + They may be administered in the same composition as the pluripotent cells and / or their progeny or in a different composition.

[0021] It will be appreciated that the methods of the invention may be practiced on any vertebrate. For example, the subject may be a mammal, such as a human, dog, cat, horse, cow, or sheep.

[0022] The methods of the invention may be used to treat or prevent back pain, age-related changes in the intervertebral disc or spinal conditions characterized by degeneration of the intervertebral disc, such as spondylolysis.

[0023] Throughout this specification the term "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.

[0024] The invention will now be described hereinafter by way of the following non-limiting Examples and with reference to the accompanying drawings. [Brief description of the drawings]

[0025] [Figure 1] FIG. 2 is a schematic representation of lumbar levels treated with STRO-1 cells in all sheep groups. [Figure 2A] Figure 1. Radiologically Determined Disc Height Index (DHI). Radiographically determined mean ± SEM Disc Height Index (DHI) at baseline and 3 months after chondroitinase ABC induced degeneration (3 months prior to STRO-1 cells) for the group of sheep receiving low dose STRO-1 cells. [Figure 2B] Figure 1: Radiologically determined disc height index (DHI). Radiographically determined mean ± SEM disc height index (DHI) at baseline and 3 months after chondroitinase ABC induced degeneration (3 months prior to STRO-1 cells) for the group of sheep receiving high dose STRO-1 cells. [Figure 3A] Figure 1 shows MRI-determined aggregate disc degeneration scores for chondroitinase ABC-injected discs. Mean ± SEM of MRI-determined aggregate disc degeneration scores following injection of chondroitinase ABC prior to treatment with low dose STRO-1 cells + HA or HA alone for 3 months. [Figure 3B] Figure 1 shows MRI-determined aggregate disc degeneration scores for chondroitinase ABC-injected discs. Mean ± SEM of MRI-determined aggregate disc degeneration scores following injection of chondroitinase ABC prior to treatment with low dose STRO-1 cells + HA or HA alone for 6 months. [Figure 4A] Figure 1 shows MRI-determined aggregate disc degeneration scores for chondroitinase ABC-injected discs. Mean ± SEM of MRI-determined aggregate disc degeneration scores following injection of chondroitinase ABC prior to treatment with high dose STRO-1 cells + HA or HA alone for 3 months. [Figure 4B] Figure 1 shows MRI-determined aggregate disc degeneration scores for chondroitinase ABC-injected discs. Mean ± SEM of MRI-determined aggregate disc degeneration scores following injection of chondroitinase ABC prior to treatment with high dose STRO-1 cells + HA or HA alone for 6 months. [Figure 5A] Figure 13: Aggregate histopathology disc degeneration score. Mean aggregate histopathology disc degeneration score at 3 months for low dose STRO-1 cells. # = significantly different from control p<0.001. Ω = p<0.01 from STRO-1+ cells. [Figure 5B] Figure 13: Aggregate histopathology disc degeneration score. Mean aggregate histopathology disc degeneration score at 6 months for low dose STRO-1 cells. # = significantly different from control p<0.001. Ω = p<0.01 from STRO-1+ cells. [Figure 6A]Figure 13: Aggregate histopathology disc degeneration score. Mean aggregate histopathology disc degeneration score at 3 months for high dose STRO-1 cells. # = significantly different from control p<0.001. Ω = p<0.01 from STRO-1+ cells. [Figure 6B] Figure 13: Aggregate histopathology disc degeneration score. Mean aggregate histopathology disc degeneration score at 6 months for high dose STRO-1 cells. # = significantly different from control p<0.001. Ω = p<0.01 from STRO-1+ cells. [Figure 7A] Figure 13: Aggregate MRI disc degeneration score. Aggregate MRI disc degeneration score for low dose STRO-1 cells at 3 months. # = significantly different from control p<0.05. Ω = p<0.05 from STRO-1+ cells. [Figure 7B] Figure 13: Aggregate MRI disc degeneration score. Aggregate MRI disc degeneration score for low dose STRO-1 cells at 6 months. # = significantly different from control p<0.05. Ω = p<0.05 from STRO-1+ cells. [Figure 8A] Figure 13: Aggregate MRI disc degeneration score. Aggregate MRI disc degeneration score for high dose STRO-1 cells at 3 months. # = significantly different from control p<0.05. Ω = p<0.05 from STRO-1+ cells. [Figure 8B] Figure 13: Aggregate MRI disc degeneration scores. Aggregate MRI disc degeneration scores for high dose STRO-1 cells at 6 months. # = significantly different from control p<0.05. Ω = p<0.05 from STRO-1+ cells. [Figure 9A] Figure 1 shows nucleus pulposus (NP) histopathology degeneration scores. Mean NP histopathology degeneration scores for low dose STRO-1 cells at 3 months. [Figure 9B] Figure 1 shows nucleus pulposus (NP) histopathology degeneration scores. Mean NP histopathology degeneration scores for low dose STRO-1 cells at 6 months. [Figure 10A] Figure 1 shows nucleus pulposus (NP) histopathology degeneration scores. Mean NP histopathology degeneration scores for high dose STRO-1 cells at 3 months. [Figure 10B]Figure 1 shows nucleus pulposus (NP) histopathological degeneration scores. Mean NP histopathological degeneration scores for high dose STRO-1 cells at 6 months. [Figure 11A] Figure 1 shows biochemically determined glycosaminoglycan (GAG) content for nucleus pulposus. Mean ± SD biochemically determined glycosaminoglycan (GAG) content for nucleus pulposus of intervertebral discs injected with low or high dose STRO-1 cells for 3 months. # = significantly different from control (p<0.05). [Figure 11B] Figure 1 shows biochemically determined glycosaminoglycan (GAG) content for nucleus pulposus. Mean ± SD biochemically determined glycosaminoglycan (GAG) content for nucleus pulposus of intervertebral discs injected with low or high dose STRO-1 cells for 6 months. # = significantly different from control (p<0.05). [Figure 12A] Figure 1: Radiologically determined disc height index (DHI). X-ray determined mean ± SEM disc height index (DHI) for chondroitinase ABC induced degenerative discs 3 and 6 months after injection with HA or HA + low dose STRO-1 cells. [Figure 12B] Figure 1: Radiologically determined disc height index (DHI). X-ray determined mean ± SEM disc height index (DHI) for chondroitinase ABC induced degenerative discs 3 and 6 months after injection with HA or HA + high dose STRO-1 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] General Techniques and Selected Definitions Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, stem cell biology, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0027] 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. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE This is described and explained throughout the literature in sources such as Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0028] As used herein, the terms "treating," "treat," or "treatment" refer to the administration of a therapeutically effective amount of STRO-1 sufficient to reduce or eliminate at least one symptom of a particular condition.+ This includes administering the pluripotent cells and / or their progeny.

[0029] As used herein, the terms "preventing," "prevent" or "prevention" refer to the administration of a therapeutically effective amount of STRO-1 sufficient to stop or hinder the onset of at least one symptom of a particular condition. + This includes administering the pluripotent cells and / or their progeny.

[0030] STRO-1 + Pluripotent cells or progeny As used herein, the phrase "STRO-1 + Multipotent cells are STRO-1 cells that can form multipotent cell colonies. + and / or TNAP + It shall be taken to mean progenitor cells.

[0031] STRO-1 + Multipotent cells are cells found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, hair follicles, intestine, lung, lymph nodes, thymus, bone, ligaments, tendons, skeletal muscles, dermis, and periosteum, and can differentiate into germ lineages such as mesoderm and / or endoderm and / or ectoderm. Thus, STRO-1 + Multipotent cells can differentiate into numerous cell types, including, but not limited to, adipose, bone, cartilage, elastic, muscle, and fibrous connective tissue. The particular lineage commitment and differentiation pathway that these cells enter depends on 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 give rise to daughter cells that are either stem cells or progenitor cells that subsequently irreversibly differentiate to give rise to the phenotypic cell.

[0032] In another embodiment, STRO-1+ The multipotent cells are enriched from a sample obtained from a subject, e.g., a subject undergoing treatment or a related or unrelated subject (whether allogeneic or xenogeneic). The terms "enriched", "enrichment" or variations thereof are used herein to refer to a cell population in which the proportion of one particular cell type or the proportion of several particular cell types is increased compared to an untreated population.

[0033] In another embodiment, the cells used in the present invention are + , VCAM-l + , THY-1 + , STRO-2 + , CD45 + , CD146 + , 3G5 + or any combination thereof.

[0034] By "individually" it is meant that the invention encompasses the recited markers or groups of markers separately, and that even though individual markers or groups of markers are not listed separately herein, the appended claims may define such markers or groups of markers separately and separately from one another.

[0035] By "in a population" it is meant that the invention encompasses any number or combination of the listed markers or groups of peptides, and that even though such number or combination of markers or groups of markers is not specifically listed herein, the appended claims may define such combinations or subcombinations separately and separately from any other combination of markers or groups of markers.

[0036] Preferably, STRO-1 + The cells are STRO-1 bright (Synonym STRO-1 bri ). Preferably, STRO-1 bright The cells were further+ , VCAM-1 + , THY-1 + , STRO-2 + and / or CD 146 + One or more of the following:

[0037] In one embodiment, STRO-1 + The multipotent cells are perivascular mesenchymal precursor cells as defined in WO 2004 / 85630.

[0038] A cell that is referred to as "positive" for a given marker may express low (lo or dim) or high (bright, bri) levels of that marker depending on the extent to which the marker is present on the cell surface, the term referring to the intensity of the fluorescence or other marker used in the cell sorting process. The distinction between lo (or dim or dull) and bri will be understood in relation to the marker used on the particular cell population being sorted. A cell that is referred to as "negative" for a given marker is not necessarily absent from the cell at all. This term means that the marker is expressed at a relatively very low level by the cell and, if detectably labeled, gives rise to a very low signal or cannot be detected above background levels.

[0039] The term "bright" as used herein refers to a marker on the cell surface that produces a relatively high signal when detectably labeled. Without wishing to be limited by theory, it is proposed that "bright" cells express more of the target marker protein (e.g., the antigen recognized by STRO-1) than other cells in the sample. For example, STRO-1 bri Cells are classified as non-clear cells (STRO-1) when labeled with FITC-conjugated STRO-1 antibody as determined by fluorescence-activated cell sorting (FACS) analysis. dull / dim) produces a fluorescent signal greater than 0.1% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. Preferably, the "bright" cells comprise 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 most brightly labeled bone marrow mononuclear cells contained in the starting sample. In a preferred embodiment ... bright Cells were cultured in the "background" medium, i.e. STRO-1 - have a surface expression of STRO-1 that is 2 log higher than that of cells that are STRO-1. dim and / or STRO-1 intermediate The cells have STRO-1 surface expression that is less than 2 logs above "background," and typically no more than about 1 log amplitude.

[0040] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, the term encompasses liver isoform (LAP), bone isoform (BAP) and kidney isoform (KAP). In a preferred embodiment, TNAP is BAP. In a particularly preferred embodiment, TNAP as used herein refers to a molecule that can bind to STRO-3 antibody produced by the hybridoma cell line deposited with ATCC under the provisions of the Budapest Treaty under deposit number PTA-7282 on December 19, 2005.

[0041] In one embodiment, STRO-1 + The pluripotent cells are capable of giving rise to clonogenic CFU-F.

[0042] STRO-1 +It is preferred that a significant proportion of the multipotent cells can differentiate into at least two different germ lineages. Non-limiting examples of lineages to which the multipotent cells may be committed include bone precursor cells, bile duct epithelial cells, and hepatocyte precursors that are multipotent to hepatocytes, neural-committed cells that can produce glial precursors that progress to oligodendrocytes and astrocytes, neuronal precursors that progress to neural cells, cardiac muscle and cardiac muscle cell precursors, and glucose-responsive insulin-secreting pancreatic beta cell lineages. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as the following progenitor cells: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular precursors, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, medulla stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, vascular, epithelial, glial, neuronal, astrocyte, and oligodendrocyte cells.

[0043] In another embodiment, STRO-1 + Multipotent cells are unable to give rise to hematopoietic cells when cultured.

[0044] In one embodiment, cells are taken from the subject to be treated, cultured in vitro using standard techniques, and used to obtain supernatants or soluble factors or expanded cells for administration to the subject as an autonomous or allogeneic composition. In an alternative embodiment, cells of one or more established human cell lines are used. In another useful embodiment of the invention, cells of a non-human animal (or from another species if the patient is not human) are used.

[0045] The present invention relates to STRO-1 produced from in vitro culture. +The use of supernatants or soluble factors obtained or derived from pluripotent cells and / or their progeny cells (the latter also referred to as expanded cells) is also contemplated. The expanded cells of the present invention may have a wide range of phenotypes depending on the culture conditions (including the number and / or type of stimulatory factors in the culture medium), the number of passages, etc. In certain embodiments, the progeny cells are obtained after about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passages from the parent population. However, the progeny cells may be obtained after any number of passages from the parent population.

[0046] The progeny cells may be obtained by culturing in any suitable culture medium. The term "medium" as used in relation to cell culture includes the components of the environment surrounding the cells. A medium may be a solid, liquid, gas, or a mixture of phases and substances. Media include liquid growth media as well as liquid media that do not support cell growth. Media also include gelatinous media such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous media include the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed. The term "medium" also refers to a substance intended for use in cell culture, even if it is not yet in contact with the cells. In other words, a nutrient-rich liquid prepared for bacterial culture is a medium. A powder mixture that, when mixed with water or other liquid, becomes suitable for cell culture may be called a "powdered medium".

[0047] In one embodiment, progeny cells useful in the methods of the invention are isolated from bone marrow using magnetic beads labeled with STRO-3 antibody, and then purified using TNAP + STRO-1 + They are obtained by isolating pluripotent cells and then culturing and expanding the isolated cells (for examples of suitable culture conditions, see Gronthos et al. Blood 85: 929-940, 1995).

[0048] In one embodiment, such expanded cells (progeny) (preferably after at least 5 passages) are - , CC9 + , HLA class I+ , HLA class II - , CD14 - , CD19 - , CD3 - , CD11a - c - , CD31 - , CD86 - , CD34 - and / or CD80 - However, under culture conditions different from those described herein, the expression of different markers may vary. Similarly, although cells of these phenotypes may predominate in the proliferating cell population, this does not mean that there is a small percentage of cells that do not have this phenotype (e.g., a small percentage of proliferating cells are CC9 - In a preferred embodiment, the expanded cells still have the capacity to differentiate into different cell types.

[0049] In one embodiment, the expanded cell population used to obtain the supernatant or soluble factors, or the cells themselves, is one in which at least 25% of the cells are CC9, more preferably at least 50%. + The present invention includes a cell which is

[0050] In another embodiment, the expanded cell population used to obtain the supernatant or soluble factors, or the cells themselves, is one in which at least 40% of the cells are STRO-1, more preferably at least 45%. + The present invention includes a cell which is

[0051] In additional embodiments, the expanded cells are selected from the group consisting of 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 β6-19, thrombomodulin, CD 10, CD 13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R, leptin-R (STRO-2=leptin-R), RANKL, STRO-1, and the like. brightand CD146, or any combination of these markers.

[0052] In one embodiment, the progeny cells are multipotent expanded STRO-1 cells as defined and / or described in WO 2006 / 032092. + Multipotent cell progeny (MEMP). STRO-1 progeny may be derived + Methods for preparing enriched populations of multipotent cells are described in WO 01 / 04268 and WO 2004 / 085630. + Multipotent cells rarely exist as completely pure preparations and will generally be present together with other cells that are tissue specific committed cells (TSCCs). WO 01 / 04268 refers to recovering such cells from bone marrow at purity levels ranging from about 0.1% to 90%. + The population containing the multipotent cells may be directly harvested from a tissue source, or alternatively, may be a population that has already been expanded ex vivo.

[0053] For example, the progeny may be substantially pure STRO-1 cells that contain 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 the cells reside. + The levels may be obtained from a harvested, non-expanded population of pluripotent cells. bright , 3G5 + This may be achieved by selecting cells which are positive for at least one marker selected individually or collectively from the group consisting of: VCAM-1, THY-1, CD146 and STRO-2.

[0054] MEMP is the marker STRO-1 bri The newly recovered STRO-1 was positive for the marker alkaline phosphatase (ALP) and negative for the marker alkaline phosphatase (ALP). +In contrast, the newly isolated STRO-1 + Multipotent cells are STRO-1 bri In a preferred embodiment of the invention, at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the administered cells are phenotype STRO-1. bri , A.L.P. - In additional preferred embodiments, the MEMPs are positive for one or more of the markers Ki67, CD44 and / or CD49c / CD29, VLA-3, α3β1. In yet additional preferred embodiments, the MEMPs do not exhibit TERT activity and / or are negative for the marker CD18.

[0055] STRO-1 + The starting population of multipotent cells may be derived from any one or more of the tissue types described in WO 01 / 04268 or WO 2004 / 085630, i.e. bone marrow, dental pulp cells, adipose tissue and skin, or, possibly 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.

[0056] In practicing the present invention, it will be understood that separation of cells bearing any cell surface marker can be accomplished by a number of different methods, but preferred methods utilize binding agents (e.g., antibodies or antigen-binding fragments thereof) to the marker of interest, followed by separation of cells that exhibit high levels of binding, low levels of binding, or no binding. The most convenient binding agents are antibodies or antibody-based molecules, preferably monoclonal antibodies or based on monoclonal antibodies, due to the specificity of these latter agents. Antibodies can be used for both steps, but other agents may also be used, and thus ligands for these markers may also be used to enrich for cells bearing or lacking the marker.

[0057] Antibodies or ligands may be attached to solid supports to allow crude separation. Separation techniques preferably maximize viability retention of the fractions collected. A variety of techniques with differing effectiveness may be used to obtain relatively crude separations. The particular technique used will depend on the separation efficiency, associated cytotoxicity, ease and speed of implementation, and the need for sophisticated equipment and / or technical skill. Separation techniques may include, but are not limited to, magnetic separation using antibody-coated magnetic beads, affinity chromatography, and "panning" using antibodies attached to a solid matrix. Techniques that provide accurate separation include, but are not limited to, FACS. Methods for performing FACS will be apparent to those skilled in the art.

[0058] Antibodies against each of the markers described herein are commercially available (e.g., monoclonal antibodies against STRO-1 are commercially available from R&D Systems, USA), available from the ATCC or other depositories, and / or can be produced using art-recognized techniques.

[0059] STRO-1 + The method for isolating pluripotent cells preferably comprises, for example, a first step, which is a solid-phase sorting step, for example using magnetic activated cell sorting (MACS) recognizing high levels of expression of STRO-1. This can then be followed, if necessary, by a second sorting step resulting in higher levels of progenitor cell expression as described in patent specification WO 01 / 14268. This second sorting step may use two or more markers.

[0060] STRO-1 +The method of obtaining multipotent cells may include harvesting the source of cells prior to the first enrichment step using known techniques. Thus, tissue will be surgically removed. The cells comprising the source tissue will then be separated into a so-called single cell suspension. This separation may be achieved by physical and / or enzymatic means.

[0061] Suitable for STRO-1 + Once a multipotent cell population is obtained, it may be cultured or expanded by any means suitable to obtain MEMPs.

[0062] In one embodiment, cells are taken from the subject to be treated, cultured in vitro using standard techniques, and used to obtain supernatant or soluble factors or expanded cells for administration to the subject as an autonomous or allogeneic composition. In an alternative embodiment, cells of one or more established human cell lines are used to obtain the supernatant or soluble factors. In another useful embodiment of the invention, cells of a non-human animal (or from another species if the patient is not human) are used to obtain the supernatant or soluble factors.

[0063] The invention can be practiced using cells from any non-human animal species, including, but not limited to, non-human primate cells, ungulates, dogs, cats, rabbits, rodents, birds, and fish cells. Primate cells used to practice the invention include, but are not limited to, chimpanzees, baboons, cynomolgus monkeys, and any other new or old world monkey cells. Ungulate cells used to practice the invention include, but are not limited to, cow, pig, sheep, goat, horse, buffalo, and bison cells. Rodent cells used to practice the invention include, but are not limited to, mouse, rat, guinea pig, hamster, and gerbil cells. Examples of rabbit species used to practice the invention include domesticated rabbits, jack rabbits, hares, cottontail rabbits, snow hares, and pikas. Chickens (Gallus gallus) are an example of an avian species used to practice the invention.

[0064] The cells useful in the method of the present invention may be stored before use or before obtaining the supernatant or soluble factors. Methods and protocols for preserving and storing eukaryotic cells, and particularly mammalian cells, are known in the art (see, for example, Pollard, JW and Walker, JM (1997) Basic Cell Culture Protocols, Second Edition, Humana Press, Totowa, NJ; Freshney, RI (2000) Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, NJ). Any method that maintains the biological activity of isolated stem cells, such as mesenchymal stem / progenitor cells, or their progeny, may be utilized in the context of the present invention. In a preferred embodiment, the cells are maintained and stored by using cryopreservation.

[0065] Administration and Compositions STRO-1 administered +The dosage of the pluripotent cells or their progeny may vary according to factors such as the individual's medical condition, age, sex and weight. The dosage regimen may be adjusted to provide an optimal therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dosage may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, "dosage unit form" refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0066] In one example, the administered STRO-1 + The dose of pluripotent cells ranged from 0.1 to 4.0 × 10 6 The dose ranges from 0.5×10 6 It may also be a cell.

[0067] STRO-1 + It should be appreciated that the multipotent cells and / or their progeny may be processed into a variety of forms (e.g., suspension, solid, porous, woven, non-interwoven, particulate, gel, paste, etc.) before being added to the intervertebral disc space.

[0068] Injection of STRO-1 into the nucleus pulposus to restore normal mechanical and / or physiological properties to damaged intervertebral discs. +Numerous biological and synthetic materials are contemplated for co-injection with the multipotent cells and / or their progeny. For example, one or more natural or synthetic glycosaminoglycans (GAGs) or mucopolysaccharides, such as hyaluronic acid (HA), chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate, galactosaminoglycuronglycan sulfate (GGGS) (see previous variations and others), including physiological salts thereof, may be injected directly into the nucleus pulposus. HA has been implicated in stimulating endogenous HA synthesis by synovial cells and proteoglycan synthesis by chondrocytes, and has previously been suggested to inhibit the release of chondrodegradative enzymes and act as a scavenger of oxygen free radicals known to be involved in cartilage deterioration. Similarly, chondroitin sulfate and glucosamine injections have been shown to inhibit the progression of articular cartilage degeneration. Arguably, other GAGs may offer similar protective or restorative properties with therapeutic value making them ideal candidates for injection into discs suffering from degenerative disc disease. Another valuable property of GAGs is their strong ability to attract and hold water. Therefore, it may be appropriate to mix GAGs with water or other aqueous substances to form a viscous gel that may then be injected into the space created from the aspiration of the nucleus pulposus, or alternatively, added to the existing nucleus pulposus as a supplement. This can form a natural "hydrogel" that can fill the three-dimensional space and act like a filler that resists collapse and allows the disc to adequately absorb the shock associated with movement.

[0069] For example, synthetic hyaluronic acid gels such as Euflexxa® (Ferring Pharmaceuticals), or Restylane® (Q-Med Aktiebolag Co., Sweden) are suitable for use in the present invention.

[0070] Examples of other injectable synthetic materials that may be used for co-administration include medical silicone, Bioplastique® (solid silicone particles suspended in a polyvinylpyrrolidone carrier; Uroplasty BV, The Netherlands), Arteplast® (microparticles of polymethylmethacrylate (PMMA) suspended in a gelatin carrier; Artcs Medical, USA), Artecoll® (smooth PMMA spheres suspended in a bovine cartilage carrier; Artepharma Pharmazeu Tische, GMBH Co., Germany). Additionally, synthetic hydrogel compositions may be used as fillers to restore normal shape to the intervertebral disc, thereby restoring normal biomechanical function.

[0071] Antioxidants with known chondroprotective capabilities are also candidates for injection into the nucleus pulposus. Examples of these include tocopherol (vitamin E), superoxide dismutase (SOD), ascorbic acid (vitamin C), catalase, and the like. Additionally, amphiphilic derivatives of sodium alginate and the like are also contemplated herein for injection. Additionally, recombinant osteogenic protein-1 (OP-1) is an excellent candidate for injection due to its ability to promote the formation of a proteoglycan-rich matrix by nucleus pulposus and annulus fibrosus cells.

[0072] The use of synthetic injectables is also contemplated. These are particularly applicable in situations where the primary objective is to restore biomechanical function to the disc. Hyaluronic acid alone or in combination with other glycosaminoglycans may be used as a carrier to deliver bioactive substances. In a preferred embodiment, hyaluronic acid and / or other GAGs are selected from the group consisting of STRO-1, STRO-2, STRO-3, STRO-4, STRO-5, STRO-6, STRO-7, STRO-8, STRO-9, STRO-10, STRO-11, STRO-12, STRO-13, STRO-14, STRO-15, STRO-16, STRO-17, STRO-18, STRO-19 ... + The concentration and viscosity of the hyaluronic acid / GAG composition are routinely adjusted to suit a given purpose.

[0073] In another example, STRO-1 +Multipotent cells or their progeny may be delivered in combination with fibrin glue. As used herein, the term "fibrin glue" refers to an insoluble matrix formed by cross-linking of fibrin polymers in the presence of calcium ions. Fibrin glue may be formed from fibrinogen, or its derivatives or metabolites, fibrin (soluble monomers or polymers) and / or complexes derived from biological tissues or fluids that form a fibrin matrix. Alternatively, fibrin glue may be formed from fibrinogen, or its derivatives or metabolites, or fibrin produced by recombinant DNA technology.

[0074] Fibrin glue may be formed by the interaction of fibrinogen with a catalyst for fibrin glue formation (e.g., thrombin and / or factor XIII). As one skilled in the art will recognize, in the presence of a catalyst (e.g., thrombin), fibrinogen is proteolytically cleaved and converted to fibrin monomers. The fibrin monomers may then form polymers that may cross-link to form a fibrin glue matrix. Cross-linking of fibrin polymers may be enhanced by the presence of a catalyst such as factor XIII. The catalyst for fibrin glue formation may be derived from plasma, cryoprecipitate, or other plasma fractions containing fibrinogen or thrombin. Alternatively, the catalyst may be produced by recombinant DNA technology.

[0075] The rate at which a clot forms depends on the concentration of thrombin mixed with fibrinogen. Since it is an enzyme-dependent reaction, the higher the temperature (up to 37°C), the faster the clot forms. The tensile strength of the clot depends on the concentration of fibrinogen used.

[0076] When fibrin clots are created in the presence of hyaluronic acid, they undergo interaction with a cross-linked matrix and become interlocked. This matrix is ​​known to play a major role in tissue regeneration and plays a cytoregulatory function 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. J Theor Biol. 119: 219-34]. The rate of degradation of hyaluronic acid is also extended in the HA-fibrin matrix, which may be beneficial in prolonging the therapeutic effect of this GAG (Wadstrom J and Tengblad A (1993) Fibrin glue reduces the dissolution rate of sodium hyaluronate. Upsala J Med Sci. 98: 159-167).

[0077] Several publications have described the use of fibrin glue for the delivery of therapeutic agents. For example, US Patent No. 4,983,393 discloses a composition for use as a vaginal insert, comprising agarose, agar, glycosaminoglycan saline, collagen, fibrin and enzymes. Furthermore, US Patent No. 3,089,815 discloses an injectable pharmaceutical preparation composed of fibrinogen and thrombin, and US Patent No. 6,468,527 discloses a fibrin glue that facilitates the delivery of various biological and non-biological agents to specific sites within the body. However, the use of fibrin+hyaluronan+ to promote chondrogenic differentiation of STRO-1+ allogeneic cells has not been described so far.

[0078] STRO-1 +The composition comprising the pluripotent cells and / or their progeny is "surgically added" to the disc space. That is, the material is added by the intervention of medical personnel, as distinct from being "added" by the body's natural growth or regeneration processes. The surgical procedure preferably involves injection with a hypodermic needle, although other surgical methods of introducing the collagen-based material into the disc may be used. For example, the material may be introduced into the disc by release through an expanded annular opening, injection with a catheter, insertion through an opening created by trauma or surgical incision, or by other means of invasive or minimally invasive deposition of the material into the disc space.

[0079] In some embodiments of the invention, it may not be necessary or desirable to immunosuppress patients prior to initiation of treatment with the cell composition. Indeed, the results presented herein show that allogeneic STRO-1 + Transplantation of pluripotent cells into sheep has been shown to be well tolerated, even in the absence of immunosuppression.

[0080] However, in other instances, it may be desirable or appropriate to pharmacologically immunosuppress the patient prior to initiating cell therapy. This may be accomplished by the use of systemic or local immunosuppressive drugs, or by delivering the cells in an encapsulation device. The cells may be encapsulated in a capsule that is permeable to the nutrients and oxygen the cells require, as well as immune humoral and therapeutic factors that are still impermeable to the cells. Preferably, the encapsulation material is hypoallergenic, easily and stably located in the target tissue, and provides additional protection to the implanted structure. These and other means for reducing or eliminating the immune response to implanted cells are known in the art. Alternatively, the cells may be genetically modified to reduce immunogenicity.

[0081] STRO-1 +It will be appreciated that the pluripotent cells or their progeny may be administered together with other beneficial drugs or biological molecules (growth factors, trophic factors). When administered together with other drugs, STRO-1 + The pluripotent cells or their progeny may be administered together in a single pharmaceutical composition or in separate pharmaceutical compositions, simultaneously with other drugs or sequentially (before or after administration of the other drugs). Bioactive factors that may be co-administered include anti-apoptotic drugs (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory drugs (e.g., p38 MAPK inhibitors, TGF-β inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and NSAIDs (non-steroidal anti-inflammatory drugs; e.g., TEPOXALIN, TOLMETIN, SUPROFEN)); immunosuppressants / immunomodulators (e.g., calcineurin inhibitors such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS); antiproliferative drugs (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); antilymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3); antithrombotic drugs (e.g., heparin, heparin derivatives, urokinase, PPack (dextrophenylalanine proline arginine chloromethyl ketone), antithrombin compounds, platelet receptor antagonists, antithrombin antibodies, antiplatelet receptor antibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandin inhibitors, and platelet aggregation inhibitors); and antioxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine), as well as local anesthetics.

[0082] Genetically modified cells In one embodiment, STRO-1 + The pluripotent cells and / or their progeny are genetically modified to express and / or secrete, for example, a protein of interest, e.g., a protein that provides a therapeutic and / or prophylactic benefit, e.g., insulin, glucagon, somatostatin, trypsinogen, chymotrypsinogen, elastase, carboxypeptidase, pancreatic lipase or amylase, or a polypeptide associated with or responsible for enhanced angiogenesis or a polypeptide associated with differentiation of cells into pancreatic or vascular cells.

[0083] Methods for genetically modifying cells will be clear to those skilled in the art.For example, the nucleic acid to be expressed in the cell is operably linked to a promoter for inducing expression in the cell.For example, the nucleic acid is linked to a promoter that can operate in various cells of interest, such as, for example, a viral promoter, for example, a CMV promoter (for example, a CMV-IE promoter) or an SV-40 promoter.Additional suitable promoters are known in the art and shall be construed mutatis mutandis to this embodiment of the present invention.

[0084] Preferably, the nucleic acid is provided in the form of an expression construct. As used herein, the term "expression construct" refers to a nucleic acid that has the ability to express the nucleic acid (e.g., reporter gene and / or counter-selectable reporter gene) to which it is operably linked in a cell. It should be understood that within the context of the present invention, an expression construct may comprise or be a plasmid, a bacteriophage, a phagemid, a cosmid, a small viral genome or genome fragment, or other nucleic acid that can maintain and / or replicate heterologous DNA in an expressible format.

[0085] Methods for the construction of expression constructs suitable for carrying out the present invention will be clear to the skilled artisan and are described, for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) or Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001). For example, each of the components of the expression construct is amplified from a suitable template nucleic acid, for example using PCR, and subsequently cloned into a suitable expression construct, such as, for example, a plasmid or phagemid.

[0086] Suitable vectors for such expression constructs are known in the art and / or described herein. For example, expression vectors suitable for the methods of the invention in mammalian cells are, for example, the pcDNA vector suite vectors provided by Invitrogen, the pCI vector suite vectors (Promega), the pCMV vector suite vectors (Clontech), the pM vectors (Clontech), the pSI vectors (Promega), the VP 16 vectors (Clontech) or the pcDNA vector suite vectors (Invitrogen).

[0087] One of skill in the art would be aware of additional vectors and sources of such vectors, such as, for example, Invitrogen Corporation, Clontech, or Promega.

[0088] Means for introducing isolated nucleic acid molecules or gene constructs containing the same into cells for expression are known to those skilled in the art. The technique used for a given organism relies on known successful techniques. Means for introducing recombinant DNA into cells include microinjection, DEAE-dextran mediated transfection, liposome-mediated transfection, such as by using Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and particle bombardment, such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA), among others.

[0089] Alternatively, the expression construct of the present invention is a viral vector. Suitable viral vectors are known in the art and are commercially available. Conventional viral-based systems for delivery of nucleic acid and its integration into host cell genome include, for example, retroviral vectors, lentiviral vectors or adeno-associated viral vectors. Alternatively, adenoviral vectors are useful for introducing episomal nucleic acid into host cells. Viral vectors are an efficient and versatile method of gene transfer in target cells and tissues. Moreover, high gene transfer efficiency has been observed in many different cell types and target tissues.

[0090] For example, retroviral vectors generally contain cis-acting long terminal repeats (LTRs) with packaging capacity for up to 6-10 kb of foreign sequence. The 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 (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, AD 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).

[0091] Various adeno-associated virus (AAV) vector systems have also 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; 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.

[0092] Additional viral vectors useful for delivering the expression constructs of the invention include, for example, viral vectors derived from the poxvirus family, such as vaccinia virus and avian poxvirus, or alphavirus or conjugate viral vectors (e.g., viral vectors described in Fisher-Hoch et al., Proc. Nall Acad. Sci. USA 56:317-321, 1989). EXAMPLES

[0093] Example 1 Experimental design To induce progressive disc degeneration, 24 sheep received injections of 1.0 IU chondroitinase ABC (cABC) (Seikagaku Corporation, Japan) into three adjacent lumbar discs (nominal L3-L4, L4-L5, and L5-L6). The remaining lumbar discs (nominal L1-L2 and L2-L3) were not injected with cABC and were considered normal controls. For 15 (±3) weeks following cABC administration, either high or low doses (4 × 10 6 or 0.5 × 10 6 STRO-l of cells + Injections of ProFreeze™ NOA Freezing Medium (Lonza Walkersville Md.) mixed with pluripotent cells or an equal amount of Euflexxa® hyaluronic acid (Ferring Pharmaceuticals) were administered directly into the nucleus pulposus of the intervertebral disc (Table 1). Animals were necropsied 3 or 6 months after injection. Figure 1 is a schematic of the spinal levels used for the experiments and the protocol for treatment.

[0094] [Table 1]

[0095] Example 2 Immunoselected STRO-l + Multipotent cell proliferation The STRO-1 used in these experiments + Multipotent cells were derived from French sheep and prepared by Lonza (USA). Bone marrow (BM) aspirates were obtained from the sheep and BM mononuclear cells (BMMNC) were prepared essentially as previously described (US 2005-0158289).

[0096] STRO-l + Multipotent cells were subsequently isolated using STRO-3 antibody by magnetic activated cell sorting as previously described (WO2006 / 108229).

[0097] Example 3 Radiology Animals underwent plain lateral radiographs of the lumbar spine under induction anesthesia 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 assessed by a blinded observer using an index of intervertebral height (DHI), calculated by averaging measurements of the anterior, middle, and posterior portions of the IVD and dividing it by the average height of the adjacent intervertebral bodies, as previously described (24).

[0098] Example 4 Magnetic Resonance Imaging (MRI) MRI showed that HA or high-dose and low-dose HA+STRO-1 were administered approximately 15 weeks after the induction of disc degeneration by injection of chondroitinase ABC. + Prior to treatment (Tx) with intradiscal injection of cells, spinal cord MRI was performed on all sheep under induction anesthesia. Three and six months after treatment, immediately prior to necropsy (Nx), spinal cord MRI was performed on all sheep.

[0099] Imaging was performed on one of two MRI scanners: STRO-1 + MRI was performed prior to cell administration using a 1.5 T Siemens VISION MRI scanner equipped with Numaris 33G software. +MRI performed after cell administration used a 1.5 Tesla Siemens AVANTRO MRI scanner with syngo B13 software. Regional scans were followed by sagittal imaging of the lumbar spine and sacrum in T1, T1 Gradient echo (TI_Flash), T2 and STIR weighting, followed by T2 weighted axial imaging of the six intervertebral disc levels L6 / SI L5 / L6, L4 / L5, L3 / L4, L2 / L3, and L1 / L2. Image sequences were provided on a CD displaying each scan as a series of 12 sagittal images. The digital images obtained for the 12 sagittal MRI-acquired sections were reviewed by two blinded qualified observers using the classification criteria of Pferrmann et al. for the disc degeneration scoring system (25) and are summarized in Table 2. The final score corresponded to the average of the scores of the two blinded examiners.

[0100] [Table 2]

[0101] Example 5 Histopathological analysis Disc units processed for histological and biochemical analysis were isolated by cutting open the adjacent cranial and sacral vertebral bodies proximal to the growth plate, respectively, with a bone saw. These spinal cord segments were fixed en bloc in Histochoice® for 56 hours and decalcified in several changes of 10% formic acid in 5% neutral buffered formalin with constant agitation for 2 weeks until complete decalcification was confirmed using a Faxitron HP43855A X-ray cabinet (Hewlett Packard, McMinnville, USA).

[0102] Decalcified specimens were processed by standard histological methods for paraffin embedding and sectioning. Paraffin sections, 4 microns thick, were mounted on Superfrost Plus glass microscope slides (Menzel-Glaser) and dried at 85°C for 30 min and then at 55°C overnight. Sections were deparaffinized in xylene (4 changes × 2 min) and rehydrated through graded ethanol washes (100–70% v / v) to tap water. All sections from whole blocks prepared from sagittal slices were stained with hematoxylin and eosin (H&E). H&E-stained tissue sections were coded and reevaluated to assess the degree of degeneration by an independent blinded histopathologist using a published (26) 4-point semiquantitative scoring method (see Table 3). Additional color stains, including Alcian Blue counterstained with neutral red, were also used to identify the distribution and assembly of matrix components in the disc sections.

[0103] [Table 3]

[0104] Example 6 Biochemical analysis of immobilized intervertebral disc tissue The annulus fibrosus (AF) and nucleus pulposus (NP) were carefully dissected from the processed demineralized disc tissue remaining after midsagittal slices were removed for histological studies. This process was more difficult in severely degenerated discs in which the demarcation boundary between the NP and AF had been lost. Finely diced NP and AF tissue aliquots were freeze-dried to constant weight, and triplicate portions (1–2 mg) of the dried tissue were hydrolyzed in 6 M HCl at 110°C for 6 h. Aliquots of the neutralized digest were assayed for hydroxyproline as a measure of tissue collagen content, as previously described (4). Triplicate portions (~2 mg) of the freeze-dried tissue were also digested with papain, as previously described (27), and aliquots were assayed for sulfated glycosaminoglycans (GAGs) using the metachromatic dye 1,9-dimethylmethylene blue.

[0105] Example 7 Statistical analysis of the data Statistical comparisons of DHI and biochemical data for all 3- and 6-month treatment groups were performed using Student's unpaired t-test, with p<0.05 considered significant. For histology and MRI aggregate degeneration and NP scores, comparisons between the various disc treatments were performed using Kruskal-Wallis or Friedman tests (nonparametric repeated measures ANOVA) with Dunn's multiple comparison post-hoc test. Statistical significance between groups was interpreted as p<0.05.

[0106] Example 8 Immunoselected STRO-1 + Results of experiments on bovine intervertebral disc regeneration using multipotent cells High dose (4.0×10 6 STRO-1 + cells) (groups 2 and 4) and low dose (0.5 × 10 6 STRO-1 + All animals in the injected groups (groups 1 and 3) maintained normal body weight over the course of the study and showed no evidence of adverse side effects.

[0107] Injection of the chemical nucleolytic drug chondroitinase ABC into the NPs of the target discs reduced the disc height index (DHI) by nearly 50% over a 3-month period, confirming significant loss of PGs and water from the disc extracellular matrix, as shown in Figure 2. The pre-DHI treatment data was supported by the MRI aggregate disc degeneration scores. As shown in Figures 3 and 4, in all groups, non-chondroitinase ABC-injected control discs provided MRI degeneration scores that were nearly 50% of the chondroitinase ABC-injected disc scores, confirming the validity of the model.

[0108] Low dose STRO-1 +The aggregate mean histopathological classification scores determined for the discs of animals sacrificed 3 months after cell injection showed a reduction in scores that was not significantly different in non-injected control discs or in discs injected with cABC or cABC+HA. However, the latter two scores were significantly higher (p<0.001) than the control disc scores (Figure 5A). + In the cell group, disc scores were significantly lower than cABC and cABC+HA injected discs (p < 0.01), but equivalent to uninjected control disc scores (Figure 5B). This pattern was consistent with the high-dose STRO-1 treatment at 3 months. + The cells were maintained in the disc (p<0.05) (Fig. 6A) but not 6 months after injection (Fig. 6B).

[0109] Non-injected controls, cABC, cABC+HA and cABC+STRO-1 in 3-month low-dose and 6-month low-dose sheep + Photomicrographs of tissue sections from cell-injected discs were analyzed along with their respective histopathological scores. This analysis highlighted the striking structural and cellular changes resulting from the various intradiscal treatments following cABC administration. This analysis demonstrated the normalization of STRO-1 disc structural integrity and deposition of new extracellular matrix six months after low-dose cell administration. + The beneficial effects mediated by the cells were also illustrated. These tissue sections were selected based on their diverse histopathology scores, which were consistent with the overall mean aggregate scores obtained for all 3-month and 6-month low-dose groups summarized in FIG. 5.

[0110] The disc histopathology scores complemented the MRI-assessed disc degeneration scores. Furthermore, in all groups, the MRI scores were significantly higher than those of the cABC+STRO-1 group, regardless of the dose used. + The same pattern of higher scores for cABC alone and cABC+HA injected discs than for cell and control disc scores followed. However, MRI summary scores were significantly higher at both 3 and 6 months low dose STRO-1 compared to cABC+HA scores (p < 0.05). +Significant differences were observed in cell-injected disc scores (Figures 7A and 7B). + Cellular MRI scores were not significantly different from control, cABC alone, or cABC+HA values ​​at 3 months, but were lower than cABC alone at 6 months (Figures 8A and 8B).

[0111] Because the experimental model of disc degeneration used in these experiments was induced by injecting the PG depolymerase chondroitinase ABC directly into the NP, the histopathological scores of this region are shown separately. In addition, the biochemical changes that occurred in response to the various treatments on the NP tissue are also shown. As is evident from Figures 9 and 10, cABC+STRO-1 + The mean NP histopathology scores for cell-injected discs followed the same pattern as the whole disc histopathology scores for cABC- and cABC+HA-injected discs showing higher degeneration scores, however the differences were not statistically significant due to interanimal sample variability within the small group size used (N = 6).

[0112] The radiologically determined disc height index (DHI) is a useful indicator of disc degeneration. (24) As previously discussed, DHI was reduced by approximately 50% 3 months after intradiscal injection of the PG depolymerase cABC.

[0113] Biochemical determination of glycosaminoglycan (GAG) content (as a marker of PGs) in disc NP tissue showed a significant loss of this component for all treated discs compared to non-injected control discs (Figure 11). Significant differences (p<0.05) in GAG levels were observed for cABC and cABC+HA tissue compared to control NP tissue for all groups, but not for the high-dose STRO-1 at 3 months. + Cell-injected discs and low-dose STRO-1 at 6 months + Cells were not significantly different from controls (Figure 11).

[0114] As can be seen in Figure 12, all injected discs showed some degree of recovery in their DHI over the 3-month post-treatment period, regardless of the treatment. However, the greatest improvement in DHI was seen with low dose STRO-1. + The improvement was most notable in the cell-injected discs, where it was shown to be statistically significant (p<0.02). + The DHI of cell-injected discs was not significantly different from the mean DHI of non-injected control discs (FIG. 12A).

[0115] Consideration The results of this study showed that low doses of STRO-1 have a beneficial effect on degenerative discs. + Intradiscal administration of cells plus HA was found to improve structural recovery to a greater extent than HA-injected discs, a conclusion supported by experimental data obtained from three independent assessments of disc integrity and matrix recovery from a baseline degenerative state corresponding to 50% of normal disc height index.

[0116] Low-dose STRO-1 for 6 months + The increase in DHI observed in the cell-injected discs is explained by the deposition of reconstituted extracellular matrix in the degenerated disc. In the healthy spine, disc height (DHI) is maintained by the presence of high concentrations of PGs and bound water molecules in the nucleus pulposus and the internal annulus (2, 4). These entities provide the disc with a high swelling pressure that maintains disc height, but also allows the disc to recover from deformation after axial compression (2, 4). In fact, the use of chondroitinase ABC to induce disc degeneration in this animal model took advantage of the ability of this enzyme to selectively degrade and remove the majority of PGs from the extracellular matrix of the NP and the internal AF (28). Histochemical experiments using Alcian Blue dye, which binds to negatively charged PGs, showed that low doses of STRO-1 +Sections from cell-injected discs revealed stronger staining of cABC+HA-injected disc sections than cABC alone, demonstrating the presence of higher concentrations of PG in these tissues. Examination of these stained sections by both white light and fluorescence microscopy also demonstrated standardization of hyaline cartilaginous endplate (CEP) morphology, as well as the lamellar structure of the AF collagen fibril aggregates. CEPs are the main pathway for nutrient diffusion into the avascular NP, and physical disruption of their structure reduces the survival of resident cells.

[0117] PG concentrations in NPs, as determined biochemically from GAG content, only partially supported the histopathological findings. + The GAG ​​content of cell-injected NPs was found to be not statistically different from control NPs, whereas other treated discs were. However, cABC+STRO-1 + No statistical difference in GAG levels between cell-injected discs and cABC or cABC+HA disc NPs could be demonstrated, suggesting comparable levels of GAGs in the tissues of these groups. The disc tissues used for biochemical analysis were pre-fixed in buffered formalin for several months prior to biochemical analysis, so it is possible that variable amounts of PGs were leached over this time. Furthermore, covalent cross-links of collagen and proteins formed during the formalin fixation process may have impaired sufficient macroscopic differentiation and dissection of the NP and AF regions of the disc from each other. Dissection was particularly difficult in degenerated discs where the border zone between NP and AF had disappeared. Disc AF has a much lower GAG content than NP, and therefore sampling errors may have significantly affected the analyzed data (1-4).

[0118] Sheep STRO-1 was administered to the nucleus pulposus of experimentally created degenerated IVDs. +The present study demonstrated that administration of multipotent cells together with a suitable carrier, such as high molecular weight hyaluronic acid (HA), accelerated the regeneration of the extracellular matrix of the intervertebral disc, assessed radiologically by the restoration of disc height. This interpretation is based on the assumption that in a loaded spine, disc height is maintained by the presence within the NP and the inner annulus of a high concentration of matrix proteoglycans, which together with the water molecules bound to it exert a high swelling pressure on this structure. Indeed, the use of chondroitinase ABC to induce disc degeneration at the beginning of the study took advantage of the ability of this enzyme to degrade and remove the majority of proteoglycans from the NP and the extracellular matrix.

[0119] The present results show that the recovery of the intervertebral disc from the degenerative state induced by the initial injection of cABC was observed at a high dose (4.0 × 10 6 ) STRO-1 + A lower dose (0.5 × 10 6 ) was shown to maintain the nutrient intake of the NP of the disc, which relies on the exchange of O2 / CO2 and metabolites between blood vessels below the CEP (1, 2). As already mentioned, even small disturbances in the interface between the NP and the CEP and in the subchondral blood supply of the vertebrae would be expected to impair the nutrient intake of the NP, as was seen to occur in degenerated discs. This nutrient deficiency could be due to the lack of nutrient intake of the injected STRO-1, which is able to survive in this oxygen-depleted environment. + It places an upper limit on the number of cells, thereby + This would result in loss of cell viability and therefore loss of therapeutic efficacy.

[0120] Low dose STRO-1 + The therapeutic mechanisms involved in the restoration of disc integrity in this animal model following administration of multipotent cells remain to be elucidated. However, the therapeutic potential of STRO-1 in the degenerative disc space remains unclear. +It is possible that the anti-inflammatory cytokines and growth factors released by multipotent cells modulate the pro-catabolic and anti-anabolic effects mediated by cytokines and other nociceptive factors released by resident disc cells in response to biochemical and biomechanical injury. + Multipotent cell-derived paracrine factors also express non-STRO-1 + This may support the normal physiological anabolic response of resident disc cells to depletion of PGs from their extracellular environment, which was occasionally observed in some cell-injected discs. + Multipotent cells may be able to engraft within the degenerated matrix and undergo differentiation into NP chondrocytes or other disc cells.

[0121] It will be appreciated by those skilled in the art that numerous changes and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0122] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0123] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of such matter forms a prior art basis or was common general knowledge in the art relevant to the present invention existing prior to the priority date of each claim of this application. [References] TIFF0007626539000004.tif202170TIFF0007626539000005.tif202170

Claims

1. A composition for treating lower back pain in a human subject, comprising: + 2. A method for the treatment of a spinal disc comprising administering to an intervertebral disc a human cell population enriched for pluripotent cells or their progeny, wherein at least 5% of the cells of said human cell population are STRO-1 + and TNAP + The composition.

2. 10. The composition of claim 1, formulated for administration to the nucleus pulposus of an intervertebral disc.

3. STRO-1 + , TNAP + 2. The composition of claim 1, wherein the multipotent cells are also positive for VCAM-1+, THY-1+, STRO-2+, CD45+, CD146+, 3G5+ or any combination thereof.

4. STRO-1 + The composition of claim 1, wherein the multipotent cells are derived from an allogeneic source.

5. 10. The composition of claim 1, further comprising a glycosaminoglycan (GAG).

6. 6. The composition of claim 5, wherein the GAG ​​is selected from the group consisting of hyaluronic acid (HA), chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparin sulfate, and galactosaminoglycan sulfate (GGGS).

7. 10. The composition of claim 1, wherein the human subject has a spinal condition characterized by degeneration of the intervertebral disc.

8. 8. The composition of claim 7, wherein the spinal condition is lower back pain, age-related changes of the intervertebral disc, or spondylolysis.

9. STRO-1 for use in the manufacture of a medicament for treating low back pain in a human subject. + Use of a human cell population enriched for pluripotent cells or their pluripotent progeny, wherein at least 5% of the cells of said human cell population are STRO-1 + and TNAP + That is, use.

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