Expanded canine progenitor cells and related methods

Abstract

One embodiment of the present invention provides expanded canine progenitor cells of non-embryonic origin that can be maintained in culture in an undifferentiated state or differentiated to form cells of multiple tissue types. The expanded cells are postnatal somatic cells that can be characterized by one or more of the following: a population doubling rate of less than about 24 hours; normal karyotype; the ability to differentiate into at least two cell types of mesoderm; extensive replication in culture and expressing markers of extensive replication such as telomerase; and expressing markers of pluripotency. Methods of isolation and culture, as well as therapeutic uses of the cells, such as treating musculoskeletal and inflammatory diseases and disorders, are also provided. Additionally, cell banks are provided that can be used to provide cells for administration to subjects, drug discovery methods, and compositions of cells, such as pharmaceutical compositions.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides expanded canine progenitor cells of postnatal origin that can be maintained in culture in an undifferentiated state or differentiated to form cells of multiple tissue types. Methods of isolation and culture, as well as therapeutic uses of the expanded canine progenitor cells, are also provided. The expanded canine progenitor cells are postnatal somatic cells, and are capable of extensive replication in culture, and may be characterized by one or more of the following through about 55 population doublings: a population doubling rate of less than about 24 hours; normal karyotype; the ability to differentiate into at least two cell types of the mesodermal germ layer; and the expression of extensive replication markers (e.g., telomerase) and pluripotency markers (e.g., oct4). The present invention also relates to methods of treating musculoskeletal disorders and inflammatory conditions using the expanded canine progenitor cells. The present invention also relates to cell banks that can be used to provide expanded canine progenitor cells for administration to canine subjects. The present invention also relates to drug discovery methods. The present invention also relates to compositions of expanded canine progenitor cells, such as compositions in pharmaceutical compositions. [Background technology]

[0002] background To date, stem cells have been used, primarily experimentally, to treat a variety of diseases in different animal species. Initially, regenerative veterinary medicine focused on orthopedic diseases, but the focus is now rapidly expanding to other areas such as oral and gastrointestinal diseases, liver, kidney, heart, respiratory, neuromuscular, skin, olfactory, and reproductive system diseases. Stem cell treatments have been most frequently used in dogs and horses for a variety of diseases in different organ systems, and in cats for renal, respiratory, and inflammatory diseases. Summary of the Invention

[0003] The present inventors have discovered canine progenitor cells of postnatal origin that can be maintained in culture in an undifferentiated state or differentiated to form cells of multiple tissue types. The canine progenitor cells are capable of extensive replication in culture and can be characterized by one or more of the following through approximately 55 population doublings: a population doubling rate of less than about 24 hours; normal karyotype; the ability to differentiate into at least two cell types of mesoderm; and expression of markers of extensive replication (e.g., telomerase) and pluripotency (e.g., oct4).

[0004] Based at least on these findings, the present invention provides canine progenitor cells and methods, including, but not limited to, expanded canine progenitor cells, compositions comprising the expanded canine progenitor cells, methods of treating musculoskeletal disorders and inflammatory conditions using the expanded canine progenitor cells, methods of establishing cell banks using the expanded canine progenitor cells, and drug discovery methods.

[0005] Thus, one embodiment of the present invention includes expanded cultured canine progenitor cells that have a population doubling rate of less than about 24 hours, have a normal karyotype, are positive for expression of telomerase and CD90, and are negative for expression of CD45 and CD34.

[0006] In one embodiment, the invention includes a method of treating an inflammatory condition in a dog, comprising administering to the dog a therapeutically effective amount of expanded canine progenitor cells, the cells having a population doubling rate of less than about 24 hours, having a normal karyotype, being positive for expression of telomerase and CD90, and being negative for expression of CD45 and CD34.

[0007] In one embodiment, the invention includes a method of treating an inflammatory condition in a dog comprising administering to the dog a therapeutically effective amount of expanded canine progenitor cells, the cells having a population doubling rate of less than about 24 hours, having a normal karyotype, being positive for expression of telomerase and CD90, being negative for expression of CD45 and CD34, and being capable of differentiating into at least two cell types of the mesoderm.

[0008] In one embodiment, the invention includes a method of treating a musculoskeletal disorder in a dog, comprising administering to the dog a therapeutically effective amount of expanded canine progenitor cells, the cells having a population doubling rate of less than about 24 hours, having a normal karyotype, being positive for expression of telomerase and CD90, and being negative for expression of CD45 and CD34.

[0009] In one embodiment, the invention includes a method of treating a musculoskeletal disorder in a dog, comprising administering to the dog a therapeutically effective amount of expanded cultured canine progenitor cells, the cells having a population doubling rate of less than about 24 hours, having a normal karyotype, being positive for expression of telomerase and CD90, being negative for expression of CD45 and CD34, and being capable of differentiating into at least two cell types of the mesoderm.

[0010] In one example, the expanded canine progenitor cells have undergone at least 40 population doublings in culture.

[0011] In another example, the expanded canine progenitor cells have undergone at least 50 population doublings in culture.

[0012] In one example, expanded canine progenitor cells have a population doubling rate in culture of about 15-24 hours.

[0013] In another example, expanded canine progenitor cells have a population doubling rate in culture of about 16 hours.

[0014] In one example, the expanded canine progenitor cells are derived from bone marrow, adipose tissue, umbilical cord blood, or placental tissue.

[0015] In one example, the expanded canine progenitor cells are positive for expression of CD29.

[0016] In one example, the expanded canine progenitor cells are negative for MHC class II expression.

[0017] In one example, the expanded canine progenitor cells are positive for expression of one or more of PTHLH, CD13, CD44, CD49c, CD73, CD90, CD105, and IL1R2.

[0018] In one example, expanded canine progenitor cells are positive for expression of IL1R2.

[0019] In one example, expanded canine progenitor cells are negative for expression of rex-1, CD34, CD45, and NOV.

[0020] In one example, expanded canine progenitor cells are positive for expression of nanog, sox-2, and oct-4.

[0021] In one example, expanded canine progenitor cells express telomerase up to approximately 55 population doublings in culture.

[0022] In one example, expanded canine progenitor cells are capable of reducing or inhibiting T cell proliferation in vivo and / or in vitro.

[0023] In one example, expanded canine progenitor cells can provide angiogenesis in vivo and / or in vitro.

[0024] In one example, expanded canine progenitor cells are capable of differentiating into at least two cell types of the mesoderm.

[0025] In another embodiment, the expanded canine progenitor cells have a population doubling rate of about 16 hours, a normal karyotype, are positive for expression of telomerase and CD90, and are negative for expression of MHC class II, CD45, and CD34. The expanded progenitor cells have undergone at least 40 population doublings in culture, are also positive for expression of one or more of PTHLH, CD13, CD44, CD49c, CD73, CD105, and IL1R2, and are derived from bone marrow.

[0026] In one example, the inflammatory condition is a chronic inflammatory condition or an acute inflammatory condition.

[0027] In one example, the acute or chronic inflammatory condition is one of dermatitis, inflammatory eye disease, inflammatory brain disease, inflammatory airway disease, and inflammatory bowel disease.

[0028] In another example, the dermatitis is atopic dermatitis.

[0029] In another example, the inflammatory eye disease is keratoconjunctivitis.

[0030] In another example, the inflammatory brain disease is meningoencephalomyelitis.

[0031] In one example, the inflammatory condition is an autoimmune disease.

[0032] In one example, the musculoskeletal disorder is one of osteoarthritis and cruciate ligament rupture.

[0033] In another example, the cruciate ligament rupture is a partial cruciate ligament rupture.

[0034] In one example, the musculoskeletal disorder is a spinal condition.

[0035] In another example, the spinal condition is one of a spinal cord injury and a disc disease.

[0036] In one embodiment, the expanded canine progenitor cells include, but are not limited to, postnatal somatic cells that have some characteristics of embryonic stem cells, but are derived from postnatal tissues and provide the effects described in this application.The expanded canine progenitor cells can achieve these effects naturally (i.e., are not genetically or pharmacologic modified).However, natural expression can be genetically or pharmacologic modified to increase efficacy.

[0037] The expanded canine progenitor cells may express pluripotency markers such as oct4. They may also express markers associated with extended replicative capacity such as telomerase. Other characteristics of pluripotency may include the ability to differentiate into different cell types of the mesoderm. Such canine progenitor cells are not tumorigenic, do not form teratomas, and are not immortalized or transformed in culture. Canine progenitor cells may be highly expanded while maintaining a normal karyotype. For example, in one embodiment, the canine progenitor cells have undergone at least 10-60 cell doublings in culture, e.g., 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, or more cell doublings, where the cells have a normal karyotype, express telomerase, express oct-4, and differentiate into at least two cell types of mesoderm. Further, the expanded canine progenitor cells have a population doubling rate of less than about 24 hours.

[0038] Expanded canine progenitor cells may be prepared by the isolation and culture conditions described herein.

[0039] Canine progenitor cells include, but are not limited to, the following numbered embodiments:

[0040] 1. Expanded cultured canine progenitor cells that have a population doubling rate of less than about 24 hours, have a normal karyotype, are capable of differentiating into at least two cell types of the mesoderm, and are postnatal somatic cells.

[0041] 2. Expanded cultured canine progenitor cells that have a population doubling rate of less than about 24 hours, have a normal karyotype, express telomerase, and are postnatal somatic cells.

[0042] 3. Expanded cultured canine progenitor cells that have a population doubling rate of less than about 24 hours, have a normal karyotype, express oct-4, and are postnatal somatic cells.

[0043] 4. Expanded cultured canine progenitor cells that have a population doubling rate of less than about 24 hours, have a normal karyotype, have undergone at least 40 population doublings in culture, and are postnatal somatic cells.

[0044] 5. The expanded cultured canine progenitor cells according to any one of 1 to 3 above, wherein the cells have undergone at least 40 population doublings in culture.

[0045] 6. The expanded cultured canine progenitor cells according to 5 above, wherein the cells have undergone at least 50 population doublings in culture.

[0046] 7. The expanded canine progenitor cells according to any one of 1 to 6 above, having a population doubling rate in culture of about 15 to 24 hours.

[0047] 8. The expanded canine progenitor cells according to any one of 1 to 7 above, which have a population doubling rate in culture of about 16 hours.

[0048] 9. The expanded canine progenitor cells according to any one of 1 to 8 above, which are derived from bone marrow, adipose tissue, umbilical cord blood, or placental tissue.

[0049] 10. The expanded canine progenitor cells according to any one of 1 to 9 above, which are positive for CD90 expression and negative for CD45 and CD34 expression.

[0050] 11. The expanded canine progenitor cells according to any one of 1 to 10 above, which are positive for CD29 expression.

[0051] 12. The expanded canine progenitor cells according to any one of 1 to 11 above, which are negative for expression of MHC class II.

[0052] 13. The expanded canine progenitor cells according to any one of 1 to 12 above, which are positive for expression of one or more of PTHLH, CD13, CD44, CD49c, CD73, CD105, and IL1R2.

[0053] 14. The expanded canine progenitor cells according to any one of 1 to 13 above, which are positive for the expression of IL1R2.

[0054] 15. The expanded canine progenitor cells according to any one of 1 to 14 above, which are negative for expression of rex-1 and NOV.

[0055] 16. The expanded canine progenitor cells according to any one of 1 to 2 and 4 to 15 above, which are positive for the expression of nanog, sox-2 and oct-4.

[0056] 17. The expanded canine progenitor cells according to any one of 1 to 16 above, which express telomerase until about 55 population doublings in culture.

[0057] 18. The expanded canine progenitor cells according to any one of 1 to 17 above, which express oct-4 for up to about 55 population doublings in culture.

[0058] 19. The expanded cultured canine progenitor cells according to any one of 1 to 18, which are allowed to differentiate into at least two cell types of mesoderm for approximately 55 population doublings in culture.

[0059] 20. The expanded canine progenitor cells according to any one of 1 to 19 above, which reduce or inhibit T cell proliferation in vitro and / or in vivo.

[0060] 21. The expanded cultured canine progenitor cells according to any one of 1 to 20 above, which are capable of providing angiogenesis in vitro and / or in vivo.

[0061] 22. The expanded canine progenitor cells according to any one of 2 to 21 above, which are capable of differentiating into at least two cell types of the mesoderm.

[0062] 23. The expanded cultured canine progenitor cells according to claim 22, which are capable of differentiating into at least two of osteoblasts, adipocytes, and chondrocytes.

[0063] 24. The expanded cultured canine progenitor cells according to any one of 1 to 23 above, wherein the cells are not tumorigenic, do not form teratomas, are not transformed, and are not immortalized.

[0064] 25. The expanded progenitor cells described in any one of 1 to 24 above, prepared by a method comprising: obtaining tissue from a dog; establishing a population of adherent cells; selecting cells that positively express CD90 and / or do not express at least one of CD45 and CD34; and expanding the selected cells in culture medium.

[0065] 26. A composition comprising the expanded precursor cells according to any one of 1 to 24 above, and a second component.

[0066] 27. A pharmaceutical composition comprising the expanded progenitor cells according to any one of 1 to 24 above, and a pharma- ceutically acceptable carrier.

[0067] 28. A kit comprising the following separately packaged components: expanded progenitor cells according to any one of 1 to 24 above; culture medium; and instructions for culturing the cells.

[0068] 29. A method for preparing the composition described in claim 26, comprising obtaining tissue from a dog; establishing a population of adherent cells; selecting cells that positively express CD90 and / or do not express at least one of CD45 and CD34; expanding the selected cells in culture medium; and adding the cells to a second component.

[0069] 30. A method for preparing the pharmaceutical composition according to claim 27, comprising mixing expanded progenitor cells with a pharma- ceutically acceptable carrier.

[0070] 31. A method for preparing expanded cultured canine progenitor cells according to any one of 1 to 24 above, the method comprising: obtaining tissue from a canine; establishing a population of adherent cells; selecting cells that positively express CD90 and / or do not express at least one of CD45 and CD34; and expanding the selected cells in culture medium.

[0071] 32. A method for establishing a cell bank, the method comprising expanding and storing the expanded progenitor cells described in any one of 1 to 24 above for future administration to a subject.

[0072] 33. A method for drug discovery, the method comprising exposing expanded progenitor cells according to any one of 1 to 24 above to an agent and evaluating one or more effects of the agent on the cells.

[0073] 34. A method for treating an inflammatory condition in a dog, comprising administering to the dog a therapeutically effective amount of a canine progenitor cell described in any one of 1 to 24 above.

[0074] 35. The method according to claim 34, wherein the inflammatory condition is a chronic inflammatory condition or an acute inflammatory condition.

[0075] 36. The method according to claim 35, wherein the acute or chronic inflammatory condition is one of dermatitis, inflammatory eye disease, inflammatory brain disease, inflammatory airway disease, and inflammatory bowel disease.

[0076] 37. The method according to claim 36, wherein the dermatitis is atopic dermatitis.

[0077] 38. The method according to claim 36, wherein the inflammatory eye disease is keratoconjunctivitis.

[0078] 39. The method according to claim 36, wherein the inflammatory brain disease is meningoencephalomyelitis.

[0079] 40. The method according to claim 34, wherein the inflammatory condition is an autoimmune disease.

[0080] 41. A method for treating a musculoskeletal disorder in a dog, comprising administering to the dog a therapeutically effective amount of a canine progenitor cell described in any one of 1 to 24 above.

[0081] 42. The method according to claim 41, wherein the musculoskeletal disorder is one of osteoarthritis and cruciate ligament rupture.

[0082] 43. The method according to claim 42, wherein the cruciate ligament rupture is a partial cruciate ligament rupture.

[0083] 44. The method according to claim 42, wherein the musculoskeletal disorder is a spinal cord condition.

[0084] 45. The method according to claim 44, wherein the spinal condition is one of spinal cord injury and intervertebral disc disease.

[0085] Canine progenitor cells include, but are not limited to, the following specific numbered embodiments:

[0086] 1. Expanded cultured canine progenitor cells with a population doubling rate of less than about 24 hours, normal karyotype, positive for expression of telomerase and CD90, and negative for expression of CD45 and CD34.

[0087] 2. The expanded canine progenitor cells according to 1 above, which have undergone at least 40 population doublings in culture.

[0088] 3. The expanded cultured canine progenitor cells according to any one of 1 to 2 above, which have undergone at least 50 population doublings in culture.

[0089] 4. The expanded canine progenitor cells according to any one of 1 to 3 above, which have a population doubling rate in culture of about 15 to 24 hours.

[0090] 5. The expanded canine progenitor cells according to any one of 1 to 4 above, which have a population doubling rate in culture of about 16 hours.

[0091] 6. The expanded canine progenitor cells according to any one of 1 to 5 above, which are derived from bone marrow, adipose tissue, umbilical cord blood, or placental tissue.

[0092] 7. The expanded canine progenitor cells according to any one of 1 to 6 above, which are positive for CD29 expression.

[0093] 8. The expanded canine progenitor cells according to any one of 1 to 7 above, which are negative for expression of MHC class II.

[0094] 9. The expanded canine progenitor cells according to any one of 1 to 8 above, which are positive for expression of one or more of PTHLH, CD13, CD44, CD49c, CD73, CD105, and IL1R2.

[0095] 10. The expanded canine progenitor cells according to any one of 1 to 9 above, which are positive for the expression of IL1R2.

[0096] 11. The expanded canine progenitor cells according to any one of 1 to 10 above, which are negative for expression of rex-1 and NOV.

[0097] 12. The expanded canine progenitor cells according to any one of 1 to 11 above, which are positive for the expression of nanog, sox-2, and oct-4.

[0098] 13. The expanded canine progenitor cells according to any one of 1 to 12 above, which express telomerase until about 55 population doublings in culture.

[0099] 14. The expanded canine progenitor cells according to any one of 1 to 13 above, which are capable of reducing or inhibiting T cell proliferation in vivo and / or in vitro.

[0100] 15. The expanded canine progenitor cells according to any one of 1 to 14 above, which are capable of providing angiogenesis in vivo and / or in vitro.

[0101] 16. The expanded canine progenitor cells according to any one of 1 to 15 above, which are capable of differentiating into at least two cell types of the mesoderm.

[0102] 17. The expanded canine progenitor cells according to any one of 1 to 16 above, which are capable of differentiating into at least two of osteoblasts, adipocytes, and chondrocytes.

[0103] 18. The expanded cultured canine progenitor cells according to any one of 1 to 17 above, which are not tumorigenic, do not form teratomas, are not transformed, and are not immortalized. [Brief description of the drawings]

[0104] [Figure 1] Figure 1 - Graph showing the proliferation curve of canine multipotent adult progenitor cells (cMAPC). Cells were counted at each passage and population doublings (PD) were calculated according to the number of cells harvested (Ch) from the number of cells initially plated (Ci) using the following formula: PDh=PDi+Log2(Ch / Ci).

[0105] [Figure 2A-B] Figures 2A-D - Flow cytometry results showing that cMAPCs are positive for CD29 (Figure 2A), cMAPCs are positive for CD90 (Figure 2B), cMAPCs are negative for CD45 (Figure 2C), and cMAPCs are negative for MHC class II (Figure 2D). [Figure 2C-D] Figures 2A-D - Flow cytometry results showing that cMAPCs are positive for CD29 (Figure 2A), cMAPCs are positive for CD90 (Figure 2B), cMAPCs are negative for CD45 (Figure 2C), and cMAPCs are negative for MHC class II (Figure 2D).

[0106] [Diagram 3] Figure 3 - Graph showing cMAPC expression levels of CD34, CD45, CD13, CD44, CD49c, CD73, CD90 and CD105 as measured by qPCR analysis. The dotted lines represent the lower limit of detection. CD34 and CD45 are undetectable. CD13, CD44, CD49c, CD73, CD90 and CD105 show positive expression.

[0107] [Figure 4A-C] Figure 4A-C - Agarose gel showing expression of pluripotency genes in cMAPC and canine mesenchymal stem cells (MSC). RNA was extracted from canine MAPC (M) and MSC (S) from three different donors. RNA was converted to cDNA. cDNA was used to perform PCR for nanog (Figure 4A), oct4 (Figure 4B) and sox2 (Figure 4C). PCR products were then loaded onto a 2% agarose gel to visualize the expression of genes. cMAPC and canine MSC show expression of nanog, oct4 and sox2.

[0108] [Diagram 5] Figure 5 - Telomerase activity in cMAPC and canine MSC. For all samples, the same number of cells was used. The assay is based on the extension of telomeric templates by endogenous telomerase in cell samples. The amount of template is then determined by quantitative PCR. Significantly more templates were extended by telomerase in cMAPC samples compared to canine MSC samples from the same donor. Numbers in brackets represent population doublings. Telomerase activity decreased as cells reached higher population doublings and therefore older age, as seen for donor 2.

[0109] [Figure 6A-C] Figure 6 - Staining results to determine the multilineage potential of cMAPCs. cMAPCs can differentiate into osteoblasts, adipocytes and chondrocytes.

[0110] [Figure 7] Figure 7 - Immunoreactivity assay results showing that cMAPC inhibited T cell proliferation. Immunoreactivity assays were performed in 96-well round-bottom plates. In each well, 100,000 canine PBMCs (peripheral mononuclear blood cells) were added to the seeded cMAPCs in serial dilutions ranging from 1:2 to 1:16. cPBMCs were stimulated with 0.5 μg / ml ConA (concanavalin A; Sigma) and the assay was analyzed after 4 days.

[0111] [Figure 8] Figure 8 - In vitro angiogenesis assay results. Conditioned medium from cMAPCs induces tube formation between human umbilical vein endothelial cells (HUVECs).

[0112] [Figure 9] Figure 9 - Cytogenic analysis showing that cMAPCs have a normal karyotype.

[0113] [Figure 10A] Figure 10A-B - Microarray data results for cMAPCs and canine MSCs. The dendrogram (Figure 10A) and PCA (principal component analysis) plot (Figure 10B) showed that cMAPCs and cMSCs formed two distinct clusters that could be considered as two different cell populations based on total gene expression. [Figure 10B] Figure 10A-B - Microarray data results for cMAPCs and canine MSCs. The dendrogram (Figure 10A) and PCA (principal component analysis) plot (Figure 10B) showed that cMAPCs and cMSCs formed two distinct clusters that could be considered as two different cell populations based on total gene expression.

[0114] [Figure 11A-C]Figure 11A-C - Marker expression cMAPC and canine MSC. RNA was extracted for cMAPC (M) and cMSC (S) from three different donors. RNA was converted to cDNA. cDNA was used to perform PCR for IL1R2 (Figure 11A) and NOV (Figure 11B). PCR products were then loaded onto a 2% agarose gel to visualize gene expression. Ribosomal protein L8 (RPL8) (Figure 11C) was used as a reference gene. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0115] Detailed Description of the Invention It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the disclosed invention, which is defined solely by the claims.

[0116] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

[0117] The methods and techniques of this application are generally carried out according to conventional methods well known in the art, unless otherwise indicated, as described in various general and more specific references cited and discussed throughout this specification.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990).

[0118] definition "A" or "an" are used herein to mean one or more than one, i.e., at least one. When the plural is used herein, it generally includes the singular.

[0119] "Autoimmune disease" refers to the inability of a subject's immune system to distinguish between self and non-self or to respond to foreign antigens. The term also encompasses an exaggerated immune response to foreign antigens, as in the case of allergic disorders. Thus, this response is present in both autoimmune and allergic disorders. Autoimmune diseases include, but are not limited to, tissue damage and inflammation caused by the production of antibodies against the organism's own tissues, impaired production of cytokines, and tissue damage caused by cytotoxic or non-cytotoxic mechanisms of action. In some embodiments, an autoimmune disease is an inappropriately regulated immune response that results in the patient's symptoms. Typically, an autoimmune response occurs when a subject's immune system recognizes a self-antigen as a foreign antigen, resulting in the production of autoreactive effector immune cells. Autoreactive effector immune cells include cells from various lineages, including, but not limited to, cytotoxic T cells, helper T cells, and B cells. Although the exact mechanism differs, the presence of autoreactive effector immune cells in a patient suffering from an autoimmune disorder can result in the destruction of the patient's tissues and cells, resulting in pathological symptoms. Non-limiting examples of autoimmune diseases include, but are not limited to, immune-mediated polyarthritis; immune-mediated thrombocytopenia; keratoconjunctivitis sicca; inflammatory encephalopathy; and pemphigus foliaceus.Similarly, the presence of cells undergoing hypersensitive reactions to foreign antigens to which normal individuals respond more suppressively indicates hypersensitivity (allergy).Examples include, but are not limited to, flea allergy dermatitis, seasonal allergies, human food allergies, dog food allergies, aeroallergens, environmental allergies, household allergies, and prescription drugs.A number of assays for determining the presence of such cells in a subject, and thus the presence of an autoimmune disorder, such as an antigen-specific autoimmune disorder or an allergic disorder, are known to those skilled in the art and can be easily adopted in the subject method.

[0120] A "cell bank" is the nomenclature of cells that have been grown and stored for future use. The cells may be stored in aliquots. They can be used directly from storage or expanded after storage. This is convenient because there are "off the shelf" cells available for administration. The cells may already be stored in a pharma- ceutically acceptable excipient, so that the cells may be administered directly or mixed with an appropriate excipient when the cells are released from storage. The cells may be frozen or otherwise stored in a form that preserves viability. In one embodiment of the present invention, a cell bank is made using cells produced by the methods described in this application.

[0121] By "co-administer" is meant the coordinate administration of two or more agents together, including simultaneous or sequential administration.

[0122] "Comprising" means including the reference without other limitations, and necessarily without any limitations or exclusions as to what else may be included. For example, "a composition comprising x and y" encompasses any composition containing x and y, even though other ingredients may be present in the composition. Similarly, "a method comprising the step of x" encompasses any method in which x is performed, regardless of whether x is the only step of the method or it is only one of the steps, no matter how many other steps there may be and how simple or complex x is in comparison thereto. "Comprised of" and similar phrases using the word root "comprise" are used herein as synonyms of "comprising" and have the same meaning.

[0123] "Comprised of" is a synonym of "comprising" (see above).

[0124] "Effective route" generally refers to a route that provides for delivery of an agent (e.g., canine progenitor cells) to a desired compartment, system, or location. For example, an effective route is one by which an agent can be administered to provide a sufficient amount of the agent to the desired site of action to produce a beneficial or desired clinical result.

[0125] When referring to the present invention, "effective time" refers to a period of time sufficient to provide a particular effect, such as treatment of an inflammatory condition or a musculoskeletal disorder.

[0126] "Immune response" refers to a patient's response to a foreign or self-antigen. The term includes cell-mediated, humoral and inflammatory responses.

[0127] The use of the term "includes" is not intended to be limiting.

[0128] By "increase" or "increasing" is meant either inducing a biological event completely or increasing the magnitude of the event.

[0129] The term "inflammatory condition" refers to a disease or disorder characterized by acute or chronic inflammation. This term can refer to inflammatory diseases, such as autoinflammatory diseases (e.g., autoimmune diseases) or other inflammatory diseases. Non-limiting examples of inflammatory conditions include dermatitis (e.g., atopic dermatitis), inflammatory eye disease, inflammatory brain disease (e.g., meningoencephalomyelitis), inflammatory airway disease, and inflammatory bowel disease. Other non-limiting examples of inflammatory conditions are disclosed in Maziarz et al., US Patent Application Publication No. 2006 / 0263337 A1, such as adverse immune reactions (e.g., those resulting from other treatments), inflammatory conditions that complicate transplantation therapy (e.g., GvHD), and innate immune disorders.

[0130] The term "ischemic condition" refers to injury due to obstructed blood flow and reperfusion injury caused by removal of the obstruction. Non-limiting examples of ischemic conditions include acute myocardial infarction, chronic heart failure, peripheral vascular disease, stroke, chronic total occlusion, renal ischemia, and acute kidney injury.

[0131] The term "isolated" refers to a cell (e.g., a canine progenitor cell) that is free from association with one or more cells in vivo, or one or more cellular components associated with the cell (e.g., a canine progenitor cell). An "enriched population" refers to a relative increase in the number of a desired cell (e.g., a canine progenitor cell) compared to one or more other cell types in vivo or in primary culture.

[0132] However, as used herein, the term "isolated" does not indicate the presence of only a particular cell (e.g., canine progenitor cell). Rather, the term "isolated" indicates that the cells (e.g., canine progenitor cell) are removed from their natural tissue environment and are present in a higher concentration compared to the normal tissue environment. Thus, an "isolated" cell population may further include cell types in addition to the particular cell (e.g., canine progenitor cell) and may include additional tissue components. This may also be expressed, for example, in terms of cell doublings. A cell (e.g., canine progenitor cell) may be capable of undergoing at least about 10, 20, 30, 40 or more doublings in vitro or ex vivo such that it is enriched compared to its original number in vivo or in its original tissue environment (e.g., bone marrow, peripheral blood, placenta, umbilical cord, umbilical cord blood, adipose tissue, etc.).

[0133] "cMAPC" is an acronym for and can be used interchangeably with "canine multipotent adult progenitor cells." Additionally, "cMAPC" can be used interchangeably with "canine progenitor cells." cMAPC refers to cells that are not embryonic stem cells or germ cells, but have many of the characteristics of these. cMAPCs can be characterized by several different characteristics, including, but not limited to, having a population doubling rate of less than about 24 hours in culture, having extended replicative capacity and normal karyotype in culture, giving rise to cell progeny of more than two cell types derived from mesoderm (e.g., osteoblasts, adipocytes, or chondrocytes) upon differentiation, and / or being postnatal somatic cells, but that they can express markers of these primitive cell types, such as nanog, sox-2, and oct-4. The expanded cMAPCs may also express one or more of parathyroid hormone-like hormone (PTHLH), CD13, CD44, CD49c, CD73, CD90, CD105, and interleukin 1 receptor type 2 (IL1R2), and may be negative for the expression of rex-1, CD34, CD45, and nephroblastoma overexpressed (NOV). In addition, the expanded cMAPCs may be surface antigen positive for CD90 and CD29, and surface antigen negative for CD45 and MHC class II. Fifth, like stem cells, cMAPCs may self-renew; i.e., have an extended replicative capacity in culture without being transformed. This means that these cells express telomerase (i.e., have telomerase activity) in culture. Sixth, the expanded MAPCs are not tumorigenic, do not form teratomas, are not transformed, and are not immortalized. Thus, the cell type designated "cMAPC" can be characterized by alternative fundamental properties that describe the cell through some of its novel properties.

[0134] The term "adult" in cMAPC is non-limiting. It refers to non-embryonic cells, such as postnatal somatic cells.

[0135] The term "musculoskeletal disorders" includes all disorders related to bones, muscles, ligaments, tendons, cartilage and joints. Treatment of musculoskeletal diseases or disorders is within the scope of regenerative medicine. For example, disorders of the vertebrae and intervertebral discs, including but not limited to disorders that require spinal fixation, spinal stabilization, repair of segmental defects of the body (such as long bones and flat bones), destruction of the intervertebral disc annulus such as annular tears, chronic inflammation of the intervertebral disc, localized disc herniation with trapped or protruding protrusion, and relative instability of the vertebrae surrounding the intervertebral disc, are musculoskeletal disorders. Musculoskeletal disorders also include sprains, strains and tears of ligaments (e.g., complete or partial cruciate ligament rupture), tendons, muscles (e.g., skeletal and cardiac muscles) and cartilage, tendonitis, tenosynovitis, fibromyalgia, osteoarthritis, rheumatoid arthritis, polymyalgia rheumatica, bursitis, and osteoporosis. Additionally, musculoskeletal disorders include genetic diseases of the musculoskeletal system as well as the musculoskeletal aspects of lysosomal storage disorders.

[0136] When referring to a protein or nucleic acid (e.g., mRNA), "negative expression" means that the protein or nucleic acid is not present in the sample and / or is not present in the sample at a level detectable by a known assay compared to a control sample.

[0137] A "pharmacologically acceptable carrier" is any pharma- ceutically acceptable medium for the canine progenitor cells used in this disclosure. Such a medium may maintain isotonicity, cell metabolism, pH, etc. It is compatible with administration to a subject in vivo and therefore can be used for cell delivery and treatment.

[0138] "Population doubling rate" refers to the amount of cell population doubling per unit time. Cell population doubling (PD) can be calculated according to the following formula: PD h =PD i +Log2(C h / C i ), in the formula, C i represents the initially seeded cells, and C hrepresents the number of cells harvested. For expanded canine progenitor cells, the population doubling rate is less than about 24 hours, e.g., about 15 to 24 hours, about 15 hours, about 16 hours (e.g., 16 hours), about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours, throughout the expansion process, e.g., from about 10 population doublings to about 55 population doublings in culture.

[0139] When referring to a protein or nucleic acid (eg, mRNA), "positive expression" means that the protein or nucleic acid is present in a sample at a level detectable by a known assay compared to a control sample.

[0140] The term "reduce" as used herein means to prevent as well as to reduce. In the context of treatment, "reduce" is either to prevent or improve one or more clinical symptoms. A clinical symptom is one (or more) that, if left untreated, will adversely affect or will affect the quality of life (health) of a subject. In the in vitro context, "reduce" is to decrease one or more analytes or biomarkers, which can be assayed and then correlated to a particular outcome or endpoint.

[0141] "Self-renewal" of stem cells refers to their ability to produce replicate daughter stem cells with the same differentiation potential as the one from which they originated.

[0142] "Subject" means an animal that is a member of the canid family, which includes wolves, jackals, foxes, coyotes, and dogs (Canis lupus familiaris). Thus, any one of the terms "dog", "canine", or "canid" can be used interchangeably when referring to the subject matter of this application. A canid can be a domestic animal, a wolf, or an animal that has some genetic contribution from two or more species of canid. Thus, a dog in this application can include any purebred or mixed breed dog.

[0143] "Substantially pure" refers to a population of canine progenitor cells (i.e., cMAPC) that is free or substantially free of other cell types. Cell purification can be achieved by any means known to those skilled in the art. For example, a substantially pure population of canine progenitor cells (i.e., cMAPC) can be achieved by growing canine progenitor cells (i.e., cMAPC) or by selecting from less pure populations. A culture of canine progenitor cells (i.e., cMAPC) is substantially pure if at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the expanded cells in the culture are canine progenitor cells (i.e., cMAPC). The presence of only a small percentage or zero percentage of other growing cell types in a culture of canine progenitor cells (i.e., cMAPC) means that the culture is a substantially pure culture of canine progenitor cells (i.e., cMAPC).

[0144] "Suppression", "inhibition" and "prevention" are used herein according to accepted definitions when used in the context of immune response. For example, "suppression" occurs when an ongoing immune response (e.g., abnormal T cell activity such as proliferation) is blocked or significantly reduced, for example, compared to the level of immune response that occurs in the absence of treatment with the cells disclosed herein. "Inhibition" refers to blocking the development of an immune response or significantly reducing such a response, for example, compared to the level of immune response that occurs in the absence of treatment with the cells disclosed herein. When administered prophylactically, such blocking can be complete so that the targeted immune response does not occur, typically referred to as "prevention" in terms of completely blocking the immune response before onset, or in the present disclosure, treatment can have a reduced effect compared to the normal untreated state, typically referred to as suppression or inhibition.

[0145] The term "therapeutically effective amount" refers to an amount of an agent (e.g., expanded canine progenitor cells) determined to produce any therapeutic response in a subject. For example, an effective anti-inflammatory therapeutic agent can prolong the survival of a subject and / or inhibit overt clinical symptoms. A therapeutically effective treatment within the meaning of the term as used herein includes a treatment that improves the quality of life of a subject, even if it does not improve the disease outcome itself. Such a therapeutically effective amount is easily ascertained by a person skilled in the art. Thus, "treat" means to deliver such an amount. In some cases, the treatment can prevent or improve any pathological symptoms of an inflammatory condition (e.g., an autoimmune disease) or a musculoskeletal disorder.

[0146] "Treat," "treating," or "treatment" are used broadly in the context of the present invention, and each such term encompasses, inter alia, preventing, ameliorating, inhibiting, or curing a deficiency, dysfunction, disease, or other deleterious process (including those that impede and / or result from treatment).

[0147] Selection and phenotype of expanded canine progenitor cells The present invention provides expanded cultured canine progenitor cells (i.e., cMAPCs) isolated from adult dogs that can differentiate to form at least two cell types of the mesoderm, such as osteoblasts, adipocytes, and chondrocytes. These cells are also capable of extensive replication in culture, exhibiting one or more of the following through approximately 55 population doublings: a population doubling rate of less than about 24 hours, a normal karyotype; and expressing markers of extensive replication (e.g., telomerase) and pluripotency (e.g., oct4).

[0148] The canine progenitor cells described herein have been isolated and expanded by the inventors, and the inventors have identified a number of specific cell surface markers and other phenotypic markers that characterize the cells.The methods described below can be used to isolate and grow canine progenitor cells from any adult canine tissue, such as bone marrow, adipose tissue, umbilical cord blood, or placental tissue.Therefore, those skilled in the art can obtain tissue from a canine and select expanded canine progenitor cells using known positive or negative selection techniques, without undue experimentation, based on the specific surface and / or genetic markers expressed (or not expressed) on these cells, as identified by the inventors.

[0149] 1. Phenotype of expanded canine progenitor cells

[0150] In one embodiment, expanded canine progenitor cells (ie, cMAPCs) are provided.

[0151] The inventors have discovered that the canine progenitor cells (i.e., cMAPCs) of the present application have a doubling rate in culture of less than about 24 hours, which is surprisingly slower than other canine progenitor cells, such as canine mesenchymal stem cells (cMSCs).Thus, in some cases, the canine progenitor cells (i.e., cMAPCs) have a population doubling rate in culture of about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours or about 24 hours, for example, less than 24 hours. In some cases, the population doubling rate is less than about 24 hours for about 10-15 population doublings in culture, about 15-20 population doublings in culture, about 20-25 population doublings in culture, about 25-30 population doublings in culture, about 30-35 population doublings in culture, about 35-40 population doublings in culture, about 40-45 population doublings in culture, about 45-50 population doublings in culture, or about 50-55 population doublings in culture.

[0152] The expanded cultured canine progenitor cells (i.e., cMAPCs) of the present application have a normal karyotype. "Karyotype" refers to the number and occurrence of chromosomes in the nucleus of a eukaryotic cell. The term is also used to indicate a complete set of chromosomes in a species or organism. Karyotype describes the number of chromosomes and their appearance under a light microscope. Karyotypes are established in terms of length, centromere position, banding pattern, and other physical characteristics. Thus, if there is no obvious aneuploidy in the chromosomes, the karyotype is considered to be normal.

[0153] Chromosomal abnormalities are established by standard procedures in the art, i.e., staining with a suitable dye such as Giemsa ("G-banding"). Such banding is obtained after limited digestion of the chromosomes with trypsin. This results in a series of faint and dark staining bands, with the dark areas tending to be heterochromatic and the light areas euchromatic. Each chromosome has a characteristic banding pattern that helps to identify it. Also, both chromosomes in a diploid nucleus will have the same banding pattern.

[0154] Chromosomal abnormalities are routinely detectable. They can be numerical, such as extra or missing chromosomes, or structural, such as translocations, inversions, large deletions, and duplications. They can be detected by a variety of routine banding techniques, such as G-banding. Occasionally, technical artifacts related to chromosomal processing can result in obvious differences between two homologs (of the same chromosome). However, these artifacts are routinely identified by analyzing an accepted number of metaphase spreads from an individual, for example, approximately 15-20. Given the level of analysis, the chances of the same technical artifact occurring repeatedly in a given specimen are very low. For a discussion of karyotyping for chromosomal abnormalities, see O'Connor, C. (2008) Karyotyping for chromosomal abnormalities. Nature Education 1(1):27.

[0155] The canine progenitor cells (i.e., cMAPCs) of the present application have a normal karyotype through about 10-15 population doublings in culture, about 15-20 population doublings in culture, about 20-25 population doublings in culture, about 25-30 population doublings in culture, about 30-35 population doublings in culture, about 35-40 population doublings in culture, about 40-45 population doublings in culture, about 45-50 population doublings in culture, or about 50-55 population doublings in culture.

[0156] Canine progenitor cells (i.e., cMAPCs) may differentiate into at least two cell types of mesoderm through about 10-15 population doublings in culture, about 15-20 population doublings in culture, about 20-25 population doublings in culture, about 25-30 population doublings in culture, about 30-35 population doublings in culture, about 35-40 population doublings in culture, about 40-45 population doublings in culture, about 45-50 population doublings in culture, or about 50-55 population doublings in culture. Mesoderm cell types into which expanded cultured canine progenitor cells (i.e., cMAPCs) of the present invention may differentiate include adipocytes, osteoblasts, and chondrocytes. In another example, canine progenitor cells (i.e., cMAPCs) may differentiate into three or more cell types of mesoderm through about 10-15 population doublings in culture, about 15-20 population doublings in culture, about 20-25 population doublings in culture, about 25-30 population doublings in culture, about 30-35 population doublings in culture, about 35-40 population doublings in culture, about 40-45 population doublings in culture, about 45-50 population doublings in culture, or about 50-55 population doublings in culture.

[0157] Canine progenitor cells (i.e., cMAPCs) are characterized by extensive replication in culture. Thus, canine progenitor cells (i.e., cMAPCs) have undergone or can undergo at least 10, at least 20, at least 30, at least 40, or at least 50 or more population doublings in culture. In one example, canine progenitor cells (i.e., cMAPCs) have undergone or can undergo 50 population doublings in culture.

[0158] The expanded canine progenitor cells (i.e., cMAPCs) are characterized by positive or negative expression of certain molecular markers, such as cell surface markers, genetic markers, and functional markers. Non-limiting examples of these markers are disclosed below. In some cases, the expanded canine progenitor cells (i.e., cMAPCs) are characterized by positive or negative expression of certain molecular markers through about 50 population doublings, such as about 30-50 population doublings, about 30-35 population doublings, about 35-40 population doublings, about 40-45 population doublings, or about 45-50 population doublings. In one example, the expanded canine progenitor cells (i.e., cMAPCs) are characterized by positive or negative expression of certain molecular markers through about 40 or 44 population doublings.

[0159] In one example, the expanded canine progenitor cells (i.e., cMAPCs) are surface antigen positive for at least one of CD90 and CD29, and / or surface antigen negative for at least one of CD34, CD45, and MHC class II.

[0160] In one example, expanded canine progenitor cells (ie, cMAPCs) are surface antigen positive for CD90 and surface antigen negative for CD34 and CD45.

[0161] In another example, the expanded canine progenitor cells (i.e., cMAPCs) are positive for expression of one or more of PTHLH, CD13, CD44, CD49c, CD73, CD90, CD105, IL1R2, nanog, oct4 and sox-2, and / or are negative for expression of rex-1, CD34, CD45, and NOV.

[0162] In another example, expanded canine progenitor cells (ie, cMAPCs) are positive for expression of IL1R2.

[0163] In another example, the expanded canine progenitor cells (i.e., cMAPCs) are positive for telomerase activity. The expanded canine progenitor cells (i.e., cMAPCs) are positive for telomerase activity through about 50 population doublings, e.g., about 20-50 population doublings, about 20-25 population doublings, about 25-30 population doublings, about 30-35 population doublings, about 35-40 population doublings, about 40-45 population doublings, or about 45-50 population doublings. In one example, the expanded canine progenitor cells (i.e., cMAPCs) are positive for telomerase activity through about 40 or 44 population doublings.

[0164] In another example, the expanded canine progenitor cells (i.e., cMAPCs) are positive for oct-4 expression. The expanded canine progenitor cells (i.e., cMAPCs) are positive for oct-4 expression through about 50 population doublings, e.g., about 20-50 population doublings, about 20-25 population doublings, about 25-30 population doublings, about 30-35 population doublings, about 35-40 population doublings, about 40-45 population doublings, or about 45-50 population doublings. In one example, the expanded canine progenitor cells (i.e., cMAPCs) are positive for oct-4 expression through about 40 or 44 population doublings.

[0165] In another example, the expanded canine progenitor cells (i.e., cMAPCs) may differentiate into at least two cell types of the mesoderm. The expanded canine progenitor cells (i.e., cMAPCs) differentiate into at least two cell types of the mesoderm through about 50 population doublings, e.g., about 20-50 population doublings, about 20-25 population doublings, about 25-30 population doublings, about 30-35 population doublings, about 35-40 population doublings, about 40-45 population doublings, or about 45-50 population doublings. In one example, the expanded canine progenitor cells (i.e., cMAPCs) differentiate into at least two cell types of the mesoderm through about 40 or 44 population doublings.

[0166] In another example, expanded canine progenitor cells (ie, cMAPCs) have the ability to reduce or inhibit T cell expansion in vivo and / or in vitro.

[0167] In another example, canine progenitor cells (ie, cMAPCs) have the ability to induce or promote angiogenesis in vivo and / or in vitro.

[0168] Thus, one skilled in the art will understand that one can ascertain the nature of the expanded canine progenitor cells (i.e., cMAPCs) and the purity of the cells based on the presence or absence of one or a combination of the markers and / or functional markers discussed above.

[0169] 2. Isolation and Expansion of Canine Progenitor Cells

[0170] In one embodiment, the canine progenitor cells (i.e., cMAPCs) of the present invention can be isolated from multiple tissue sources, including but not limited to bone marrow, placenta, umbilical cord and umbilical cord blood, muscle, brain, liver, spinal cord, blood, or skin.In one example, bone marrow aspirate is obtained from the femur or tibia of an adult canine subject using a syringe (e.g., Jamshidi needle).In an exemplary material, the canine progenitor cells (i.e., cMAPCs) are derived from bone marrow.

[0171] In some cases, cells obtained from tissue sources can be fractionated using, for example, Histopaque density centrifugation. The mononuclear fraction can be collected and the total cell count determined. The cells of the mononuclear fraction can then be inoculated at a desired density onto or into a static (e.g., protein-coated flask) or non-static culture vessel (e.g., stirred tank bioreactor) and cultured in culture medium under conditions sufficient to expand the cells. In one embodiment, the cells of the mononuclear fraction are plated onto CPPT-coated flasks. The cells of the mononuclear fraction can be cultured at approximately 500 cells / cm. 2 ~about 300,000 cells / cm 2 or more, e.g., about 100,000 cells / cm 2 ~about 250,000 cells / cm2 , about 40,000 cells / cm 2 ~about 100,000 cells / cm 2 , or approximately 2,000 cells / cm 2 ~about 5,000 cells / cm 2 In another embodiment, the cells of the mononuclear fraction can be inoculated into a non-static culture vessel, such as a hollow fiber bioreactor, at a desired density and cultured in culture medium under conditions sufficient to expand the cells. One example of a hollow fiber bioreactor is described in U.S. Patent Application Publication No. 2012 / 0308531 A1 to Pinxteren et al., and is also commercially available as the Quantum® Cell Expansion System (Terumo, BCT, Lakewood, Colorado).

[0172] The cells of the mononuclear fraction can be cultured in a culture medium containing serum and other supplements necessary for cell growth and survival (e.g., growth factors, amino acids, sugars, hormones, buffers, vitamins, etc.). In one example, the cells of the mononuclear fraction can be cultured in a static culture vessel (e.g., protein-coated flasks) using a culture medium containing the following components: about 20-60% MCDB-201 medium (e.g., about 40%); about 20-60% αMEM medium (e.g., about 35-50%); about 1-5 mM ultraglutamine (e.g., about 2 mM); about 5-20% FBS (e.g., about 10-18%); about 0.5-2x ITS (insulin-transferrin-selenium) (e.g., about 1x); about 0.1-2x LA-BSA (linoleic acid-bovine serum albumin (e.g., about 0.5x); about 5-150 µM L-ascorbic acid-2-phosphate (e.g., about 100 μM); about 5-20 ng / ml human / canine PDGF-BB (e.g., about 10 ng / ml); about 10-75 mM dexamethasone (e.g., about 50 nM); about 5-20 ng / ml canine EGF (e.g., about 10 ng / ml); and about 0.5-20 ng / ml hFGF2 (e.g., about 1-10 ng / ml).

[0173] In another embodiment, the cells of the mononuclear fraction can be cultured in a non-static culture vessel (e.g., a hollow fiber bioreactor) using a culture medium containing the following components: about 20-60% MCDB-201 medium (e.g., about 40%); about 20-60% αMEM medium (e.g., about 35-50%); about 1-5 mM ultraglutamine (e.g., about 2 mM); about 5-20% FBS (e.g., about 10-18%); about 0.5-2x ITS (insulin-transferrin-selenium) (e.g., about 1x); about 0.1-2x LA-BSA (linoleic acid-bovine serum albumin (e.g., about 0.5x); about 5-150 µM L-ascorbic acid-2-phosphate (e.g., about 100 μM); about 5-20 ng / ml human / canine PDGF-BB (e.g., about 10 ng / ml); about 10-75 mM dexamethasone (e.g., about 50 nM); about 5-20 ng / ml canine EGF (e.g., about 10 ng / ml); about 0.5-20 ng / ml hFGF2 (e.g., about 1-10 ng / ml); and about 0.5-20 ng / ml TGFβ1 (e.g., about 1-10 ng / ml).

[0174] The cells of the mononuclear fraction can be incubated in a humidified incubator under conditions sufficient to expand the cells to a desired confluence. In some cases, the cells are incubated at about 38°C for 1-2 days, 2-3 days, 3-4 days, 4-5 days, or 5 days or more. The cells are also incubated at a desired CO2 concentration (e.g., about 1-2%, about 2-3%, about 3-4%, about 4-5%, or about 5% or more) and a desired O2 concentration (e.g., about 1-10%, e.g., about 3-5%). Once the desired confluence is reached, the cells can be pulled and passaged. In some cases, cells can be lifted and passaged at less than 100% confluency, for example, about 10-20%, about 20-30%, about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-99% confluency. In one example, cells are lifted and passaged at 50-80% confluency.

[0175] In one embodiment, canine progenitor cells are isolated and expanded as follows: Bone marrow aspirate can be obtained from the femur or tibia of a dog. The bone marrow aspirate can be drawn into a syringe using a Jamshidi needle. The mononuclear fraction can be isolated by Histopaque density centrifugation. Total cell count can be determined, and the cells are then plated at approximately 100,000-250,000 cells / cm in CPPT coated flasks containing culture medium. 2 Plate at a density of 100-200 μM. The culture medium can contain the following components: about 40% MCDB-201 medium; about 35-50% αMEM medium; about 2 mM ultraglutamine; about 10-18% FBS; about 1× ITS (insulin-transferrin-selenium); about 0.5× LA-BSA (linoleic acid-bovine serum albumin); about 100 μM L-ascorbic acid-2-phosphate; about 10 ng / ml human / canine PDGF-BB; about 50 nM dexamethasone; about 10 ng / ml canine EGF; about 1-10 ng / ml hFGF2; and about 1-10 ng / ml TGFβ1. The flasks can be incubated at 38°C, 5% CO2, 5% O2 in a humidified incubator. After about 3-5 days, clonal expansion of the cells is visible. Once the clones reach about 50-80% confluence, the cells can be pulled and passaged.

[0176] The cells can then be washed with PBS and then detached from the plate using trypsin. The trypsinization reaction can be stopped by adding Dulbecco's Phosphate Buffered Saline (DPBS) to the flask. The cell solution can then be transferred to a conical tube and centrifuged at about 500 x g for about 5 minutes. The supernatant can then be removed and the cell pellet resuspended in DPBS. Cell counts can then be determined and the cells plated at about 2,000 cells / cm onto CPPT-coated flasks in culture medium as above. 2 Cells can be incubated at 38 °C, 5% CO2, 5% O2 and passaged every 2-3 days.

[0177] In one embodiment, the expanded cells are subjected to one or a combination of known positive or negative selection techniques that rely on molecular markers and / or potency (discussed above) expressed (or not) or exhibited in these cells. Thus, the expanded cultured canine progenitor cells (i.e., cMAPCs) of the present invention can be selected based on the presence and / or absence of one or more markers (as disclosed herein) and / or potency using the selection techniques discussed below.

[0178] Both positive and negative selection techniques are available to one of skill in the art, and many monoclonal and polyclonal antibodies suitable for negative selection purposes are also available in the art (e.g., Leukocyte Typing V, Schlossman et al., Eds. (1995) Oxford University Press) and commercially available from a number of sources.

[0179] Techniques for separating mammalian cells from mixtures of cell populations have also been described in Schwartz et al., U.S. Pat. No. 5,759,793 (magnetic separation), Basch et al., 1983 (immunoaffinity chromatography) and Wysocki and Sato, 1978 (fluorescence activated cell sorting, FACS).

[0180] In one embodiment, the expanded canine progenitor cells are selected that express at least one of CD90 (positive) and / or do not express at least one of CD45 and CD34 (negative).In some cases, FACS is used to detect the presence or absence of cell surface antigens.As described in the following examples, for example, FACS can be used to detect the presence or absence of CD90, CD45 and CD34, as well as MHC class II and CD29.

[0181] In another embodiment, the expanded canine progenitor cells are selected that express one or more of PTHLH, CD13, CD44, CD49c, CD73, CD90, CD105, IL1R2, nanog, oct4, and sox-2 (positive) and / or do not express rex-1, CD34, CD45, and NOV (negative). In some cases, PCR (e.g., semi-quantitative PCR, sqPCR) is used to detect the presence or absence of gene markers. As described in the following examples, for example, sqPCR can be used to detect the presence or absence of PTHLH, CD13, CD44, CD49c, CD73, CD90, CD105, IL1R2, nanog, oct4, sox-2, rex-1, CD34, CD45, and NOV.

[0182] In another embodiment, the expanded canine progenitor cells positive for telomerase activity are selected.The assay for detecting telomerase activity is known in the art.It is important that for any assay of telomerase activity, positive and negative controls must be included.In one example, telomerase activity can be determined using a commercially available kit, such as TRAPEze RT telomerase detection kit (Merck), as described in the following examples.

[0183] In another embodiment, the expanded canine progenitor cells are selected to have the ability to reduce or inhibit T cell proliferation in vivo and / or in vitro. The ability of cells to inhibit or reduce T cell proliferation can be determined using immunocompetence assays, examples of which are known in the art. An example of an immunocompetence assay used to determine the ability of cells to reduce or inhibit T cell proliferation is provided in the following examples. Other examples of immunocompetence assays, such as mixed lymphocyte reaction (MLR), are disclosed in Maziarz et al., US Patent Application Publication No. 2006 / 0263337 A1.

[0184] In another embodiment, the expanded cultured canine progenitor cells are selected to have the ability to induce or promote angiogenesis in vivo and / or in vitro.The assay for determining the ability of cells to induce or promote angiogenesis is known in the art.In one example, as described in Woda et al., US Patent Application Publication No. 2014 / 0242629 A1 and in the following examples, HUVEC tube formation assay can be used to determine the ability of cells to induce or promote angiogenesis.

[0185] In some embodiments, the purity of the selected and expanded canine progenitor cells (i.e., cMAPCs) is about 100% (substantially pure). In other embodiments, it is 95%-100%. In some embodiments, it is 85%-95%. In further embodiments, the percentage can be about 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%, 80%-90%, or 90%-95%. In another embodiment, the purity can be expressed in terms of cell doublings, where the canine cells (i.e., cMAPCs) have undergone, for example, 1-5, 5-10, 10-20, 20-30, 30-40, 40-50 or more cell doublings in culture. In one example, the canine progenitor cells (ie, cMAPCs) have undergone at least 40, preferably at least 50 population doublings in culture.

[0186] The selected canine progenitor cells (ie, cMAPCs) may be further cultured in static or non-static culture vessels as described above.

[0187] composition In one embodiment, a composition may comprise expanded cultured canine progenitor cells (i.e., cMAPCs) of the present invention and a second component (e.g., an additive, vehicle or carrier, such as a culture medium or multiple culture media).

[0188] In another embodiment, the expanded canine progenitor cells (ie, cMAPCs) of the present invention may be formulated as a pharmaceutical composition.

[0189] No. 7,015,037 is incorporated by reference for teaching pharmaceutical formulations. In certain embodiments, the expanded cultured canine progenitor cells (i.e., cMAPCs) of the present invention are present in a composition that is suitable and suitable for delivery, i.e., physiologically compatible.

[0190] In some embodiments, the purity of the expanded cultured canine progenitor cells (i.e., cMAPCs) for administration to a subject is about 100% (substantially pure). In other embodiments, it is 95%-100%. In some embodiments, it is 85%-95%. In particular, in the case of an admixture with other cells, the percentage can be about 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%, 80%-90%, or 90%-95%. Alternatively, the isolation / purity can be expressed in terms of cell doublings, where the canine cells have undergone, for example, 1-5, 5-10, 10-20 or more cell doublings in culture.

[0191] The choice of formulation for administering the expanded cultured canine progenitor cells (i.e., cMAPCs) of the present invention for a given application will depend on a variety of factors. Prominent among these will be the subject's species, the nature of the inflammatory condition or musculoskeletal disorder being treated, its condition and distribution in the subject, the nature of other therapies and agents being administered, the optimal route for administration, survivability through the route, administration regimen, and other factors that will be apparent to those skilled in the art. For example, the selection of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form.

[0192] The final formulation of an aqueous suspension of expanded cultured canine progenitor cells (i.e., cMAPCs) / medium typically involves adjusting the ionic strength of the suspension to isotonic (i.e., about 0.1-0.2) and physiological pH (i.e., pH about 6.8-7.5). The final formulation also typically contains a fluid lubricant.

[0193] In some embodiments, the expanded cultured canine progenitor cells (i.e., cMAPCs) of the present invention are formulated in an injectable unit dosage form, such as a solution, suspension or emulsion.The pharmaceutical preparation suitable for cell injection is typically a sterile aqueous solution and dispersion.The carrier for the injectable preparation may be, for example, a solvent or dispersion medium containing water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0194] One of skill in the art can readily determine the amount of expanded cultured canine progenitor cells (i.e., cMAPCs) and optional additives, vehicles and / or carriers in the compositions administered in the methods of the present invention. Typically, any additives (in addition to the cells) are present in an amount of 0.001 to 50% by weight in a solution such as phosphate buffered saline. The active ingredient is present in the order of micrograms to milligrams, for example, about 0.0001 to about 5% by weight, preferably about 0.0001 to about 1% by weight, most preferably about 0.0001 to about 0.05% by weight or about 0.001 to about 20% by weight, preferably about 0.01 to about 10% by weight, most preferably about 0.05 to about 5% by weight.

[0195] In some embodiments, the expanded canine progenitor cells (i.e., cMAPCs) of the present invention are encapsulated for administration, especially when encapsulation enhances the efficacy of treatment or provides advantages in handling and / or shelf life. The expanded canine progenitor cells (i.e., cMAPCs) can be encapsulated by membranes and capsules before transplantation. It is contemplated that any of the many available cell encapsulation methods can be used.

[0196] A wide variety of materials may be used in various embodiments for microencapsulation of canine cells, including, for example, polymer capsules, alginate-poly-L-lysine-alginate microcapsules, poly-L-lysine alginate barium capsules, barium alginate capsules, polyacrylonitrile / polyvinyl chloride (PAN / PVC) hollow fibers, and polyethersulfone (PES) hollow fibers.

[0197] Techniques for microencapsulation of cells that can be used for administration of cells are known to those of skill in the art and are described, for example, in Chang, P et al., 1999; Matthew, HW et al., 1991; Yanagi, K. et al., 1989; Cai ZH et al., 1988; Chang, TM, 1992 and U.S. Pat. No. 5,639,275 (describes, for example, biocompatible capsules for the long-term maintenance of cells stably expressing biologically active molecules). Further methods of encapsulation are described in European Patent Application Publication No. 301,777 and U.S. Patent Nos. 4,353,888; 4,744,933; 4,749,620; 4,814,274; 5,084,350; 5,089,272; 5,578,442; 5,639,275; and 5,676,943, all of which are incorporated herein by reference in their relevant portions with respect to encapsulation of cells.

[0198] Certain embodiments incorporate expanded canine progenitor cells (i.e., cMAPCs) into a polymer, such as a biopolymer or synthetic polymer. Examples of biopolymers include, but are not limited to, fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. Other factors, such as cytokines discussed above, can also be incorporated into the polymer. In other embodiments of the present invention, expanded canine progenitor cells (i.e., cMAPCs) can be incorporated into the interstices of a three-dimensional gel. Typically, large polymers or gels are surgically implanted. Polymers or gels that can be formulated into sufficiently small particles or fibers can be administered by other common, more convenient, non-surgical routes.

[0199] The dosage of expanded canine progenitor cells (i.e., cMAPC) will vary within a wide range and will be adapted to the individual requirements in each specific case. The number of cells will vary according to the weight and condition of the recipient, the number or frequency of administration, and other variables known to those skilled in the art. Expanded canine progenitor cells (i.e., cMAPC) can be administered by a route suitable for tissue or organ. For example, expanded canine progenitor cells (i.e., cMAPC) can be administered by systemic administration, i.e., intravenous administration, or can be administered intrathecally to target specific tissue or organ, such as the brain or spinal cord.

[0200] The expanded cultured canine progenitor cells of the present invention (i.e., cMAPCs) are cultured at a concentration of about 0.01 to about 5 × 10 6 The cells can be suspended in a suitable excipient at a concentration of cells / ml or more. Suitable excipients for injection solutions are those that are biologically and physiologically compatible with the cells and the recipient, such as buffered saline or other suitable excipients. The compositions for administration can be formulated, manufactured, and stored according to standard methods that meet the appropriate sterility and stability requirements.

[0201] Administration The dosage for a canine subject can be determined without undue experimentation by those skilled in the art from this disclosure, the documents cited herein, and knowledge in the art. The dosage of expanded cultured canine progenitor cells (i.e., cMAPCs) suitable for use according to various embodiments of the present invention will depend on a number of factors. The parameters that determine the optimal dosage to be administered for primary and adjunctive therapy generally include some or all of the following: the inflammatory or musculoskeletal condition being treated and its stage; the subject's health, sex, age, weight, and metabolic rate; the subject's immune competency; other therapies being administered; and potential complications expected from the subject's medical history or genotype. Parameters may also include whether the expanded cultured canine progenitor cells (i.e., cMAPCs) are syngeneic, autologous, allogeneic, or xenogeneic; the site and / or distribution that the cells / media must target to be effective; and such characteristics of the site, such as the accessibility of the cells / media and / or the engraftment of the cells. Further parameters include co-administration with other factors (such as growth factors and cytokines). The optimal dose in a given situation will also take into account how the expanded canine progenitor cells (i.e., cMAPCs) are formulated, how they are administered, and the extent to which the expanded canine progenitor cells localize to the target site following administration.

[0202] In various embodiments, the expanded canine progenitor cells (i.e., cMAPC) of the present invention can be administered at an initial dose and then maintained by further administration. The expanded canine progenitor cells (i.e., cMAPC) can be administered initially by one method and then by the same method or one or more different methods. Levels can be maintained by continued administration of the expanded canine cells (i.e., cMAPC). Various embodiments administer the expanded canine progenitor cells (i.e., cMAPC) initially, maintain their levels in the subject, or both by intravenous injection. In various embodiments, other forms of administration are used depending on the subject's condition and other factors, as discussed elsewhere herein.

[0203] The expanded canine progenitor cells (i.e., cMAPCs) can be administered at many frequencies over a wide range of times. Generally, the length of treatment will be proportional to the length of the disease process, the effectiveness of the therapy being applied, and the condition and response of the subject being treated.

[0204] use Administering the expanded canine progenitor cells (i.e., cMAPCs) of the present invention is useful for reducing any of the overt symptoms of inflammatory conditions or musculoskeletal disorders described in this application. This may be based on the underlying effects of the cells, such as treating inflammatory conditions (e.g., autoimmune diseases) by reducing or inhibiting abnormal T cell proliferation; treating ischemic conditions (e.g., myocardial infarction) by promoting angiogenesis; treating musculoskeletal disorders (e.g., cruciate ligament rupture) by differentiating into connective tissues such as ligaments, tendons, bones, muscles, and cartilage.

[0205] In one example, the compositions and methods disclosed herein relate to treating an inflammatory condition in a subject by administering an expanded canine progenitor cell (i.e., cMAPC) composition disclosed herein. In some embodiments, the subject is suffering from an autoimmune disease, and the cell composition is used to treat the autoimmune disease.

[0206] In another example, the compositions and methods disclosed herein relate to treating a musculoskeletal disorder in a subject by administering the expanded canine progenitor cell (i.e., cMAPC) composition disclosed herein. In one embodiment, the subject suffers from osteoarthritis, and the expanded canine progenitor cell (i.e., cMAPC) composition is used to treat the osteoarthritis. In another embodiment, the subject suffers from cruciate ligament rupture, and the expanded canine progenitor cell (i.e., cMAPC) composition is used to treat the cruciate ligament rupture. In yet another embodiment, the subject suffers from a spinal cord condition, and the expanded canine progenitor cell (i.e., cMAPC) composition is used to treat the spinal cord condition.

[0207] In another example, the compositions and methods disclosed herein relate to treating an ischemic condition in a subject by administering the expanded cultured canine progenitor cell (i.e., cMAPC) composition disclosed herein. In one embodiment, the subject suffers from acute myocardial infarction, chronic heart failure, peripheral vascular disease, stroke, chronic total occlusion, renal ischemia, and / or acute kidney injury, and the expanded cultured canine progenitor cell (i.e., cMAPC) composition is used to treat acute myocardial infarction, chronic heart failure, peripheral vascular disease, stroke, chronic total occlusion, renal ischemia, and / or acute kidney injury.

[0208] The compositions disclosed herein are used to treat, alleviate or ameliorate symptoms, or inhibit a wide variety of inflammatory conditions, ischemic conditions, and musculoskeletal disorders, such as those described above.

[0209] In some embodiments, the expanded cultured canine progenitor cells (i.e., cMAPCs) of the present invention can be used to deliver inhibitory or other biological factors, including but not limited to, cytokines, stem cell growth factors, and angiogenesis regulators, to a site of disease or injury (e.g., a site of inflammation or ischemia). For example, in some embodiments, the expanded cultured canine progenitor cells (i.e., cMAPCs) can be transduced with a gene encoding a desired biological factor, which the cells then produce once within the subject, e.g., at the site of inflammation or ischemia.

[0210] In some embodiments, the expanded cultured canine progenitor cell (i.e., cMAPC) compositions disclosed herein can be used to treat, for example, infectious diseases where the pathogenesis of infection is not the result of the cytopathic effect of the pathogen, but rather the result of tissue damage caused by an immune inflammatory response to the infectious agent. In diseases such as hepatitis B or C or HSV-induced corneal inflammation, treatment with the expanded cultured canine progenitor cell (i.e., cMAPC) disclosed herein provides a unique opportunity to control virus-induced immune inflammatory diseases. Viruses such as Coxsackie are known to cause pancreatitis and are associated with the development of type 1 diabetes. Thus, the expanded cultured canine progenitor cell (i.e., cMAPC) compositions as disclosed herein can be used to inhibit local tissue damage caused by infection and reduce inflammation that induces the development of autoimmune disorders.

[0211] The subject method is used to treat a variety of different conditions and transplant situations. In order to maintain the expanded canine progenitor cells (i.e., cMAPC) at the site until the completion of the surgical procedure, in some embodiments, it is convenient to administer the expanded canine progenitor cells (i.e., cMAPC) in a pharma- ceutically acceptable carrier, such as an artificial gel, or in clotted plasma, or by utilizing other controlled release mechanisms known in the art.

[0212] Further, other uses provided by the present invention include screening one or more agents or compounds for their ability to affect the capacity or efficacy of expanded canine progenitor cells (i.e., cMAPCs) to provide one or more of the effects discussed above, such as angiogenesis, and reduce or inhibit T cell expansion in vitro and / or in vivo. Such screening methods include (i) contacting expanded canine progenitor cells (i.e., cMAPCs) with an agent or compound, and (ii) evaluating the capacity or efficacy of the cells to have an effect. Such agents include, but are not limited to, small organic molecules, antisense nucleic acids, siRNAs, DNA aptamers, peptides, antibodies, non-antibody proteins, cytokines, chemokines, and chemoattractants. The agent can then be used to increase the capacity or efficacy of expanded canine progenitor cells (i.e., cMAPCs) to achieve the evaluated effect. Evaluation can be in vivo or in vitro. In one example, a HUVEC tube formation assay is used to screen for agents that modulate the ability of expanded canine progenitor cells (e.g., cMAPCs) to provide angiogenesis in vivo and / or in vitro. In another example, an in vitro proliferation assay (e.g., MLR) is used to screen for agents that modulate the ability of expanded canine progenitor cells (i.e., cMAPCs) to inhibit or reduce T cell proliferation in vitro and / or in vivo.

[0213] A further application of the present invention is the establishment of a cell bank to provide expanded canine progenitor cells (i.e., cMAPCs) for clinical administration. The construction of a cell bank can be performed by preparing and expanding canine progenitor cells (i.e., cMAPCs) as described herein, and then storing expanded cells from the population for future administration to subjects. The expanded canine cells (i.e., cMAPCs) can be used directly from the bank or expanded before use.

[0214] Therefore, the present invention also relates to a diagnostic procedure carried out before administering these cells to a subject, which comprises evaluating the efficacy or ability of the cells to achieve one or more of the effects discussed above and / or to show one or more of the genotypic or phenotypic markers discussed above. The expanded canine progenitor cells (i.e., cMAPCs) can be taken from a cell bank and used directly or expanded before administration. In either case, the expanded canine progenitor cells (i.e., cMAPCs) are evaluated for the efficacy or ability of the cells to achieve one or more of the effects and / or to show one or more genotypic or phenotypic markers.

[0215] Although the expanded cultured canine progenitor cells (i.e., cMAPCs) selected for efficacy are necessarily assayed during the selection procedure, it may be preferable and advisable to assay the cells again before administering them to a subject for treatment to ensure that the cells are still effective at the desired level. This is particularly preferable if the expanded cultured canine progenitor cells (i.e., cMAPCs) have been stored for any period of time, such as in a cell bank, where the cells will most likely be frozen during storage.

[0216] For treatment methods with expanded canine progenitor cells (i.e., cMAPC), there may be multiple (i.e., sequential) assays to ensure that the cells can still achieve the desired level of efficacy and / or exhibit one or more genotypic or phenotypic markers after manipulations performed within this time frame between the initial isolation of the cells and administration to the subject. For example, assays may be performed after each expansion of the canine progenitor cells (i.e., cMAPC). If the expanded canine progenitor cells (i.e., cMAPC) are stored in a cell bank, they may be assayed after release from storage. If frozen, they may be assayed after thawing. If the expanded canine progenitor cells (i.e., cMAPC) are subsequently expanded one or more times from a cell bank, they may be assayed after (each) expansion. Preferably, a portion of the final cell product (that is physically administered to the subject) may be assayed.

[0217] In another embodiment, the expanded canine progenitor cells (i.e., cMAPCs) of the present invention can be provided in a kit with suitable packaging materials.For example, the kit can include the following separately packaged components: expanded canine progenitor cells (i.e., cMAPCs); medium or mediums; and instructions for culturing expanded canine progenitor cells (i.e., cMAPCs).In some cases, expanded canine progenitor cells (i.e., cMAPCs) can be provided as frozen stocks with suitable factors and media separately packaged for culturing as described herein.

[0218] The present invention also provides a kit that contains an effective amount of suitable factors for isolating and culturing canine progenitor cells.For example, when obtaining bone marrow aspirate from a dog, the technician only needs to select canine progenitor cells using the method described herein with suitable reagents provided in the kit, and then use the culture medium provided as kit components to culture the cells as described by the method of the present invention.The composition of the culture medium is described herein.

[0219] All patent and scientific literature cited herein is incorporated by reference for its teachings.

[0220] The following examples are for illustrative purposes only and are not intended to limit the scope of the claims appended hereto. EXAMPLES

[0221] Experiments were conducted to characterize the novel canine progenitor cells of the present application, hereinafter referred to as "Canine multi-potent adult progenitor cells" or "cMAPCs."

[0222] cMAPC isolation

[0223] Bone marrow aspirates were obtained from the femur or tibia of young donor dogs under informed consent. Bone marrow aspirates were drawn into a syringe using a Jamshidi needle. The mononuclear fraction was isolated by Histopaque density centrifugation. Total cell numbers were determined (NC-200, Chemometec) and cells were plated in protein-coated flasks at 100,000–250,000 cells / cm. 2The culture medium had the following composition: 40% MCDB-201 medium (Sigma), 35-50% αMEM medium (Lonza), 2 mM ultraglutamine (Lonza), 10-18% FBS (Gibco), 1×ITS (insulin-transferrin-selenium, Lonza), 0.5×LA-BSA (linoleic acid-bovine serum albumin, Sigma), 100 μM L-ascorbic acid-2-phosphate (Sigma), 10 ng / ml human / canine PDGF-BB (Biotechne / KingfisherBiotech), 50 nM dexamethasone (Sigma), 10 ng / ml canine EGF (Sino Biological), 1-10 ng / ml hFGF2 (Biotechne), and 1-10 ng / ml TGFβ1 (Biotechne). Flasks were incubated at 38 °C, 5% CO2, 5% O2 in a humidified incubator, and clonal expansion of cMAPCs was visible after 3–5 days. When clones reached 50–80% confluency, cells were harvested and passaged.

[0224] cMAPC cell culture

[0225] The cells were washed with PBS and then detached from the plate using 1x TrypLE Select (Gibco). The trypsinization reaction was stopped by adding DPBS to the flask. The cell solution was transferred to a conical tube and centrifuged at 500xg for 5 min. The supernatant was then removed and the cell pellet was resuspended in DPBS. The cells were counted and the cells were resuspended at 2,000 cells / cm. 2 Cells were seeded onto protein-coated flasks at a density of 1000 x g / ml. The composition of the culture medium was as described above. Cells were incubated at 38 °C, 5% CO2, 5% O2, and passaged every 2–3 days.

[0226] As cells are counted every passage, a growth curve showing population doublings can be generated using the following formula: PD h =PD i +Log2(C h / C i ), in the formula, PD his the population doubling at the time of recovery, and PD i is the initial population doubling at seeding, and C h is the cell number at harvest, and C i is the initial cell number seeded. When cells were expanded to senescence, cMAPCs were able to exceed 40 population doublings, whereas canine mesenchymal stem cells (cMSCs) began senescence already after 20 population doublings (Figure 1). Tables 1A and 1B show the doubling rate / time, the number of hours required for one population doubling, for cMAPCs and cMSCs, respectively, by passage. [Table 1A] [Table 1B]

[0227] Canine mesenchymal stem cell (cMSC) isolation

[0228] The mononuclear fraction from fresh bone marrow aspirates was isolated using Ficoll density centrifugation. Isolated mononuclear cells were plated at 200,000–350,000 cells / cm on tissue culture treated plastic. 2 The cells were seeded at a density of 100-200 μg / ml. Clonal expansion of cMSCs was visible after 3–5 days. When clones reached 50–80% confluence, the cells were harvested and passaged.

[0229] cMSC culture

[0230] T75 tissue culture treated flasks were used for cMSC expansion. After removing the medium from the flasks, each flask was washed with 5 ml of PBS. 4 ml of 1x Tryple Select was then added to each flask. The flasks were incubated at room temperature for 2-5 minutes. If all the cells did not detach, the flasks were gently tapped. 5 ml of DPBS was then added to each flask and the contents were transferred to a conical tube. The tubes were centrifuged at 500 x g for 5 minutes. After removing the supernatant from each tube and resuspending, the cells were counted. The cells were then plated at 5,000 cells / cm in a T75 culture flask in 10 ml of commercial MSC medium (e.g., Lonza). 2 The flasks were incubated at 38° C., 5.5% CO2 and 20% O2.

[0231] Flow cytometry

[0232] Immunophenotypic analysis of cMAPCs shows that the cells express CD29 (Biolegend) and CD90 (eBioscience) and are negative for CD45 (Serotec) and HLA class II (eBioscience) (Figure 2A-D). As an isotype control, non-specific IgG (Becton Dickinson or Bio-Rad) was used instead of the primary antibody. cMAPCs were diluted in FACS buffer (PBS + 2% BSA) to a concentration of 1E+06 cells / ml. 100 μl of cell sample was used for each staining. After adding the antibodies, the cells were incubated for 30 min on ice and in the dark. The cells were then washed by adding 2 ml of FACS buffer and centrifuged at 1000 × g for 5 min. The supernatant was decanted and the cells were resuspended. The cells were measured on a FACS Celesta (Becton Dickinson).

[0233] Marker analysis by PCR

[0234] Total RNA is isolated from 100,000-500,000 cells using the HighPure RNA Isolation Kit (Roche) according to the manufacturer's instructions. RNA concentration is measured using Nanodrop, and 250ng-500ng of RNA is used as template to synthesize cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions. The resulting cDNA is diluted 5-10 times.

[0235] The primers used for transcription of the selected gene set are shown in Table 2 below. [Table 2]

[0236] Ribosomal protein L8 (RPL8) was used as a reference gene. GeNorm analysis confirmed that this gene was stably expressed in all cMAPC and cMSC conditions. RPL8 was used to confirm the quality of the cDNA.

[0237] For CD marker gene expression analysis, 5 μl of diluted cDNA was taken and qPCR reaction was performed using the LightCycler 480 SYBRGreen 1 kit (Roche) according to the manufacturer's instructions. The primer sequences of the selected surface markers are shown in Table 3. [Table 3]

[0238] The temperature program was 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The mRNA expression levels were based on Cq (quantification cycle) values. Markers with Cq values ​​less than 35 were considered positive, and those greater than 35 were considered negative. Melting curve analysis was performed to test primer-dimer formation and amplicon specificity. The results are shown in Figure 3, where the red dotted line is the lower limit of detection, CD34 and CD45 were undetectable, and CD13, CD44, CD49c, CD73, CD90, and CD105 showed good expression.

[0239] Markers specific for cMAPC and cMSC were analyzed by semi-quantitative PCR. 5 μl of diluted cDNA was used to perform PCR reactions using the iTaq DNA polymerase kit (BioRad) according to the manufacturer's instructions. The following program was used: initial denaturation at 95°C for 2 min, followed by 28–32 cycles of amplification (95°C for 15 s, 60°C for 15 s, 72°C for 30 s); and a final extension step at 72°C for 5 min.

[0240] Transcription of pluripotency genes was analyzed in a PCR reaction (iTaq DNA Polymerase Kit (BioRad)) using 5 μl of diluted cDNA and the following program: initial denaturation at 95 °C for 2 min, followed by 40-45 cycles of amplification (15 s at 95 °C, 15 s at 60 °C, 30 s at 72 °C); and a final extension step at 72 °C for 5 min. The resulting PCR products of cMAPC markers, cMSC markers, and pluripotency markers were separated on a 2% agarose gel (Invitrogen) to assess the presence and specificity of the products (NANOG (Figure 4A), Oct4 (Figure 4B), SOX2 (Figure 4C)). Bands were stained by immersing the gel in water containing GelRed Nuclear Acid Gel Stain (Biotium). Photographs of the gel were taken using a BioRad Chemidoc XRS.

[0241] Telomerase activity

[0242] Telomerase activity of cMAPC was measured using TRAPeze RT Telomerase Detection Kit (Merck). Briefly, 1,000,000 cells were lysed in 100 μl of CHAPS buffer and incubated on ice for 30 min. Samples were centrifuged at 12,000 × g to remove cell debris. 2 μl of sample was added to the reaction solution. The reaction solution was incubated at 37 ° C for 90 min. During this step, active telomerase in the sample started to elongate the telomere template. Telomerase was then inactivated by incubating the sample at 95 ° C for 5 min. The generated telomeres were finally quantified by real-time qPCR (Figure 5). cMAPC showed significant telomerase activity even at more than 25 population doublings; whereas cMSC showed no telomerase activity at more than 23 population doublings.

[0243] Adipogenic lineage cell differentiation

[0244] Cells were cultured at 40,000 cells / cm in control medium. 2 The cells were seeded at a density of 100 μg / l and cultured for 24 h. The composition of the adipogenic control medium was DMEM high glucose (4.5 g / l; Lonza), 1% L-glutamine (Lonza), and 3% FBS. The medium was then replaced with adipogenic differentiation medium (adipogenic control medium supplemented with 1 μM dexamethasone, 0.5 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma), 2 μM bovine pancreatic insulin (Sigma), 33 μM biotin (Sigma), 17 μM pantothenate (Sigma), 5 μM rosiglitazone (Sigma), and 5% rabbit serum (Thermo Scientific)). The medium was replaced twice a week, and the cells were harvested after 9 days. Lipid droplets were visualized by Oil Red O (ORO) staining. The cells were fixed with citrate-buffered acetone solution (Sigma) for 30 seconds and stained with ORO (Sigma) for 10 minutes at 37° C. (FIG. 6A).

[0245] osteogenic differentiation

[0246] Cells were plated at 40,000 cells / cm in control medium (DMEM high glucose, 1% L-glutamine and 5% FBS). 2 and cultured for 24 h. The medium was then changed to osteogenic differentiation medium consisting of osteogenic control medium supplemented with 50 μM L-ascorbic acid-2P, 50 nM dexamethasone, and 10 mM β-glycerophosphate (Sigma). The medium was changed twice a week, and cells were harvested after 7 days for alkaline phosphatase (ALP) staining. For staining, cells were fixed using a citrate-buffered acetone solution (Sigma), protected from light, and stained with alkaline dye mixture (Sigma) at 37 °C for 10 min (Figure 6B).

[0247] chondrogenic differentiation

[0248] Cells were seeded at a density of 300,000 cells in 15 ml conical tubes containing 1 ml total volume of chondrogenic control or differentiation medium and centrifuged at 350 × g for 5 min. The composition of chondrogenic control medium was DMEM high glucose with 10% FBS. The composition of chondrogenic basal medium was DMEM high glucose, 0.625 × ITS, 100 nM dexamethasone, 125 μM L-ascorbic acid-2P, 2 mM L-glutamine, 1.25 × LA-BSA, 400 μg / ml proline (Sigma) and 1 mg / ml sodium pyruvate (Sigma). The following chondrogenic inducers were added to the medium: 10 ng / ml transforming growth factor β1 (TGF-β1) and bone morphogenetic protein 2 (BMP2; Biotechne). The medium was changed every 3–4 days and pellets were harvested for Alcian blue staining after 24 days. Frozen tissue blocks were prepared from the pellets. 5 μm cryosections were cut and mounted on glass slides. Slides were first stained with 0.5% Alcian Blue solution (Sigma) for 30 min, followed by Nuclear Fast Red (Vector Labs) staining for 5 min (Figure 6C).

[0249] Immunocompetence assay

[0250] Immunocompetence assays were performed in 96-well round-bottom plates. 100,000 canine PBMCs (peripheral mononuclear blood cells) were added to each well in which cMAPCs were seeded at serial dilutions ranging from 1:2 to 1:16. cPBMCs were stimulated with 0.5 μg / ml ConA (concavalin A; Sigma) and assays were analyzed 4 days later. Briefly, plates were centrifuged at 1000 × g for 5 min at room temperature. The supernatant was aspirated and cells were resuspended in DPBS-FACS buffer, after which the plates were centrifuged again (1000 × g, 5 min, room temperature). The DPBS-FACS buffer was removed and a mixture of CD3 / IgG2b antibodies (Abcam) was added. The mixture was incubated for 30 min at 4 °C in the dark. The plates were then centrifuged (1000 × g, 5 min, room temperature), the antibody mixture was removed and the cells were washed with DPBS-FACS buffer. The plates were centrifuged, the supernatant was removed, and a mixture of GAM (goat anti-mouse)-APC antibody and 7-ADD (7-aminoactinomycin D; Becton Dickinson) was added. The mixture was incubated for 15 min at 4° C. in the dark. The cells were washed with DBPS-FACS buffer, centrifuged (1000×g, 5 min, room temperature), resuspended in DBPS-FACS buffer, and the plates were measured using a FACS Celesta (Becton Dickinson). As shown in FIG. 7, cMAPC could substantially inhibit or reduce T cell proliferation.

[0251] Luminal formation (angiogenic ability)

[0252] Briefly, 55,000 human umbilical vein endothelial cells (hUVECs) were seeded in 24-well plates coated with Matrigel (Corning). Conditioned medium from cMAPCs was added and each well was photographed approximately 18 hours later. The number of tubes formed was counted for each well. Statistical analysis between conditions was performed using one-way ANOVA. Conditioned medium from cMAPCs was able to induce tube formation (Figure 8).

[0253] Cytogenetic analysis

[0254] Cells were prepared for cytogenetic analysis by G-Banding. Briefly, for colcemid treatment of cMAPCs, demecolcine (0.1 μg / ml; Sigma) and ethidium bromide (10 μg / ml; Sigma) were added to the medium, followed by an incubation step at 37°C for 1 h. . Afterwards, for hypotonic treatment, hypotonic solution (Rainbow Scientific) was added to the medium and incubated at 37°C for 40 min. Cells were then harvested and transferred to a 15 ml conical tube. Cells were fixed by adding 500 μl of fixative (methanol:acetic acid; 3:1) per 10 ml of supernatant. The solution was centrifuged at 500 × g for 10 min, after which the supernatant was removed, the cell pellet was loosened and ice-cold methanol:acetic acid fixative was added up to 15 ml. Cells were gently mixed well in the fixative. This fixation step was repeated three times. One ml of the cell suspension (after removal of the supernatant) was transferred to a microcentrifuge tube, then filled with fixative and karyotype analysis was performed by G-banding in an external laboratory (Figure 9).

[0255] Microarrays

[0256] RNA was extracted from frozen cell pellets, and quality control of each sample and quantification of the corresponding RNA were performed on an Agilent BioAnalyzer. RNA was analyzed on Affymetrix Canine Genome 2.0 arrays (Affymetrix). RMA normalization and differential expression analysis between different conditions were performed using R language with Oligo and Limma packages (Bioconductor). Differential expression was calculated using moderated t-statistics with a Bayesian adjusted denominator. Using dendrograms (Figure 10A) and PCA (principal component analysis) plots (Figure 10B), we showed that cMAPCs and cMSCs formed two separate clusters and could be considered as two distinct cell populations based on total gene expression.

[0257] In another microarray experiment to confirm the cMAPC and cMSC specific markers identified from the microarray, RNA was extracted from cMAPC (M) and cMSC (S) from three different donors. The RNA was converted to cDNA. The cDNA was used to perform PCR for the genes IL1R2 (Figure 11A) and NOV (Figure 11B). The primer sequences for the marker genes are shown in Table 4. [Table 4]

[0258] The PCR products were then loaded onto a 2% agarose gel to visualize gene expression. It could be confirmed that cMAPCs show expression of the gene IL1R2, but this expression was not observed in cMSCs. Expression of NOV was confirmed in cMSCs but not in cMAPCs. Ribosomal protein L8 (RPL8) (Figure 11C) was used as a reference gene. GeNorm analysis confirmed that this gene was stably expressed in all conditions for MAPCs and MSCs. Gel staining of RPL8 confirmed that equal amounts of cDNA were loaded onto the gel for all conditions.

Claims

1. Expanded cultured canine progenitor cells that have a population doubling rate of less than about 24 hours, have a normal karyotype, are capable of differentiating into at least two cell types of the mesoderm, and are postnatal somatic cells.

2. Expanded canine progenitor cells that have a population doubling rate of less than about 24 hours, have a normal karyotype, express telomerase, and are postnatal somatic cells.

3. Expanded canine progenitor cells that have a population doubling rate of less than about 24 hours, have a normal karyotype, express oct-4, and are postnatal somatic cells.

4. Expanded canine progenitor cells having a population doubling rate of less than about 24 hours, having a normal karyotype, having undergone at least 40 population doublings in culture, and being postnatal somatic cells.

5. The expanded canine progenitor cells of any one of claims 1 to 3, wherein the cells have undergone at least 40 population doublings in culture.

6. 6. The expanded canine progenitor cells of claim 5, wherein the cells have undergone at least 50 population doublings in culture.

7. The expanded canine progenitor cells of any one of claims 1 to 4, having a population doubling rate in culture of about 15 to 24 hours.

8. The expanded canine progenitor cells of any one of claims 1 to 4, having a population doubling rate in culture of about 16 hours.

9. The expanded canine progenitor cells according to any one of claims 1 to 4, which are derived from bone marrow, adipose tissue, umbilical cord blood, or placental tissue.

10. The expanded canine progenitor cells according to any one of claims 1 to 4, which are positive for CD90 expression and negative for CD45 and CD34 expression.

11. The expanded canine progenitor cells according to any one of claims 1 to 4, which are positive for CD29 expression.

12. The expanded canine progenitor cells according to any one of claims 1 to 4, which are negative for the expression of MHC class II.

13. 5. The expanded canine progenitor cells of any one of claims 1 to 4, which are positive for expression of one or more of PTHLH, CD13, CD44, CD49c, CD73, CD105, and IL1R2.

14. The expanded canine progenitor cells according to any one of claims 1 to 4, which are positive for IL1R2 expression.

15. The expanded canine progenitor cells according to any one of claims 1 to 4, which are negative for the expression of rex-1 and NOV.

16. The expanded canine progenitor cells according to any one of claims 1 to 2 and 4, which are positive for the expression of nanog, sox-2 and oct-4.

17. The expanded canine progenitor cells of any one of claims 1 to 4, which express telomerase up to about 55 population doublings in culture.

18. The expanded canine progenitor cells of any one of claims 1 to 4, which express oct-4 for up to about 55 population doublings in culture.

19. 5. The expanded canine progenitor cells of claim 1, which differentiate into at least two cell types of the mesoderm for up to about 55 population doublings in culture.

20. The expanded canine progenitor cells of any one of claims 1 to 4, which reduce or inhibit T cell proliferation in vitro and / or in vivo.

21. The expanded canine progenitor cells according to any one of claims 1 to 4, which are capable of providing angiogenesis in vitro and / or in vivo.

22. Expanded canine progenitor cells described in any one of claims 2 to 4, which are capable of differentiating into at least two cell types of the mesoderm.

23. 23. The expanded canine progenitor cells of claim 22, which are capable of differentiating into at least two of osteoblasts, adipocytes, and chondrocytes.

24. The expanded canine progenitor cells of any one of claims 1 to 4, wherein the cells are not tumorigenic, do not form teratomas, are not transformed, and are not immortalized.

25. The expanded progenitor cells of any one of claims 1 to 4, prepared by a method comprising obtaining tissue from a dog; establishing a population of adherent cells; selecting cells that positively express CD90 and / or do not express at least one of CD45 and CD34; and expanding the selected cells in a culture medium.

26. A composition comprising the expanded progenitor cells according to any one of claims 1 to 4 and a second component.

27. A pharmaceutical composition comprising the expanded progenitor cells according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

28. A kit comprising the following separately packaged components: expanded progenitor cells according to any one of claims 1 to 4; a culture medium; and instructions for culturing the cells.

29. 27. A method for preparing the composition of claim 26, comprising obtaining tissue from a dog; establishing a population of adherent cells; selecting cells that positively express CD90 and / or do not express at least one of CD45 and CD34; expanding the selected cells in culture medium; and adding the cells to the second component.

30. 28. A method for preparing the pharmaceutical composition of claim 27, comprising mixing the expanded progenitor cells with the pharmaceutically acceptable carrier.

31. 5. A method for preparing expanded canine progenitor cells according to any one of claims 1 to 4, the method comprising: obtaining tissue from a canine; establishing a population of adherent cells; selecting cells that positively express CD90 and / or do not express at least one of CD45 and CD34; and expanding the selected cells in a culture medium.

32. 10. A method for establishing a cell bank, said method comprising expanding and storing the expanded progenitor cells of any one of claims 1 to 4 for future administration to a subject.

33. 10. A method for drug discovery, the method comprising exposing expanded progenitor cells of any one of claims 1 to 4 to an agent and assessing one or more effects of the agent on the cells.

34. A method for treating an inflammatory condition in a dog, comprising administering to the dog a therapeutically effective amount of canine progenitor cells according to any one of claims 1 to 4.

35. 35. The method of claim 34, wherein the inflammatory condition is a chronic inflammatory condition or an acute inflammatory condition.

36. 36. The method of claim 35, wherein the acute inflammatory condition or the chronic inflammatory condition is one of dermatitis, inflammatory eye disease, inflammatory brain disease, inflammatory airway disease, and inflammatory bowel disease.

37. 37. The method of claim 36, wherein the dermatitis is atopic dermatitis.

38. 37. The method of claim 36, wherein the inflammatory eye disease is keratoconjunctivitis.

39. 37. The method of claim 36, wherein the inflammatory brain disease is meningoencephalomyelitis.

40. 35. The method of claim 34, wherein the inflammatory condition is an autoimmune disease.

41. A method for treating a musculoskeletal disorder in a dog, comprising administering to the dog a therapeutically effective amount of the canine progenitor cells according to any one of claims 1 to 4.

42. 42. The method of claim 41, wherein the musculoskeletal disorder is one of osteoarthritis and cruciate ligament rupture.

43. 43. The method of claim 42, wherein the cruciate ligament rupture is a partial cruciate ligament rupture.

44. 42. The method of claim 41, wherein the musculoskeletal disorder is a spinal condition.

45. 45. The method of claim 44, wherein the spinal condition is one of a spinal cord injury and an intervertebral disc disease.

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