阅读说明：本技术 包含人胎盘灌注液细胞、其亚群的组合物及其用途 (Compositions comprising human placental perfusate cells, subpopulations thereof, and uses thereof ) 是由 乔迪·P·格尼 张小葵 斯泰西·赫布 罗伯特·J·哈黎里 于 2014-11-14 设计创作，主要内容包括：本文提供了包含来自人胎盘灌注液的单核细胞的组合物和使用该细胞的方法,包括使用该细胞和造血细胞以例如建立嵌合体,降低移植物抗宿主疾病的严重程度或持续时间,治疗或减轻肌肉减少症、代谢紊乱和血液学疾病例如恶性血液病的症状,和治疗或减轻缺血性脑病(例如,缺氧缺血性脑病)及其它中枢神经系统损伤的症状。(Provided herein are compositions comprising mononuclear cells from human placental perfusate and methods of using the cells, including using the cells and hematopoietic cells, for example, to establish chimerism, reduce the severity or duration of graft versus host disease, treat or alleviate symptoms of sarcopenia, metabolic disorders, and hematologic diseases such as hematologic malignancies, and treat or alleviate symptoms of ischemic encephalopathy (e.g., hypoxic ischemic encephalopathy) and other central nervous system injuries.)

1. Composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 6 x10⁵CD34⁺A cell.

2. The composition of claim 1, wherein the composition further comprises a 2-fold greater amount of CD34⁺A cell.

3. The composition of claim 1, wherein the composition further comprises a 10-fold greater amount of CD34⁺A cell.

4. The composition of claim 1, wherein the composition further comprises a 50-fold greater amount of CD34⁺A cell.

5. The composition of claim 1, wherein the composition comprises substantially pure human placental perfusate CD34⁺A cell.

6. Composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 5x10⁵CD34⁺CD45^-A cell.

7. The composition of claim 6, wherein the composition further comprises a 2-fold greater amount of CD34⁺CD45^-A cell.

8. The composition of claim 6, wherein the composition further comprises a 10-fold greater amount of CD34⁺CD45^-A cell.

9. The composition of claim 6, wherein the composition further comprises a 50-fold greater amount of CD34⁺CD45^-A cell.

10. The composition of claim 6, wherein the composition comprises substantially pure human placental perfusate CD34⁺CD45^-A cell.

11. Composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 6 x10⁵CD34⁺CD31⁺A cell.

12. The composition of claim 11, wherein the composition further comprises a 2-fold greater amount of CD34⁺CD31⁺A cell.

13. The composition of claim 11, wherein the composition further comprises a 10-fold greater amount of CD34⁺CD31⁺A cell.

14. The composition of claim 11, wherein the composition further comprises a 50-fold greater amount of CD34⁺CD31⁺A cell.

15. The composition of claim 11, wherein the composition comprises substantially pure human placental perfusate CD34⁺CD31⁺A cell.

16. Composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 5x10⁵CD34⁺KDR⁺A cell.

17. The composition of claim 16, wherein the composition further comprises a 2-fold greater amount of CD34⁺KDR⁺A cell.

18. The composition of claim 16, wherein the composition further comprises a 10-fold greater amount of CD34⁺KDR⁺A cell.

19. The composition of claim 16, wherein the composition further comprises a 50-fold greater amount of CD34⁺KDR⁺A cell.

20. The composition of claim 16, wherein the composition comprises substantially pure human placental perfusate CD34⁺KDR⁺A cell.

21. Composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 5x10⁵CD34⁺CXCR4⁺A cell.

22. The composition of claim 21, wherein the composition further comprises a 2-fold greater amount of CD34⁺CXCR4⁺A cell.

23. The composition of claim 21, wherein the composition further comprises a 10-fold greater amount of CD34⁺CXCR4⁺A cell.

24. The composition of claim 21, wherein the composition further comprises a 50-fold greater amount of CD34⁺CXCR4⁺A cell.

25. The composition of claim 21, wherein the composition comprises substantially pure human placental perfusate CD34⁺CXCR4⁺A cell.

26. Composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 6 x10⁵CD34⁺CD38^-A cell.

27. The composition of claim 26, wherein the composition further comprises a 2-fold greater amount of CD34⁺CD38^-A cell.

28. The composition of claim 26, wherein the composition further comprises a 10-fold greater amount of CD34⁺CD38^-A cell.

29. The composition of claim 26, wherein the composition further comprises a 50-fold greater amount of CD34⁺CD38^-A cell.

30. The composition of claim 26, wherein the composition comprises substantially pure human placental perfusate CD34⁺CD38^-A cell.

31. Composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 7 x10⁵CD34⁺CD117^-A cell.

32. The composition of claim 31, wherein the composition further comprises a 2-fold greater amount of CD34⁺CD117^-A cell.

33. The composition of claim 31, wherein the composition further comprises a 10-fold greater amount of CD34⁺CD117^-A cell.

34. The composition of claim 31, wherein the composition further comprises a 50-fold greater amount of CD34⁺CD117^-A cell.

35. The composition of claim 31, wherein the composition comprises substantially pure human placental perfusate CD34⁺CD117^-A cell.

36. Composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 6 x10⁵CD34⁺CD140a⁺A cell.

37. The composition of claim 36, wherein the composition further comprises a 2-fold greater amount of CD34⁺CD140a⁺A cell.

38. The composition of claim 36, wherein the composition is further oneThe step contains 10 times more amount of CD34⁺CD140a⁺A cell.

39. The composition of claim 36, wherein the composition further comprises a 50-fold greater amount of CD34⁺CD140a⁺A cell.

40. The composition of claim 36, wherein the composition comprises substantially pure human placental perfusate CD34⁺CD140a⁺A cell.

41. Composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 3 x10⁵CD34⁺Nestin⁺A cell.

42. The composition of claim 40, wherein the composition further comprises a 2-fold greater amount of CD34⁺Nestin⁺A cell.

43. The composition of claim 40, wherein the composition further comprises a 10-fold greater amount of CD34⁺Nestin⁺A cell.

44. The composition of claim 40, wherein the composition further comprises a 50-fold greater amount of CD34⁺Nestin⁺A cell.

45. The composition of claim 40, wherein said composition is substantially pure human placental perfusate CD34⁺Nestin⁺A cell.

46. Composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 3 x10⁴CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

47. The composition of claim 46, wherein the composition further comprises 2-fold moreNumber of CDs 3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

48. The composition of claim 46, wherein the composition further comprises a 10-fold greater amount of CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

49. The composition of claim 46, wherein the composition further comprises a 50-fold greater amount of CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

50. The composition of claim 46, wherein said composition is substantially pure human placental perfusate CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

51. The composition of any one of claims 1-50, wherein human placental perfusate is isolated from a single placental perfusate.

52. A method of treating a central nervous system injury, disease, or disorder in a subject, comprising administering to the subject the composition of any one of claims 1-50 and hematopoietic cells from another source.

53. The method of claim 52, wherein the central nervous system injury, disease or disorder is hypoxic ischemic encephalopathy.

54. A method of treating sarcopenia in a subject comprising administering to the subject the composition of any one of claims 1 to 50 and hematopoietic cells from another source.

55. A method of inducing chimerism in a subject comprising administering to the subject the composition of any one of claims 1 to 50 and hematopoietic cells from another source.

56. A method for cell transplantation in a subject, comprising administering to the subject the composition of any one of claims 1-50 and hematopoietic cells from another source.

57. A method of reducing the severity or duration of Graft Versus Host Disease (GVHD) in a subject, comprising administering to the subject the composition of any one of claims 1-50 and hematopoietic cells from another source.

58. A method of treating a metabolic disorder in a subject, comprising administering to the subject the composition of any one of claims 1-50 and hematopoietic cells from another source.

59. A method of treating a hematologic disease or hematologic malignancy in a subject, comprising administering to the subject the composition of any one of claims 1-50 and hematopoietic cells from another source.

60. A composition as defined in any one of claims 1 to 50 for use in a method comprising:

(a) treating a central nervous system injury, disease, or disorder in a subject, preferably the central nervous system injury, disease, or disorder is hypoxic ischemic encephalopathy;

(b) inducing a chimera in a subject;

(c) for cell transplantation;

(d) for reducing the severity or duration of Graft Versus Host Disease (GVHD) in a subject;

(e) treating a metabolic disorder in a subject;

(f) treating a hematologic or hematologic malignancy in a subject; or

(g) Treating sarcopenia in a subject.

61. The composition for use in claim 60, wherein the composition further comprises hematopoietic cells from another source.

1. Field of the invention

Provided herein are compositions comprising mononuclear cells from human placental perfusate and methods of using the cells, including use of the cells with hematopoietic cells, e.g., for the establishment of chimeras (chimerasm); reducing the severity or duration of graft versus host disease; treating or alleviating the symptoms of sarcopenia, metabolic disorders and hematological diseases such as hematological malignancies; and treating or alleviating symptoms of ischemic encephalopathy (e.g., hypoxic ischemic encephalopathy) and other central nervous system injuries.

2. Background of the invention

Placental perfusate comprises a collection of placental cells obtained by passing perfusate through the placental vasculature and a collection of perfusate from the vasculature, from the maternal surface of the placenta, or from both. Methods of perfusing mammalian placenta are described, for example, in U.S. Pat. No. 7,045,146 and U.S. Pat. No. 7,255,879. The population of placental cells obtained by perfusion is heterogeneous, including, inter alia, CD34⁺Cells, nucleated cells such as granulocytes, monocytes and macrophages, and tissue culture matrix-adherent placental stem cells.

3. Summary of the invention

Provided herein are compositions comprising isolated human placental perfusate. In a particular embodiment, the human placental perfusate comprises at least 6 x10⁵CD34⁺A cell. In some embodiments, the human placental perfusate further comprises a 2-fold greater amount of CD34⁺A cell. In some embodiments, the human placental perfusate further comprises a 10-fold greater amount of CD34⁺A cell. In some embodiments, the human placental perfusate further comprises a 50-fold greater amount of CD34⁺A cell. In a more specific embodiment, the human placental perfusate comprises substantially pure human placental perfusate CD34⁺A cell.

In other particular embodiments, the human placental perfusate comprises at least 5x10⁵CD34⁺CD45^-A cell. In some embodiments, the human placental perfusate further comprises a 2-fold greater amount of CD34⁺CD45^-A cell. In some embodiments, the human placental perfusate further comprises a 10-fold greater amount of CD34⁺CD45^-A cell. In some embodiments, the human placental perfusate further comprises a 50-fold greater amount of CD34⁺CD45^-A cell. In a more specific embodiment, the human placental perfusate comprises substantially pure human placental perfusate CD34⁺CD45^-A cell.

In other particular embodiments, the human placental perfusate comprises at least 6 x10⁵CD34⁺CD31⁺A cell. In some embodiments, the human placental perfusate further comprises a 2-fold greater amount of CD34⁺CD31⁺A cell. In some embodiments, the human placental perfusate further comprises a 10-fold greater amount of CD34⁺CD31⁺A cell. In some embodiments, the human placental perfusate further comprises a 50-fold greater amount of CD34⁺CD31⁺A cell. In a more specific embodiment, the human placental perfusate comprisesSubstantially pure human placental perfusate CD34⁺CD31⁺A cell.

In other particular embodiments, the human placental perfusate comprises at least 5x10⁵CD34⁺KDR⁺A cell. In some embodiments, the human placental perfusate further comprises a 2-fold greater amount of CD34⁺KDR⁺A cell. In some embodiments, the human placental perfusate further comprises a 10-fold greater amount of CD34⁺KDR⁺A cell. In some embodiments, the human placental perfusate further comprises a 50-fold greater amount of CD34⁺KDR⁺A cell. In a more specific embodiment, the human placental perfusate comprises substantially pure human placental perfusate CD34⁺KDR⁺A cell.

In other particular embodiments, the human placental perfusate comprises at least 5x10⁵CD34⁺CXCR4⁺A cell. In some embodiments, the human placental perfusate further comprises a 2-fold greater amount of CD34⁺CXCR4⁺A cell. In some embodiments, the human placental perfusate further comprises a 10-fold greater amount of CD34⁺CXCR4⁺A cell. In some embodiments, the human placental perfusate further comprises a 50-fold greater amount of CD34⁺CXCR4⁺A cell. In a more specific embodiment, the human placental perfusate comprises substantially pure human placental perfusate CD34⁺CXCR4⁺A cell.

In other particular embodiments, the human placental perfusate comprises at least 6 x10⁵CD34⁺CD38^-A cell. In some embodiments, the human placental perfusate further comprises a 2-fold greater amount of CD34⁺CD38^-A cell. In some embodiments, the human placental perfusate further comprises a 10-fold greater amount of CD34⁺CD38^-A cell. In some embodiments, the human placental perfusate further comprises a 50-fold greater amount of CD34⁺CD38^-A cell. In a more specific embodiment, the human placental perfusate comprises substantially pure human placental perfusate CD34⁺CD38^-Cells。

In other particular embodiments, the human placental perfusate comprises at least 7 x10⁵CD34⁺CD117^-A cell. In some embodiments, the human placental perfusate further comprises a 2-fold greater amount of CD34⁺CD117^-A cell. In some embodiments, the human placental perfusate further comprises a 10-fold greater amount of CD34⁺CD117^-A cell. In some embodiments, the human placental perfusate further comprises a 50-fold greater amount of CD34⁺CD117^-A cell. In a more specific embodiment, the human placental perfusate comprises substantially pure human placental perfusate CD34⁺CD117^-A cell.

In other particular embodiments, the human placental perfusate comprises at least 6 x10⁵CD34⁺CD140a⁺A cell. In some embodiments, the human placental perfusate further comprises a 2-fold greater amount of CD34⁺CD140a⁺A cell. In some embodiments, the human placental perfusate further comprises a 10-fold greater amount of CD34⁺CD140a⁺A cell. In some embodiments, the human placental perfusate further comprises a 50-fold greater amount of CD34⁺CD140a⁺A cell. In a more specific embodiment, the human placental perfusate comprises substantially pure human placental perfusate CD34⁺CD140a⁺A cell.

In other particular embodiments, the human placental perfusate comprises at least 3 x10⁵CD34⁺Nestin⁺A cell. In some embodiments, the human placental perfusate further comprises a 2-fold greater amount of CD34⁺Nestin⁺A cell. In some embodiments, the human placental perfusate further comprises a 10-fold greater amount of CD34⁺Nestin⁺A cell. In some embodiments, the human placental perfusate further comprises a 50-fold greater amount of CD34⁺Nestin⁺A cell. In a more specific embodiment, the human placental perfusate comprises substantially pure human placental perfusate CD34⁺Nestin⁺A cell.

In other particular embodiments, the human placental perfusate comprises at least 3 x10⁴CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell. In some embodiments, the human placental perfusate further comprises a 2-fold greater amount of CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell. In some embodiments, the human placental perfusate further comprises a 10-fold greater amount of CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell. In some embodiments, the human placental perfusate further comprises a 50-fold greater amount of CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell. In a more specific embodiment, the human placental perfusate comprises substantially pure human placental perfusate CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

In some embodiments, the human placental perfusate is isolated from perfusion of a single placenta.

Also provided herein are methods of treating a central nervous system injury, disease, or disorder in a subject, comprising administering to the subject a composition comprising isolated human placental perfusate provided herein and hematopoietic cells from another source. In a specific embodiment, the central nervous system injury, disease, or disorder is an ischemic encephalopathy (e.g., hypoxic ischemic encephalopathy).

Also provided herein are methods of treating sarcopenia in a subject comprising administering to the subject a composition comprising isolated human placental perfusate provided herein and hematopoietic cells from another source.

Provided herein are methods of inducing chimerism in a subject comprising administering to the subject a composition comprising isolated human placental perfusate provided herein and hematopoietic cells from another source.

Provided herein are methods for cell transplantation in a subject comprising administering to the subject a composition comprising isolated human placental perfusate provided herein and hematopoietic cells from another source.

Provided herein are methods for reducing the severity or duration of Graft Versus Host Disease (GVHD) in a subject comprising administering to the subject a composition comprising isolated human placental perfusate provided herein and hematopoietic cells from another source.

Provided herein are methods of treating a metabolic disorder in a subject comprising administering to the subject a composition comprising isolated human placental perfusate provided herein and hematopoietic cells from another source.

Provided herein are methods of treating a hematologic disease or hematologic malignancy in a subject, comprising administering to the subject a composition comprising isolated human placental perfusate provided herein and hematopoietic cells from another source.

Provided herein are compositions comprising isolated human placental perfusate or human placental perfusate cells for use in a method comprising: (a) for use in treating a central nervous system injury, disease or disorder in a subject, preferably the central nervous system injury, disease or disorder is hypoxic ischemic encephalopathy; (b) for inducing chimerism in a subject; (c) for cell transplantation; (d) for reducing the severity or duration of Graft Versus Host Disease (GVHD) in a subject; (e) for treating a metabolic disorder in a subject; (f) for treating a hematologic or hematologic malignancy in a subject; or (g) for treating sarcopenia in a subject.

Also provided herein are compositions comprising isolated human placental perfusate or human placental perfusate cells for use in a method comprising: (a) for use in treating a central nervous system injury, disease or disorder in a subject, preferably the central nervous system injury, disease or disorder is hypoxic ischemic encephalopathy; (b) for inducing chimerism in a subject; (c) for cell transplantation; (d) for reducing the severity or duration of Graft Versus Host Disease (GVHD) in a subject; (e) for treating a metabolic disorder in a subject; (f) for treating a hematologic or hematologic malignancy in a subject; or (g) for treating sarcopenia in a subject, wherein the composition further comprises hematopoietic cells from another source.

4. Brief description of the drawings

Fig. 1 depicts the total nucleated cell count of the 43-person placental perfusate and cord blood unit pairs.

FIGS. 2A-2C depict human placental perfusate cells (A) gated preferential CD45⁺Cells (B) and Gate-preferential CD34⁺FACS analysis of cells (C).

FIGS. 3A-3E depict human placental perfusate (A) and umbilical cord blood (B) gated preferential CD34⁺Comparison of cells. Then human placental perfusate cell gate CD34⁺Cells (C) can be sorted to isolate CD34⁺CD45^-(D) And CD34⁺CD45⁺(E) A cell.

FIG. 4 depicts expression specific CD34 in Human Placental Perfusate (HPP) or umbilical cord blood (HUCB)⁺Percent nucleated cells of phenotype.

Figure 5 depicts lipoprotein uptake experiments using human placental perfusate endothelial cells (upper panel) and microtubule formation observed in HUVECs and Human Placental Perfusate (HPP) cells (lower panel).

Figure 6 depicts the percentage of nucleated cells expressing CD34 and/or nestin in Human Placental Perfusate (HPP) or umbilical cord blood (HUCB).

Figure 7 depicts the percentage of nucleated cells expressing specific HLA antigens in Human Placental Perfusate (HPP) or umbilical cord blood (HUCB).

Figure 8 depicts the percentage of nucleated cells expressing CD3 with or without CD4 and with or without CD8 in human placental perfusate or umbilical cord blood (HUCB).

5. Detailed description of the invention

In various aspects, provided herein are methods of producing mononuclear cells (HPCs) from human placental perfusate, e.g., human placental perfusate, compositions comprising the cells, and uses of the cells in treating individuals having a central nervous system injury, disease, disorder, or condition. In a more specific embodiment, the disease, disorder, or condition is an ischemic encephalopathy (e.g., hypoxic ischemic encephalopathy). Also provided herein are methods of administering HPCs (e.g., human placental perfusate) to a subject (e.g., a human subject) to reduce the severity of graft-versus-host disease and to treat or reduce symptoms of metabolic and hematologic diseases (e.g., hematologic malignancies). Also provided herein are methods of administering HPCs (e.g., human placental perfusate) to a subject (e.g., a human subject) to treat or alleviate symptoms of sarcopenia.

5.1 compositions comprising placental perfusate cells and methods of using the same

Placental perfusate comprises total mononuclear cells obtained from perfusate solution flowing through the placenta as described herein. Typically, placental perfusate from a single placental perfusate comprises from about 100 million to about 500 million nucleated cells. In certain embodiments, placental perfusate from a single placental perfusate comprises from about 100 million to about 400 million nucleated cells, from about 100 million to about 300 million nucleated cells, or from about 100 million to about 200 million nucleated cells.

Mononuclear cells (HPCs) from human placental perfusate, e.g., human placental perfusate, for use according to the present disclosure can be collected in any medically or pharmaceutically acceptable manner and can be present in a composition, e.g., a pharmaceutical composition. In certain embodiments, a composition provided herein (e.g., a pharmaceutical composition, i.e., a pharmaceutical grade solution suitable for administration to a human) comprises human placental perfusate.

In certain embodiments, the placental perfusate or perfusate cells comprise CD34⁺Cells, for example, hematopoietic stem or progenitor cells or endothelial progenitor cells. In a more specific embodiment, such cells may comprise CD34⁺CD45^–Stem or progenitor cells, CD34⁺CD45⁺Stem or progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, and/or erythroid progenitor cells.

In other embodiments, the placental perfusate and placental perfusate cells comprise, for example, endothelial progenitor cells, osteoprogenitor cells, and/or natural killer cells.

In certain embodiments, the placental perfusate from which red blood cells are collected and removed from the placenta, or perfusate cells isolated from the perfusate, comprise about 60-90%, e.g., about 60%, 65%, 70%, 80%, 85%, or 90%, e.g., about 60-90%, 65-90%, 70-90%, or about 75-90% white blood cells. In some embodiments, fromPlacental perfusate from which erythrocytes are collected and removed from the placenta, or perfusate cells isolated from the perfusate, comprise about 2-11%, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11%, e.g., about 5-8%, or about 6-7% natural killer cells (CD 3)^–，CD56⁺) (ii) a And/or about 7-37%, e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37%, e.g., about 20-25%, about 22-24%, or about 22-23% T cells (CD 3)⁺) (ii) a And/or about 5-15%, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, e.g., about 8-12%, or about 10-11% B cells (CD 19)⁺) (ii) a And/or monocytes (CD 14) in the range of about 20-32%, e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32%, e.g., about 22-28%, 25-28%, or about 26-27%⁺) (ii) a And/or about 1-5%, e.g., about 1, 2, 3, 4, or 5%, e.g., about 2-4% or about 2-3% endothelial progenitor cells (e.g., CD34)⁺，CD31⁺) (ii) a And/or about 0.5-5%, e.g., about 0.5, 1, 2, 3, 4, or 5%, e.g., about 2-4% or about 2-3% of neural progenitor cells (nestin)⁺) (ii) a And/or about 1-7%, e.g., about 1, 2, 3, 4, 5, 6, or 7%, e.g., about 2-4% or about 3-4% hematopoietic progenitor cells (CD 34)⁺) (ii) a And/or about 1-5%, e.g., about 1, 2, 3, 4, or 5%, e.g., about 2-4%, about 2-3%, or about 1-2% adherent placental stem cells (e.g., CD34)^–，CD117^–，CD105⁺And CD44⁺) As determined, for example, by flow cytometry such as FACS analysis.

In certain embodiments, the placental perfusate cells comprise CD34⁺A cell. In a more specific embodiment, the CD34⁺The cell is CD34⁺CD45^–A cell. In another embodiment, the CD34⁺Cells were isolated from placenta. In yet another embodiment, the population of placental perfusate cells further comprises additional isolated CD34 that is not isolated from the perfusate⁺Cells (e.g., isolated from cord blood)Placental blood, peripheral blood, bone marrow, etc.). In another embodiment, the additional CD34⁺The cells are isolated from umbilical cord blood, placental blood, peripheral blood, or bone marrow.

In other embodiments, CD34⁺The cells are additionally CD117^-. In certain embodiments, CD34⁺The cells are additionally CD31⁺，CXCR4⁺And/or KDR⁺. In certain embodiments, CD34⁺The cells are additionally CD140a⁺. In certain embodiments, CD34⁺The cells are additionally nestin⁺. In certain embodiments, said human placental perfusate cells, e.g., said CD34⁺The cells contain more CD117 than equivalent amounts of cord blood cells^-A cell. In certain embodiments, the CD34⁺The cells contain more CD31 than equivalent cord blood cells⁺，CXCR4⁺And/or KDR⁺A cell. In certain embodiments, any of the CDs 34⁺The cell is CD34⁺CD45^–A cell. In certain embodiments, said human placental perfusate cells, e.g., said CD34⁺The cells contain more CD140a than equivalent cord blood cells⁺A cell. In certain embodiments, said human placental perfusate cells, e.g., said CD34⁺The cells contain more nestin than equivalent amount of cord blood cells⁺A cell.

In another specific embodiment, said placental perfusate cells, e.g., said CD34⁺Cells in specific equivalent amounts of CD34 from cord blood⁺Higher levels of cells produce large amounts of one or more angiogenesis-related markers. In particular embodiments, the marker comprises CD31, KDR and/or CXCR 4. In one embodiment, the CD34⁺The cell is CD45^–. In a more specific embodiment, the CD34⁺Cells or CD34⁺CD45^–Cell specific equivalent of CD34 from cord blood⁺The cell expresses a higher level of at least one CD31, CXCR4, or KDR. In certain embodiments, the placental perfusate cells are, for example,the CD34⁺Placental cells express higher levels of nestin than equivalent amounts of cord blood cells.

In another specific embodiment, said placental perfusate is enriched in CD34⁺A cell. In certain embodiments, the placental perfusate is enriched in CD45^-A cell. In certain embodiments, the placental perfusate is enriched in CD34⁺CD45^-A cell. In certain embodiments, the placental perfusate is enriched in CD31⁺，KDR⁺And/or CXCR4⁺A cell. In certain embodiments, the placental perfusate is enriched in CD34⁺CD31⁺，CD34⁺KDR⁺And/or CD34⁺CXCR4⁺A cell. In certain embodiments, the placental perfusate is enriched in CD140a⁺A cell. In certain embodiments, the placental perfusate is enriched in CD34⁺CD140a⁺A cell. In certain embodiments, the placental perfusate is enriched for CD117^-A cell. In certain embodiments, the placental perfusate is enriched in CD34⁺CD117^-In certain embodiments, the placental perfusate is enriched in CD38^-A cell. In certain embodiments, the placental perfusate is enriched in CD34⁺CD38^-A cell. In certain embodiments, the placental perfusate is enriched for nestin⁺A cell. In certain embodiments, the placental perfusate is enriched in CD34⁺Nestin⁺A cell. In certain embodiments, the placental perfusate is enriched in CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

With respect to enrichment, a particular cell population can be enriched for one or more cell types, e.g., cells exhibiting a specific cell surface marker phenotype, by, e.g., introducing the cell type into the cell population, adding an additional number of cell types to the cell population, and/or removing (removing some or all) one or more cell types, e.g., cells exhibiting a different specific cell surface marker phenotype, from the cell population.

In some embodiments, enriching a particular cell population or cell subpopulation in the placental perfusate or placental perfusate cells is accomplished by one or more rounds of cell sorting, e.g., FACS cell sorting. In some embodiments, enriching a particular cell population or cell subpopulation in the placental perfusate or placental perfusate cells is accomplished by removing one or more cell populations or cell subpopulations. In some embodiments, enriching a particular cell population or cell subpopulation in the placental perfusate or placental perfusate cells is accomplished by adding a cell population or cell subpopulation that has been isolated from placental perfusate. In some embodiments, enriching a particular cell population or cell subpopulation in the placental perfusate or placental perfusate cells is accomplished by adding a cell population or cell subpopulation that has been isolated from another source (e.g., umbilical cord blood). In some embodiments, enriching a particular cell population or cell subpopulation in the placental perfusate or placental perfusate cells is accomplished by adding placental perfusate that has been enriched for that cell population or cell subpopulation. In other embodiments, enriching a particular cell population or cell subpopulation in the placental perfusate or placental perfusate cells is accomplished by expanding the cell population or cell subpopulation. In some embodiments, enriching a particular cell population or cell subpopulation in the placental perfusate or placental perfusate cells is accomplished by increasing the total number of those cells in the placental perfusate or placental perfusate cells. In some embodiments, enriching a particular cell population or cell subpopulation in the placental perfusate or placental perfusate cells is accomplished by increasing the proportion of those cells in the placental perfusate or placental perfusate cells. In some embodiments, enriching a particular cell population or cell subpopulation in the placental perfusate or placental perfusate cells is accomplished by culture expansion of the particular cell population or cell subpopulation. In some embodiments, removing a particular cell population or cell subpopulation from the placental perfusate or placental perfusate cells is accomplished by culture expansion of another particular cell population or cell subpopulation. Enrichment or isolation of a particular cell population or cell subpopulation can be performed after expansion of the particular cell population or cell subpopulation, or on total nucleated cells from placental perfusate.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 2 x10⁶，3×10⁶，4×10⁶，5×10⁶，6×10⁶，7×10⁶，8×10⁶Or 9X 10⁶CD34⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 6 x10⁵To 3X 10⁷CD34⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁶To 1X10⁷CD34⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁵To 1X10⁸CD34⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁴To 1X10⁸CD34⁺A cell. In a specific embodiment, said CD34 is obtained by cell sorting total nucleated cells of placental perfusate with an antibody against CD34⁺A cell. In some embodiments, the CD34⁺Cells have been isolated from placental perfusate or from cells of said placental perfusate. In some embodiments, the CD34+ cells of the placental perfusate have been expanded in culture.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 10% CD34⁺A cell. In another embodiment, the placental perfusate or the placental perfusate cells comprise 8% to 12% CD34⁺A cell.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 2 x10⁶，3×10⁶，4×10⁶，5×10⁶，6×10⁶，7×10⁶，8×10⁶Or 9X 10⁶CD34⁺CD45^-A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 5x10⁵To 1X10⁷CD34⁺CD45^-A cell. In another embodiment, said placental perfusate or said placental perfusate isThe cells contained 1X10⁶To 1X10⁷CD34⁺CD45^-A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁵To 1X10⁸CD34⁺CD45^-A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁴To 1X10⁸CD34⁺CD45^-A cell. In one embodiment, the CD34⁺CD45^-Cells have been obtained by cell sorting of total nucleated cells of placental perfusate with antibodies against CD34, followed by cell sorting of total nucleated cells of placental perfusate with antibodies against CD 45. In another specific embodiment, said CD34⁺CD45^-Cells have been obtained by cell sorting of total nucleated cells of placental perfusate with antibodies against CD45, followed by cell sorting of total nucleated cells of placental perfusate with antibodies against CD 34. In some embodiments, the CD34⁺CD45^-Cells have been isolated from placental perfusate or from cells of said placental perfusate.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 2 x10⁶，3×10⁶，4×10⁶，5×10⁶，6×10⁶，7×10⁶，8×10⁶Or 9X 10⁶CD34⁺CD31⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 6 x10⁵To 3X 10⁷CD34⁺CD31⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁶To 1X10⁷CD34⁺CD31⁺In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10 cells⁵To 1X10⁸CD34⁺CD31⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁴To 1X10⁸CD34⁺CD31⁺A cell. In a concrete exampleIn one embodiment, the CD34⁺CD31⁺Cells have been obtained by cell sorting of total nucleated cells of placental perfusate with antibodies against CD34, followed by cell sorting of total nucleated cells of placental perfusate with antibodies against CD 31. In another specific embodiment, said CD34⁺CD31⁺Cells have been obtained by cell sorting of total nucleated cells of placental perfusate with antibodies against CD31, followed by cell sorting of total nucleated cells of placental perfusate with antibodies against CD 34. In some embodiments, the CD34⁺CD31⁺Cells have been isolated from placental perfusate or from cells of said placental perfusate.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 2 x10⁶，3×10⁶，4×10⁶，5×10⁶，6×10⁶，7×10⁶，8×10⁶Or 9X 10⁶CD34⁺KDR⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 5x10⁵To 2X 10⁷CD34⁺KDR⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁶To 1X10⁷CD34⁺KDR⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁵To 1X10⁸CD34⁺KDR⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁴To 1X10⁸CD34⁺KDR⁺A cell. In one embodiment, the CD34⁺KDR⁺Cells have been obtained by cell sorting of total nucleated cells of placental perfusate with an antibody against CD34, followed by cell sorting of total nucleated cells of placental perfusate with an antibody against KDR. In another specific embodiment, said CD34⁺KDR⁺Cells total nucleated cells of placental perfusate have been cell sorted with antibodies against KDR, followed by placental perfusion with antibodies against CD34And (3) carrying out cell sorting on the total nucleated cells of the liquid. In some embodiments, the CD34⁺KDR⁺Cells have been isolated from placental perfusate or from cells of said placental perfusate.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 2 x10⁶，3×10⁶，4×10⁶，5×10⁶，6×10⁶，7×10⁶，8×10⁶Or 9X 10⁶CD34⁺CXCR4⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 6 x10⁵To 3X 10⁷CD34⁺CXCR4⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁶To 1X10⁷CD34⁺CXCR4⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁵To 1X10⁸CD34⁺CXCR4⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁴To 1X10⁸CD34⁺CXCR4⁺A cell. In one embodiment, the CD34⁺CXCR4⁺Cells have been obtained by cell sorting of total nucleated cells of placental perfusate with antibodies against CD34, followed by cell sorting of total nucleated cells of placental perfusate with antibodies against CXCR 4. In another specific embodiment, said CD34⁺CXCR4⁺Cells have been obtained by cell sorting of total nucleated cells of placental perfusate with antibodies against CXCR4, followed by cell sorting of total nucleated cells of placental perfusate with antibodies against CD 34. In some embodiments, the CD34⁺CXCR4⁺Cells have been isolated from placental perfusate or from cells of said placental perfusate.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 2 x10⁶，3×10⁶，4×10⁶，5×10⁶，6×10⁶，7×10⁶，8×10⁶Or 9X 10⁶CD34⁺CD38^-A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 6 x10⁵To 3X 10⁷CD34⁺CD38^-A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁶To 1X10⁷CD34⁺CD38^-A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁵To 1X10⁸CD34⁺CD38^-A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁴To 1X10⁸CD34⁺CD38^-A cell. In one embodiment, the CD34⁺CD38^-Cells have been obtained by cell sorting of total nucleated cells of placental perfusate with antibodies against CD34, followed by cell sorting of total nucleated cells of placental perfusate with antibodies against CD 38. In another specific embodiment, said CD34⁺CD38^-Cells have been obtained by cell sorting of total nucleated cells of placental perfusate with antibodies against CD38, followed by cell sorting of total nucleated cells of placental perfusate with antibodies against CD 34. In some embodiments, the CD34⁺CD38^-Cells have been isolated from placental perfusate or from cells of said placental perfusate.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 2 x10⁶，3×10⁶，4×10⁶，5×10⁶，6×10⁶，7×10⁶，8×10⁶Or 9X 10⁶CD34⁺CD117^-A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 7 x10⁵To 2X 10⁷CD34⁺CD117^-A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁶To 1X10⁷CD34 CD117^-A cell. In another embodimentSaid placental perfusate or said placental perfusate cells comprise 1x10⁵To 1X10⁸CD34⁺CD117^-A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁴To 1X10⁸CD34⁺CD117^-A cell. In one embodiment, the CD34⁺CD117^-The cells have been obtained by cell sorting of total nucleated cells of the placental perfusate with an antibody against CD34, followed by cell sorting of total nucleated cells of the placental perfusate with an antibody against CD 117. In another specific embodiment, said CD34⁺CD117^-The cells have been obtained by cell sorting of total nucleated cells of the placental perfusate with an antibody against CD117, followed by cell sorting of total nucleated cells of the placental perfusate with an antibody against CD 34. In some embodiments, the CD34⁺CD117^-Cells have been isolated from placental perfusate or from cells of said placental perfusate.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 2 x10⁶，3×10⁶，4×10⁶，5×10⁶，6×10⁶，7×10⁶，8×10⁶Or 9X 10⁶CD34⁺CD140a⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 6 x10⁵To 2X 10⁷CD34⁺CD140a⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁶To 1X10⁷CD34⁺CD140a⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁵To 1X10⁸CD34⁺CD140a⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁴To 1X10⁸CD34⁺CD140a⁺A cell. In one embodiment, the CD34⁺CD140a⁺Cells have been foetal with antibodies against CD34Total nucleated cells of the disc perfusate were obtained by cell sorting followed by cell sorting of total nucleated cells of the placental perfusate with antibodies against CD140 a. In another specific embodiment, said CD34⁺CD140a⁺Cells have been obtained by cell sorting of total nucleated cells of placental perfusate with antibodies against CD140a, followed by cell sorting of total nucleated cells of placental perfusate with antibodies against CD 34. In some embodiments, the CD34⁺CD140a⁺Cells have been isolated from placental perfusate or from cells of said placental perfusate.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 2 x10⁶，3×10⁶，4×10⁶，5×10⁶，6×10⁶，7×10⁶，8×10⁶Or 9X 10⁶CD34⁺Nestin⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 6 x10⁵To 2X 10⁷CD34⁺Nestin⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁶To 1X10⁷CD34⁺Nestin⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁵To 1X10⁸CD34⁺Nestin⁺A cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁴To 1X10⁸CD34⁺Nestin⁺A cell. In one embodiment, the CD34⁺Nestin⁺The cells have been obtained by cell sorting of total nucleated cells of the placental perfusate with an antibody against CD34, followed by cell sorting of total nucleated cells of the placental perfusate with an antibody against nestin. In another specific embodiment, said CD34⁺Nestin⁺Cells total nucleated cells of placental perfusate have been cell sorted with antibodies against nestin, followed by refinement of total nucleated cells of placental perfusate with antibodies against CD34And (4) cell sorting. In some embodiments, the CD34⁺Nestin⁺Cells have been isolated from placental perfusate or from cells of said placental perfusate.

In another specific embodiment, said placental perfusate or said placental perfusate cells comprise about 2 x10⁶，3×10⁶，4×10⁶，5×10⁶，6×10⁶，7×10⁶，8×10⁶Or 9X 10⁶CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 4 x10⁴to 5×10⁶CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁶To 1X10⁷CD3⁺CD4⁺CD8^-CD25^hiCD127^lowIn another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10 cells⁵To 1X10⁸CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell. In another embodiment, said placental perfusate or said placental perfusate cells comprise 1x10⁴To 1X10⁸CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell. In one embodiment, the CD3⁺CD4⁺CD8^-CD25^hiCD127^lowThe cells have been isolated. In a more specific embodiment, the CD3⁺CD4⁺CD8^-CD25^hiCD127^lowThe cells have used human CD4⁺CD25^hiCD127^lowRegulatory T cells (Cat #15861, Stem Cell) were isolated using the complete kit.

In certain embodiments, enriched CD34⁺Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺Cells are 3 times as fine as the non-enriched placenta perfusate or placenta perfusateAnd (4) cells. In certain embodiments, enriched CD34⁺The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure CD34⁺A population of cells.

In certain embodiments, enriched CD45^-Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD45^-Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD45^-The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure CD45^-A population of cells.

In certain embodiments, enriched CD34⁺CD45^-Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺CD45^-Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺CD45^-The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are a pure or substantially pure population of CD34⁺CD45^-A cell.

In certain embodiments, enriched CD31⁺，KDR⁺And/or CXCR4⁺Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD31⁺，KDR⁺And/or CXCR4⁺Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD31⁺，KDR⁺And/or CXCR4⁺The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodimentWherein the placental perfusate cells are pure or substantially pure CD31⁺，KDR⁺And/or CXCR4⁺A population of cells.

In certain embodiments, enriched CD34⁺CD31⁺，CD34⁺KDR⁺And/or CD34⁺CXCR4⁺Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺CD31⁺，CD34⁺KDR⁺And/or CD34⁺CXCR4⁺Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺CD31⁺，CD34⁺KDR⁺And/or CD34⁺CXCR4⁺The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure CD34⁺CD31⁺，CD34⁺KDR⁺And/or CD34⁺CXCR4⁺A population of cells.

In certain embodiments, enriched CD117^-Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD117^-Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD117^-The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure CD117^-A population of cells.

In certain embodiments, enriched CD34⁺CD117^-Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺CD117^-Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺CD117^-The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 or 50 times as many as(ii) non-enriched placental perfusate or placental perfusate cells. In another embodiment, the placental perfusate cells are pure or substantially pure CD34⁺CD117^-A population of cells.

In certain embodiments, enriched CD38^-Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD38^-Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD38^-The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure CD38^-A population of cells.

In certain embodiments, enriched CD34⁺CD38^-Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺CD38^-Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺CD38^-The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure CD34⁺CD38^-A population of cells.

In certain embodiments, enriched CD140a⁺Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD140a⁺Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD140a⁺The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure CD140a⁺A population of cells.

In certain embodiments, enriched CD34⁺CD140a⁺Cell size 2 times that of non-enriched placentaPerfusate or placental perfusate cells. In certain embodiments, enriched CD34⁺CD140a⁺Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺CD140a⁺The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure CD34⁺CD140a⁺A population of cells.

In certain embodiments, the enriched nestin⁺Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, the enriched nestin⁺Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, the enriched nestin⁺The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure nestin⁺A population of cells.

In certain embodiments, enriched CD34⁺Nestin⁺Cells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺Nestin⁺Cells were 3 times greater than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD34⁺Nestin⁺The cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure CD34⁺Nestin⁺A population of cells.

In certain embodiments, enriched CD3⁺CD4⁺CD8^-CD25^hiCD127^lowCells were 2 times larger than the cells of the unenriched placental perfusate or placental perfusate. In certain embodiments, enriched CD3⁺CD4⁺CD8^-CD25^hiCD127^lowCells3 times of the cells of the non-enriched placental perfusate or placental perfusate. In certain embodiments, enriched CD3⁺CD4⁺CD8^-CD25^hiCD127^lowThe cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times as many as the cells of the unenriched placental perfusate or placental perfusate. In another embodiment, the placental perfusate cells are pure or substantially pure CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA population of cells.

In certain embodiments, the placental perfusate cells, e.g., the CD34⁺Cells, express lower levels of CD3 than equivalent amounts of cord blood cells. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells, express lower levels of CD3 and CD8 than equivalent amounts of cord blood cells. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells, express lower levels of CD3 and CD4 than equivalent amounts of cord blood cells.

In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD8⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺A cell. In certain embodiments, the placental perfusate or the placental perfusate cells have been depleted of CD3⁺A cell. In certain embodiments, the placental perfusate or the placental perfusate cells have been depleted of CD3⁺CD8⁺A cell. In certain embodiments, the placental perfusate or the placental perfusate cells have been depleted of CD3⁺CD4⁺A cell.

In certain embodiments, CD3 is removed⁺The cells result in a more undigested placental perfusate or2-fold less CD3 in placental perfusate cells⁺A cell. In certain embodiments, CD3 is removed⁺The cells resulted in 3-fold less CD3 than in unremoved placental perfusate or placental perfusate cells⁺A cell. In certain embodiments, CD3 is removed⁺The cells result in less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20-fold less CD3 than in unremoved placental perfusate or placental perfusate cells⁺A cell.

In certain embodiments, CD3 is removed⁺CD8⁺The cells resulted in 2-fold less CD3 than in unremoved placental perfusate or placental perfusate cells⁺CD8⁺A cell. In certain embodiments, CD3 is removed⁺CD8⁺The cells resulted in 3-fold less CD3 than in unremoved placental perfusate or placental perfusate cells⁺CD8⁺A cell. In certain embodiments, CD3 is removed⁺CD8⁺The cells result in 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 fold less CD3 than in unremoved placental perfusate or placental perfusate cells⁺CD8⁺A cell.

In certain embodiments, CD3 is removed⁺CD4⁺The cells resulted in 2-fold less CD3 than in unremoved placental perfusate or placental perfusate cells⁺CD4⁺A cell. In certain embodiments, CD3 is removed⁺CD4⁺The cells resulted in 3-fold less CD3 than in unremoved placental perfusate or placental perfusate cells⁺CD4⁺A cell. In certain embodiments, CD3 is removed⁺CD4⁺The cells result in 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 fold less CD3 than in unremoved placental perfusate or placental perfusate cells⁺CD4⁺A cell.

In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25^hiCD127^lowA cell. In certain embodiments, the placental perfusate cells are, for example,the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25^hiCD127^lowCD45RA⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25^hiCD127^lowCD45RA^-A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25hiCD127lowCD45RA^-HLADR⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25^+/-CD127^+/-A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25^+/-CD127^+/-CD45RA⁺HLADR^-A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25^+/-CD127^+/-CD45RA^-CCR7⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25^+/-CD127^+/-CD45RA^-CCR7^-A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25^+/-CD127^+/-CD45RA⁺CCR7^-A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25^+/-CD127^+/-CD45RA^-HLADR⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8^-CD25^+/-CD127^+/-CD45RA^-CD69⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4^-CD8⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4^-CD8⁺CD45RA⁺HLADR^-CCR7⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4^-CD8⁺CD45RA^-CCR7⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4^-CD8⁺CD45RA⁺CCR7^-A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4^-CD8⁺CD45RA^-CCR7^-A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4^-CD8⁺CD45RA^-HLADR⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4⁺CD8⁺A cell. In certain embodiments, the placental perfusate cells, e.g., the placental perfusate cellsCD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4^-CD8^-A cell. In certain embodiments, the placental perfusate cells, e.g., the CD34⁺The cells contain less CD3 than the equivalent amount of cord blood cells⁺CD4^-CD8^-CD69⁺A cell.

In certain embodiments, any CD34 described herein⁺Cells or CD34⁺The cell population is expanded. In certain embodiments, any CD34 described herein⁺Cells or CD34⁺The cell population is enriched. In certain embodiments, any CD3 described herein⁺Cells, e.g. CD34⁺CD3⁺Cells, were removed.

In certain embodiments, the placental perfusate or the placental perfusate cells are treated to inhibit CD3⁺And (5) cell proliferation. In certain embodiments, the placental perfusate or the placental perfusate cells are treated to inhibit CD3⁺CD8⁺And (5) cell proliferation. In certain embodiments, the placental perfusate or the placental perfusate cells are treated to inhibit CD3⁺CD4⁺And (5) cell proliferation. In one embodiment, CD3⁺CD4⁺Inhibition of cell proliferation by addition of isolated CD3⁺CD4⁺CD8^-CD25^hiCD127^lowAnd (4) cells.

Placental perfusate, placental perfusate cells, and any population and subpopulation thereof, can be combined. In one embodiment, one or more of said placental perfusate cell populations or subpopulations are combined with total nucleated cells from placental perfusate. In another embodiment, the one or more populations or subpopulations of placental perfusate cells are combined with each other. In a specific embodiment, the one or more populations or subpopulations have been enriched for cells of one or more specific phenotypes. In another specific embodiment, said one or more populations or subpopulations have been isolated from placental perfusate or placental perfusate cells. In another specific embodiment, said one or more populations or subpopulations are obtained by one or more rounds of cell sorting. In another embodiment, the one or more populations or subpopulations have been depleted of one or more specific phenotypes of cells.

In yet another embodiment, the placental perfusate or perfusate cell population is contacted with a plurality of CD34⁺And (4) combining the cells. The CD34⁺The cells can be contained, for example, in untreated placenta, umbilical cord blood, or peripheral blood; placental, umbilical cord or peripheral blood; isolated placental, umbilical cord or peripheral blood CD34⁺A population of cells; untreated bone marrow; bone marrow total nucleated cells; isolated bone marrow CD34⁺Cell populations, and the like. In one embodiment, the hematopoietic stem cell is CD34⁺Placental endothelial progenitor cells.

In one aspect, provided herein is a method of treating a subject having a central nervous system injury, disease, or disorder, comprising administering to the subject placental perfusate, or any population or subpopulation of cells presented herein, or any combination thereof, in an amount sufficient to produce a detectable improvement or reduction in worsening of one or more symptoms of the central nervous system injury, disease, or disorder. In a specific embodiment, the central nervous system injury, disease, or disorder is an ischemic encephalopathy (e.g., hypoxic ischemic encephalopathy). In another specific embodiment, the placental perfusate cells are total nucleated cells from placental perfusate. In another embodiment, the placental perfusate cells are any population, subpopulation, or combination comprising placental perfusate cells described herein. In another specific embodiment, the population of placental perfusate cells comprises placental perfusate cells isolated from a single placental perfusion. In another specific embodiment, said population of placental perfusate cells comprises isolated CD34 that is not isolated from said perfusate⁺A cell. In a more specific embodiment, the CD34⁺Cells were isolated from placenta. In another more specific embodiment, said CD34⁺The cells are isolated from umbilical cord blood, placental blood, peripheral blood, or bone marrow. In another more specific embodiment, said CD34⁺Cell specific equivalent of cord blood CD34⁺Higher levels of cellular expressionNestin of (a).

In another aspect, provided herein are methods of administering HPCs (e.g., human placental perfusate) to a subject (e.g., a human subject) to reduce the severity of graft-versus-host disease and/or to treat or reduce symptoms of metabolic and hematologic diseases (e.g., hematologic malignancies).

In another aspect, provided herein are methods of administering HPC (e.g., human placental perfusate) to a subject (e.g., a human subject) to treat or alleviate symptoms of sarcopenia. Further embodiments of the method are described in detail hereinafter.

5.2 isolation, sorting and characterization of placental perfusate cells

Provided herein are methods of obtaining placental perfusate and placental perfusate cells from a mammalian placenta. In all embodiments described herein, the preferred perfusate is human placental perfusate and the preferred perfusate cells are human placental perfusate cells. Also described herein are methods for isolating and characterizing cell populations and subpopulations, and combinations thereof.

Mononuclear cells (HPCs) from human placental perfusate, e.g., human placental perfusate, for use according to the present disclosure can be collected in any medically or pharmaceutically acceptable manner and can be present in a composition, e.g., a pharmaceutical composition. In certain embodiments, a composition provided herein (e.g., a pharmaceutical composition, i.e., a pharmaceutical grade solution suitable for administration to a human) comprises human placental perfusate. In certain embodiments, the composition comprises human placental perfusate obtained from a partially bled placenta. In certain embodiments, the composition comprises human placental perfusate obtained from a bled placenta. In certain embodiments, the composition comprises cells, such as stem cells, isolated from human placental perfusate. In certain embodiments, the composition comprises nucleated cells isolated from human placental perfusate, e.g., monocytes or total nucleated cells.

In one embodiment, the HPCs, such as human placental perfusate, are sterile.

In one embodiment, red blood cells and/or granulocytes are removed by standard methodsHPCs or human placental perfusate are processed to produce a population of nucleated cells. The enriched cell population may be used without freezing, or frozen for later use. If the cell population is to be frozen, a standard cryoprotectant (e.g., DMSO, glycerol, Epilife) can be added to the enriched cell population prior to freezing thereof^TMCell cryogens (Cascade Biologics)).

In certain embodiments, the cells obtained from placental perfusate comprise mononuclear cells from placental perfusate. In certain embodiments, the cells obtained from placental perfusate comprise total nucleated cells from placental perfusate. In particular embodiments, the perfusate may be processed to remove or substantially remove red blood cells by adding hydroxyethyl starch to the perfusate and subsequent gravity sedimentation.

In certain embodiments, the cells obtained from placental perfusate are obtained from a single placenta. In certain embodiments, the cells obtained from placental perfusate are obtained from more than one placenta. In certain embodiments, the cells obtained from placental perfusate are obtained from two placentas. In embodiments where the cells are obtained from more than one placenta, the cells from different placentas may not necessarily be related or matched to each other.

As described herein, placental perfusate can be obtained from a placenta that has been drained of cord blood and perfused to remove residual blood prior to obtaining placental cells from perfusion. Placental perfusate can be obtained from a placenta that has been drained of cord blood but not perfused to remove residual blood. Placental perfusate can be obtained from a placenta isolated from as much as 0.5 to 6.0 inches, e.g., 0.5 to 1.0, 1.0 to 1.5, 1.5 to 2.0, 2.0 to 2.5, 2.5 to 3.0, 3.0 to 3.5, 3.5 to 4.0, or 4.0 to 6.0 inches of umbilical cord, where the umbilical cord can contain residual umbilical cord blood, a portion of which can enter the placental perfusate during perfusion and thereby be contained in the placental perfusate. Placental perfusate can be obtained from a placenta without draining the cord blood or perfusing to remove residual blood. In the latter two embodiments, the placental cells, e.g., nucleated cells from placental perfusate, e.g., HPCs, comprise nucleated cells from placental blood and/or umbilical cord blood. In a specific embodiment, the placental perfusate according to the present disclosure is free of umbilical cord blood. In another specific embodiment, the placental perfusate according to the present disclosure is substantially free of umbilical cord blood, e.g., the placental perfusate comprises less than 10%, less than 5%, less than 1%, less than 0.5%, or less than 0.1% umbilical cord blood. Typically, if cord blood cells are included in the perfusate cells, then for purposes of the methods provided herein, the cells are considered to be part of the HPC population, rather than HT cells, such as UCB cells.

Placental perfusate can be collected from a single individual (i.e., as a single unit) for administration, or mixed with other units, e.g., from the same individual or from one or more other individuals. In certain embodiments, the placental perfusate or cells thus obtained are stored prior to administration. In certain embodiments, a unit of placental perfusate comprises a sufficient number of cells to administer at least about 1.0x10 per kilogram body weight of the subject⁵，0.5x10⁶，1.0x10⁶，1.5x10⁶，2.0x10⁶，2.5x10⁶，3.0x10⁶，4.0x10⁶，5.0x10⁶Or 1.0x10⁷Cells obtained from placental perfusate, e.g., total nucleated cells. In certain embodiments, a unit of placental perfusate or cells thus obtained is administered. In certain embodiments, less than one unit is administered. In certain embodiments, more than one unit is administered.

The placenta used to obtain placental perfusate can be recovered after successful production and placental removal. In certain embodiments, the placenta is from term production. In certain embodiments, the placenta is from a premature birth. In some embodiments, the placenta is the placenta of an infant born at about 23 to about 25 weeks of gestation. In some embodiments, the placenta is the placenta of an infant born at about 26 to about 29 weeks of gestation. In some embodiments, the placenta is the placenta of an infant born at about 30 to about 33 weeks of gestation. In some embodiments, the placenta is the placenta of an infant born at about 34 to about 37 weeks of gestation. In some embodiments, the placenta is the placenta of an infant born at about 37 to about 42 weeks of gestation.

In particular embodiments, the placenta may be stored for about 1 hour to about 72 hours or about 4 to about 24 hours before perfusing the placenta to remove any residual cord blood, or before perfusing the placenta without removing residual cord blood. The placenta can be stored in an anticoagulation solution at about 5 ℃ to about 25 ℃, e.g., room temperature. Suitable anticoagulant solutions are well known in the art. For example, heparin or warfarin sodium solution may be used. In one embodiment, the anticoagulation solution comprises heparin solution (1% w/w in 1:1000 solution). In certain embodiments, the placenta is stored for no more than 36 hours prior to collection of the HPCs, e.g., human placental perfusate.

The human placental perfusate or cells thus obtained for use according to the present disclosure are generally unrelated to the subject receiving the cells. The human placental perfusate or cells thus obtained for use according to the present disclosure are typically not matched or partially mismatched to the subject receiving the cells.

The human placental perfusate or cells thus obtained for use according to the present disclosure may be obtained by any method. Placental perfusate can be obtained, for example, as disclosed in U.S. Pat. No. 7,045,148, U.S. Pat. No. 7,255,879, and/or U.S. Pat. No. 8,057,788, each of which is incorporated herein by reference in its entirety. The perfusion may be, for example, perfusion by the pan (pan) method, in which the perfusate is forced through the placental vessels, and the perfusate that seeps out of the placenta (usually the maternal side) is collected in a pan containing the placenta. Perfusion may also be, for example, closed loop perfusion, where the perfusate flows through and is collected from the fetal vessels of the placenta only. See, for example, U.S. patent No. 8,057,788, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the perfusion may be continuous, i.e., two or more passes of perfusate flowing through the placenta prior to separating cells obtained from the placental perfusate (e.g., HPCs or total nucleated cells from the placental perfusate).

In certain embodiments, a placenta is perfused with about 0.5-2 liters of perfusate, e.g., about 0.5-1 liter, or about 750 mL. In particular embodiments, the perfusion of the placenta is completed within about 15 minutes to 2 hours, e.g., about 30 minutes to 1.5 hours, about 30 minutes to 1 hour, or about 30 minutes.

The number and type of cells collected from the mammalian placenta can be monitored by, for example, detecting changes in morphology and cell surface markers using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., staining with tissue-specific or cell marker-specific antibodies), Fluorescence Activated Cell Sorting (FACS), Magnetically Activated Cell Sorting (MACS), detecting cell morphology using optical or confocal microscopy, and/or detecting changes in gene expression using techniques well known in the art such as PCR and gene expression profiling. These techniques can also be used to identify cells that are positive for one or more specific markers. For example, using antibodies to CD34, one can use the techniques described above to determine whether a cell contains a detectable amount of CD 34; if contained, the cell is CD34⁺. Similarly, a cell is positive for a particular marker if it produces enough of the marker RNA, or significantly more of the marker RNA, that it can be detected by RT-PCR than an adult cell. The sequences of antibodies and specific genes for cell surface markers (e.g., CD markers such as CD34) are well known in the art.

In another embodiment, placental cells, e.g., placental perfusate or perfusate cells, can be identified and characterized by colony forming unit analysis. Colony forming unit assays are well known in the art.

The placental perfusate or perfusate cells may additionally be assessed for viability, proliferation potential and longevity using standard techniques known in the art, such as trypan blue exclusion analysis, fluorescein diacetate uptake analysis, propidium iodide uptake analysis (to assess viability) and thymidine uptake analysis, MTT cell proliferation analysis (to assess proliferation). Longevity can be determined by techniques well known in the art, for example by determining the maximum population doubling in extended culture.

The cells can be sorted, for example, using a Fluorescence Activated Cell Sorter (FACS). Fluorescence Activated Cell Sorting (FACS) is a well-known method for separating particles, including cells, based on their fluorescent properties (Kamarch, 1987, Methods Enzymol, 151: 150-. The laser excites the fluorescent moieties in the individual particles to produce small charges, allowing the positive and negative particles to be electromagnetically separated from the mixture. In one embodiment, the cell surface marker-specific antibody or ligand is labeled with a different fluorescent label. Cells were treated with a cell sorter, allowing for the separation of cells based on their ability to bind the antibody used. FACS sorted particles can be placed directly into individual wells of a 96-well or 384-well plate to facilitate isolation and cloning.

In another embodiment, the magnetic beads can be used to separate or sort cells, and/or remove cell populations. Cells can be sorted, for example, using magnetic bead activated cell sorting (MACS), a method of separating particles based on their ability to bind to magnetic beads (0.5-100 μm diameter). A variety of useful modifications can be made on magnetic beads, including the covalent addition of antibodies that specifically recognize particular cell surface molecules or haptens. The magnetic beads are then mixed with the cells to bind them. The cells are then passed through a magnetic field to isolate cells having specific cell surface markers. In one embodiment, these cells may then be isolated and re-mixed with magnetic beads coupled with antibodies to additional cell surface markers. The cells are then passed through a magnetic field to separate the cells that bind the two antibodies. The cells can then be diluted into separate trays, such as microtiter trays, for clonal isolation.

Placental perfusate cells can be isolated using other methods known in the art, e.g., selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection); based on differential cell agglutination in the mixed population, for example, using soybean agglutinin; freezing and thawing process; filtering; conventional and zonal centrifugation; centrifugal elutriation (countercurrent centrifugation); separating by unit gravity; distributing the reflux; electrophoresis; and so on.

5.3 methods of Using hematopoietic cells such as cord blood cells and human placental perfusate cells

In one aspect, provided herein are methods of transplanting hematopoietic cells in a subject, e.g., a human subject, comprising administering hematopoietic cells in combination with mononuclear cells (HPCs) from human placental perfusate, e.g., human placental perfusate. The HPCs can be human placental perfusate, total nucleated cells from placental perfusate, or any population, subpopulation, or combination of mononuclear cells from human placental perfusate described herein, including those enriched or depleted for a particular population or subpopulation. Sources of hematopoietic cells that can be used in the methods of transplanting hematopoietic cells described herein include, for example, bone marrow or cells therein, peripheral blood or cells therein, and umbilical cord blood or cells therein. As used herein, hematopoietic cells of these sources are collectively referred to as "HT cells.

In one embodiment, provided herein is a method of transplanting HT cells, e.g., human umbilical cord blood cells (UCB) cells, e.g., human umbilical cord blood, in a subject, e.g., a human subject, comprising administering HT cells, e.g., human umbilical cord blood cells (UCB) cells, e.g., human umbilical cord blood, and combining mononuclear cells (HPCs) from human placental perfusate, e.g., human placental perfusate. In one embodiment, the HT cells, e.g., human UCB, are not associated with a subject. In a specific embodiment, HT cells, e.g., UCB cells, e.g., human UCB, are not matched to the subject moiety. In another embodiment, HPCs, such as human placental perfusate, are not associated with the subject. In one embodiment, HPCs, such as human placental perfusate, are not matched to portions of the subject. In another embodiment, HPCs, such as human placental perfusate, are not matched to the subject. In yet another embodiment, the HT cells, e.g., human UCB, are not associated with the subject and the HPCs, e.g., human placental perfusate, are not associated with the subject. In yet another embodiment, the HT cells, e.g., human UCB, are not associated with the subject and are not partially matched and the HPCs, e.g., human placental perfusate, are not associated with the subject and are not partially matched or are not matched to the subject. In one embodiment, HPCs, e.g., human placental perfusate, are unrelated and mismatched to HT cells, e.g., human UCB cells, e.g., UCB. In one embodiment, HPCs, e.g., human placental perfusate, are unrelated and mismatched to HT cells, e.g., human UCB cells such as UCB, and the subject.

As used herein, "related" in the context of UCB or HPC refers to autologous or primary or secondary blood relatives, unless otherwise indicated. For example, UCB associated with a subject refers to UCB from the subject's own body, or UCB from a subject's primary or secondary blood relative. In another embodiment, the UCB associated with HPC refers to the UCB and HPC from the same donor, or a donor of one or two blood relatives. Similarly, "unrelated" in these contexts refers to relationships further than the second blood relative, unless otherwise indicated.

As used herein, "matched" in the context of UCBs or cells from human placental perfusate (e.g., HPCs) refers to HLA matched unless otherwise indicated. Furthermore, "partial matching" as used herein in the context of UCBs or cells from human placental perfusate (e.g., HPC) refers to the case of HLA locus matching at 3/6, 4/6, or 5/6. Likewise, "mismatch," as used herein, in the context of UCB or cells from human placental perfusate (e.g., HPC), refers to HLA locus matching at 0/6, 1/6, or 2/6, unless otherwise indicated. "match," "partial match," and "mismatch" may refer, for example, to the relationship between HT cells, e.g., UCB cells, and HPCs; relationship between HT cells, e.g. UCB cells, units; and/or the relationship between HT cells, e.g., UCB cells, and/or HPCs and the subject that is the recipient of the cells.

In certain embodiments, the method comprises administering one unit of UCB or cells therein. In another embodiment, the methods presented herein comprise administering multiple units of UCB or cells therein. For example, the methods presented herein can include administering 2, 3, or 4 units of UCB or cells therein. In cases where more than one unit of HT cells, e.g., UCB cells, is used, in certain embodiments, at least a portion of the HT cells, e.g., UCB cells, may be unrelated to the subject, to the HPCs, and/or to other portions of the HT cells, e.g., UCB cells (e.g., other UCB cell units). In cases where more than one unit of HT cells, e.g., UCB cells, is used, in certain embodiments, at least a portion of the HT cells, e.g., UCB cells, may not match or partially match the subject, the HPCs, and/or other portions of the HT cells, e.g., UCB cells (e.g., other UCB cell units). In another embodiment, the methods presented herein can comprise administering less than one unit of HT cells or UCB, or cells therein. For example, the methods presented herein can include administering 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 units of HT cells or UCB, or cells therein. In particular embodiments, the methods presented herein may comprise administering a specific number of units (less than 1, 1, or greater than 1) in multiple administrations.

In another aspect, provided herein is a method for inducing chimerism in a subject, comprising administering to the subject a combination of HT cells, e.g., UCB cells, such as UCB, and HPCs, e.g., human placental perfusate, wherein at least a portion of the HT cells, e.g., UCB cells, are not partially matched to the subject, and/or the HPCs are not matched or partially matched to the subject, thereby inducing chimerism in the subject. As used herein, unless otherwise indicated, "chimera" refers to the presence of non-autologous DNA in a subject, e.g., DNA from cells that are not matched or partially matched relative to the recipient subject.

In one embodiment of the method, more than one unit of HT cells, e.g., UCB cells, is administered to the subject, e.g., 2, 3, or 4 units of HT cells, e.g., UCB cells, are administered to the subject. In particular embodiments where more than one unit of HT cells, e.g., UCB cells, are administered to a subject, the method of inducing chimerism may result in multiple chimerism, i.e., a chimerism may be produced that involves greater than 1 up to all of the administered HT cells, e.g., UCB cell units or progeny thereof.

In another embodiment of the method, a chimera involving HPCs or progeny thereof may be produced. . In yet another embodiment, chimeras involving HT cells, e.g., UCB cells (including multiple chimeras in cases where more than one unit of HT cells, e.g., UCB cells, is administered), or progeny thereof, and HPCs, or progeny thereof, may be produced.

In yet another embodiment of the method, the HT cells, e.g., UCB cells, are not subject-related. In the case where more than one unit of HT cells, e.g., UCB, is administered, one or more units of HT cells, e.g., UCB cells, may be unrelated to the subject. In one embodiment of the method, HPCs are independent of the subject and may additionally be independent of HT cells, such as UCB cells. In yet another embodiment of the method, both HT cells, e.g., UCB cells and HPCs, are independent of the subject.

In certain embodiments of the method, chimeras (including HT cells such as UCB cells or progeny thereof, or one or both of HPC or progeny thereof) are first detected in the subject within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 days or by administering a combination of more HT cells such as UCB cells to the subject.

Chimeras can be detected using methods well known in the art. For example, blood samples can be used to detect chimeras. In one embodiment, chimeras are detected using a Polymerase Chain Reaction (PCR) -based method, for example, by short tandem repeat assays. In one embodiment, the detection of chimeras following hematopoietic stem cell transplantation involves identifying the genetic profile of the recipient and donor and then evaluating the extent of the mixture in the recipient's blood, bone marrow or other tissue. Chimerism testing by DNA (transplantation assay) was done using methods commonly used in human identity identification and by analysis of genomic polymorphisms called Short Tandem Repeat (STR) loci. In one embodiment, quantification (e.g., using short tandem repeat experiments) of peripheral blood donor chimeras (UCB/s and perfusate cells) (whole blood, NK and T cells) is assessed at days 7,14, 30, 60, 100 and 180 (+/-10 days), and quantification of peripheral blood recipient chimeras (e.g., using short tandem repeat experiments) is assessed at baseline together with donor cell (UCB and perfusate cell) chimeras at baseline.

In yet another aspect, provided herein is a method for cell transplantation in a subject, comprising administering to the subject HT cells, e.g., human UCB cells such as UCB, and HPCs, e.g., human placental perfusate, in combination, wherein at least a portion of the HT cells, e.g., UCB cells, partially match the subject, and/or the HPCs are not matched or partially matched to the subject, such that cell transplantation occurs in the subject. In certain embodiments, cell transplantation comprises transplantation of HT cells, such as UCB cells, or progeny thereof. In certain other embodiments, the cell transplantation comprises transplantation of HPCs or progeny thereof. In other embodiments, transplantation includes transplantation of HT cells, e.g., UCB cells or progeny thereof, and HPCs or progeny thereof.

In one embodiment of the method, the HT cells, e.g., UCB cells, are not associated with the subject. In a specific embodiment, HT cells, such as UCB cells, are not matched to the subject moiety. In another embodiment, HPCs are independent of the subject and may be otherwise independent of HT cells, e.g., UCB cells. In one embodiment, the HPC does not match the object portion. In another embodiment, the HPC does not match the object. In yet another embodiment, the UCB cells are not subject-related and HPCs are not subject-related. In yet another embodiment, HT cells, such as UCB cells, are not subject-independent and partially mismatched and HPCs are not subject-independent and partially mismatched or are not subject-mismatched. In certain embodiments, the methods presented herein exhibit enhanced engraftment relative to administration of HT cells, e.g., UCB cells, alone.

Transplantation can be detected using methods known in the art. For example, in one embodiment, the absolute neutrophil count can be from day 0 to 3 days after nadir is reached>500/mm³And until a platelet count of ≥ 20,000/mm is measured over 3 consecutive days, independent of platelet infusion for a minimum of 7 days, on 3 different days³Whole blood differential counts were performed every 1-3 days. As used herein, "neutrophil transplantation" refers to an absolute neutrophil count of greater than 500/mm³The first 3 days after the nadir of the neutrophils. As used herein, "platelet transplantation" to blood cells ≧ 20,000/mm without infusion³After 7 days, the platelet count was 20,000/mm or more³For 3 consecutive days.

In certain embodiments, cell transplantation is detected in the subject by administering more HT cells, e.g., a UCB cell combination HPC, to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 days, or 2 months, 2.5 months, 3 months, or more to the subject.

In certain embodiments, the methods presented herein comprise administering one unit of HT cells, e.g., UCB cells such as UCB. In another embodiment, the methods presented herein comprise administering multiple units of HT cells, e.g., UCB cells such as UCB. For example, the methods presented herein can include administering 2, 3, or 4 units of HT cells, e.g., UCB cells such as UCB.

In yet another aspect, provided herein is a method for reducing the duration or severity of GVHD in a subject, comprising administering to the subject a combination of HT cells, e.g., human UCB cells such as UCB, and HPCs, e.g., human placental perfusate, wherein at least a portion of the HT cells, e.g., UCB cells, partially match the subject, and/or the HPCs are not matched or partially matched with the subject, whereby a reduction in the duration or severity of GVHD in the subject occurs.

In one embodiment of the method, the HT cells, e.g., UCB cells, are not associated with the subject. In a specific embodiment, HT cells, such as UCB cells, are not matched to the subject moiety. In another embodiment, HPCs are independent of the subject and may be otherwise independent of HT cells, e.g., UCB cells. In one embodiment, the HPC does not match the object portion. In another embodiment, the HPC does not match the object. In yet another embodiment, the UCB cells are not subject-related and HPCs are not subject-related. In yet another embodiment, HT cells, such as UCB cells, are not subject-independent and partially mismatched and HPCs are not subject-independent and partially mismatched or are not subject-mismatched. In certain embodiments, the methods presented herein demonstrate reduced duration or severity of GVHD as compared to administration of HT cells, e.g., UCB cells, alone.

In certain embodiments, the methods presented herein comprise administering one unit of HT cells, e.g., UCB cells such as UCB. In another embodiment, the methods presented herein comprise administering multiple units of HT cells, e.g., UCB cells such as UCB. For example, the methods presented herein can include administering 2, 3, or 4 units of HT cells, e.g., UCB cells such as UCB.

In another aspect, provided herein is a method for treating an individual having sarcopenia, comprising administering to the individual placental perfusate or any of the cell populations or subpopulations set forth herein, or any combination thereof, in an amount sufficient to produce a detectable improvement or decrease in the extent of worsening in one or more symptoms of sarcopenia, comprising administering to the subject a combination of HT cells, e.g., human UCB cells such as UCB, and HPCs, e.g., human placental perfusate.

In yet another aspect, provided herein is a method for treating an individual having a central nervous system injury, disease, or disorder, comprising administering to the individual placental perfusate, or any of the cell populations or subpopulations given herein, or any combination thereof, in an amount sufficient to produce a detectable improvement or decrease in the extent of worsening in one or more symptoms of the central nervous system injury, disease, or disorder, comprising administering to the subject a combination of HT cells, e.g., human UCB cells such as UCB, and HPCs, e.g., human placental perfusate. In a specific embodiment, the central nervous system injury, disease, or disorder is an ischemic encephalopathy (e.g., hypoxic ischemic encephalopathy).

In certain embodiments, the methods presented herein comprise administering HT cells, e.g., UCB cells such as UCB, concurrently with HPCs, e.g., human placental perfusate. In one embodiment, the cells are administered to the subject simultaneously. In another embodiment, the HT cells, e.g., UCB cells and HPCs, are administered to the subject within 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 18, or 24 hours or more, or within 1, 2, 3, 4, 5, 6, or 7 days or more. In one embodiment, HT cells, e.g., UCB cells such as UCB, are administered to a subject, followed by administration of HPCs, e.g., human placental perfusate, e.g., within 1 hour of administration of UCB, or within a minimum time required to confirm that the subject does not exhibit an adverse reaction to UCB administration.

The methods provided herein can exhibit advantages including, for example, reducing the time to cell transplantation, limiting the time to neutropenia in a subject, limiting the time to thrombocytopenia in a subject, establishing chimeras, and reducing the severity or duration of GVHD, or preventing GVHD, relative to administration of HT cells, e.g., UCB cells such as UCB, alone.

The ratio of administration of HT cells, e.g., UCB cells, and HPCs can vary. The proportion of administration of HT cells, e.g., UCB cells, and HPCs can be determined at the discretion of a person skilled in the art. In certain embodiments, the ratio of HT cells, e.g., UCB cells, to HPCs is about 100,000,000:1, 50,000,000:1, 20,000,000:1, 10,000,000:1, 5,000,000:1, 2,000,000:1, 1,000,000:1, 500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1, 10,000:1, 5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1: 1; 1: 2; 1: 5; 1: 10; 1: 100; 1: 200; 1: 500; 1:1,000; 1:2,000; 1:5,000; 1:10,000; 1:20,000; 1:50,000; 1:100,000; 1:500,000; 1:1,000,000; 1:2,000,000; 1:5,000,000; 1:10,000,000; 1:20,000,000; 1:50,000,000 or about 1:100,000,000. In certain embodiments, the ratio of HT cells, e.g., UCB cells, to HPCs is about 20:1 to about 1:20, or about 1:10, about 1:5, about 1:1, about 5:1, or about 10: 1.

Administration of HT cells, such as UCB cells and HPCs, can be performed using any cell administration technique known in the art. In one embodiment, administration is intravenous, e.g., intravenously, e.g., via IV, PICC line, midline, etc. For example, HT cells such as UCB cells and HPCs can be administered to a subject in any pharmaceutically or medically acceptable manner, including injection or infusion, in separate compositions or in a single composition. In certain embodiments, the compositions may be formulated as injectable compositions (e.g., WO96/39101, incorporated herein by reference in its entirety).

In certain embodiments, HT cells, such as UCB cells or HPCs, are administered parenterally to a subject. The term "parenteral" as used herein includes subcutaneous injections, intravenous, intramuscular, intraarterial injection or infusion techniques. In certain embodiments, HT cells, such as UCB cells or HPCs, are administered to a subject intravenously. In certain other embodiments, HT cells, such as UCB cells or HPC cells, are administered to a subject intraventricularly.

HT cells, such as UCB cells and HPCs, can be packaged individually or in combination in any pharmaceutically acceptable carrier. HT cells, such as UCB cells or HPCs, can be loaded, stored or transported in any pharmaceutically or medically acceptable container, such as a blood bag, transfer bag, plastic tube, syringe, vial, and the like.

Administration of HT cells, e.g., UCB cells and/or HPCs, to a subject may be performed one or more times. In certain embodiments, the administration is performed once. In certain embodiments, administration is performed a plurality of times, e.g., 2, 3, 4, or more times. In certain embodiments, HT cells, e.g., UCB cells, are administered multiple times. In certain embodiments, the HPCs are administered multiple times.

In certain embodiments, the amount of cord blood or cells obtained therefrom (e.g., total nucleated cells of cord blood) administered to a subject according to the methods described herein can be determined based on the amount of cells present in the cord blood. The amount or number of UCB administered to a subject or cells obtained therefrom (e.g., total nucleated cells of umbilical cord blood) and/or human placental perfusate or HPC or total nucleated cells obtained therefrom depends on the source of, the severity or nature of, the disorder or condition of, the age, weight, and physical condition of the subject, etc., the umbilical cord blood or cells obtained therefrom (e.g., total nucleated cells of umbilical cord blood) and/or human placental perfusate or HPC or total nucleated cells obtained therefrom. In certain embodiments, about 0.01 to about 0.1, about 0.1 to about 1, about 1 to about 10, about 10 to about 10, per kilogram body weight of the subject is administered²About 10²To about 10³About 10³To about 10⁴About 10⁴To about 10⁵About 10⁵To about 10⁶About 10⁶To about 10⁷About 10⁷To about 10⁸Or about 10⁸To about 10⁹Cord blood cells (e.g., total nucleated cells of cord blood), human placental perfusate or cells obtained therefrom (e.g., HPCs or total nucleated cells from placental perfusate), or total cord blood cells and cells obtained from placental perfusate (e.g., HPCs or total nucleated cells). In various embodiments, at least about 0.1, 1, 10, or 10 per kilogram body weight of the subject is administered²、10³、10⁴、10⁵、10⁶、10⁷、10⁸Or 10⁹The cord blood cells (e.g.,total nucleated cells from umbilical cord blood), cells obtained from placental perfusate (e.g., HPCs or total nucleated cells from placental perfusate), or umbilical cord blood cells and cells obtained from placental perfusate.

In a specific embodiment, at least about 0.5x10 is administered per kilogram of body weight of the subject⁶、1.0x10⁶、1.5x10⁶、2.0x10⁶、2.5x10⁶、3.0x10⁶、3.5x10⁶、4.0x10⁶、4.5x10⁶、5.0x10⁶、5.5x10⁶、6.0x10⁶、6.5x10⁶、7.0x10⁶、7.5x10⁶、8.0x10⁶、8.5x10⁶、9.0x10⁶、9.5x10⁶、1.0x10⁷、1.5x10⁷、2.0x10⁷、2.5x10⁷、3.0x10⁷、3.5x10⁷、4.0x10⁷、4.5x10⁷、5.0x10⁷、5.5x10⁷Or 6.0x10⁷Cord blood cells (e.g., total nucleated cells from cord blood), cells obtained from placental perfusate (e.g., HPCs or total nucleated cells from placental perfusate), or cord blood cells and cells obtained from placental perfusate (e.g., HPCs or total nucleated cells from placental perfusate). In a more specific embodiment, at least about 0.5x10 is administered per kilogram of body weight of the subject⁶、1.0x10⁶、1.5x10⁶、2.0x10⁶、2.5x10⁶、3.0x10⁶、3.5x10⁶、4.0x10⁶、4.5x10⁶Or 5.0x10⁶Cells obtained from placental perfusate (e.g., HPCs or total nucleated cells from placental perfusate). In a more specific embodiment, at least about 1.5x10 is administered per kilogram of body weight of the subject⁷，2.0x10⁷，2.5x10⁷，3.0x10⁷，3.5x10⁷，4.0x10⁷，4.5x10⁷，5.0x10⁷，5.5x10⁷Or 6.0x10⁷Cord blood cells (e.g., total nucleated cells of cord blood). In various embodiments, up to about 10 is administered per kilogram of body weight of the subject⁴、10⁵、10⁶、10⁷、10⁸Or 10⁹Umbilical cord bloodCells, cells obtained from placental perfusate (e.g., HPCs or total nucleated cells from placental perfusate), or umbilical cord blood cells and cells obtained from placental perfusate (e.g., HPCs or total nucleated cells from placental perfusate). In a specific embodiment, up to about 0.5x10 is administered per kilogram of body weight of the subject⁶、1.0x10⁶、1.5x10⁶、2.0x10⁶、2.5x10⁶、3.0x10⁶、3.5x10⁶、4.0x10⁶、4.5x10⁶、5.0x10⁶、5.5x10⁶、6.0x10⁶、6.5x10⁶、7.0x10⁶、7.5x10⁶、8.0x10⁶、8.5x10⁶、9.0x10⁶、9.5x10⁶、1.0x10⁷、1.5x10⁷、2.0x10⁷、2.5x10⁷、3.0x10⁷、3.5x10⁷、4.0x10⁷、4.5x10⁷、5.0x10⁷、5.5x10⁷Or 6.0x10⁷Cord blood cells (e.g., total nucleated cells from cord blood), cells obtained from placental perfusate (e.g., HPCs or total nucleated cells from placental perfusate), or cord blood cells and cells obtained from placental perfusate (e.g., HPCs or total nucleated cells from placental perfusate). In a more specific embodiment, up to about 0.5x10 is administered per kilogram of body weight of the subject⁶、1.0x10⁶、1.5x10⁶、2.0x10⁶、2.5x10⁶、3.0x10⁶、3.5x10⁶、4.0x10⁶、4.5x10⁶Or 5.0x10⁶Cells obtained from placental perfusate (e.g., HPCs or total nucleated cells from placental perfusate). In a more specific embodiment, up to about 1.5x10 is administered per kilogram of body weight of the subject⁷、2.0x10⁷、2.5x10⁷、3.0x10⁷、3.5x10⁷、4.0x10⁷、4.5x10⁷、5.0x10⁷、5.5x10⁷Or 6.0x10⁷Cord blood cells (e.g., total nucleated cells of cord blood).

In particular embodiments of the above embodiments, the cord blood cells (e.g., total nucleated cells of cord blood) or cells obtained from placental perfusate(e.g., total HPCs or nucleated cells from placental perfusate) are CD34+ cells. In certain embodiments, at least about 10 is administered per kilogram of body weight⁴To about 10⁷CD34+ cells. The CD34+ cells may be derived from cord blood alone, or may be derived from cord blood and placental perfusate.

HT cells, e.g., UCB cells such as UCB and HPCs, e.g., placental perfusate, can be delivered in a volume appropriate to the size of the subject. Typical adult blood volumes are about 85-100mL/kg body weight. Thus, adult blood volumes range from about 40mL to about 300 mL. In various embodiments, therefore, HT cells, e.g., UCB cells such as UCB, and HPCs, e.g., placental perfusate, are administered in an amount of about 0.5mL, 1.0mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 11mL, 12mL, 13mL, 14mL, 15mL, 16mL, 17mL, 18mL, 19mL, 20mL, 21mL, 22mL, 23mL, 24mL, 25mL, 26mL, 27mL, 28mL, 29mL, or about 30mL, or more, of the total volume. The administration of the volume may be a single administration or multiple administrations. The volume of cord blood or cord blood cell count, or the time of administration of the human placental perfusate or cells obtained therefrom (e.g., HPCs or total nucleated cells from placental perfusate) may be, for example, 0.5 hour, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, or more.

In some embodiments, small infusions of 20mL or less are performed using a syringe. Large volume infusions can be administered by infusion sets, for example, over 1-4 hours.

The methods provided herein can be performed on any subject in need thereof. In one aspect, the subject is in need of hematopoietic reconstitution, partial reconstitution, or augmentation. In some embodiments, the subject is a human subject. In certain embodiments, the subject is an adult subject. In certain embodiments, the subject is 25 years old or younger. In some embodiments, the subject is an infant.

In certain embodiments, prior to the methods set forth herein, e.g., transplantation, methods of inducing chimerism, and/or methods of transplantation, the subject has been modulated for spinal cord inhibition using, e.g., TBI, clofarabine, and/or Ara-C1; attenuation modulation using, for example, busulfan, fludarabine and/or alemtuzumab; or radiation therapy or other treatments such as immunosuppressive treatments or treatments to reduce blood cell counts.

In a particular aspect, the methods provided herein can be used as a method for treating a metabolic disorder, such as a subject in need of an inborn errors of metabolism, adrenoleukodystrophy, mucopolysaccharidosis, niemann-pick disease, metachromatic leukodystrophy, wolman disease, krabbe disease, gaucher's disease, fucosidosis, or Batten disease.

In another particular aspect, the methods provided herein can be used as a method of treating a hematologic disorder or malignancy, for example, a subject having a lymphohematopoietic malignancy, myelodysplastic syndrome, oligomegakaryocytic thrombocytopenia, leukemia, such as Acute Lymphoblastic Leukemia (ALL) and Acute Myeloid Leukemia (AML), granulocytopenia, sickle cell disease, such as sickle cell anemia, β -thalassemia (e.g., b-thalassemia gravis), severe combined immunodeficiency disease, bone marrow failure, or anemia, such as severe aplastic anemia or Diamond-Blackfan anemia, in need thereof.

The term "treatment" as used herein refers to a reduction or amelioration of the progression, severity and/or duration of a disease or disorder or any parameter or symptom of the disease or disorder. A treatment may be considered effective if the subject is alive, or if the disorder or condition to be treated has a measurable improvement in any way as a result of the treatment. Such an improvement can be manifested, for example, by one or more measurable indicators, including, for example, a detectable change in a physiological condition or a set of physiological conditions associated with a particular disease, disorder, or condition. A treatment is also considered effective if the indicator shows a response to the treatment by a change to within or near normal values for individuals of, for example, similar age, as compared to the expected value of the indicator or indicators in the absence of the treatment.

In certain embodiments of the methods provided herein, the methods provided herein may be used as a first therapy in combination with one or more second therapies in the treatment of a disorder or condition. The second therapy includes, but is not limited to, surgery, hormonal therapy, immunotherapy, phototherapy, or treatment with certain drugs. Example therapies that may be combined with the methods provided herein include control of ambient temperature; supplying oxygen; a ventilator or ventilator; peripheral blood transfusion; supplementing iron; vein nutrition; phototherapy; performing surgery; therapeutic agents for metabolic disorders or hematological diseases (including hematological tumors); antibiotics or antiviral drugs; anti-inflammatory agents (e.g., steroidal anti-inflammatory compounds, non-steroidal anti-inflammatory (NSAID) compounds); nitric oxide; an antihistamine; an immunosuppressant; and immunomodulatory compounds (e.g., TNF- α inhibitors).

5.4 cord blood cells

Cord blood (also referred to herein as UCB or "cord blood") for use according to the present disclosure may be collected in any medically or pharmaceutically acceptable manner and may be present in a composition, e.g., a pharmaceutical composition. Various methods have been described for collecting cord blood. See, for example, U.S. patent nos. 6,102,871; U.S. patent No. 6,179,819 and U.S. patent No. 7,147,626, the contents of each of which are incorporated by reference in their entirety. Conventional techniques for collecting cord blood are based on the use of a needle or cannula, which is used with the aid of gravity. Cord blood may be collected, for example, into a blood bag, transfer bag, or sterile plastic tubing.

In some embodiments, the cord blood is obtained from a commercial cord blood bank (e.g., lifebank usa, etc.). In another embodiment, cord blood is collected from the postpartum mammalian umbilical cord and used immediately (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after collection). In other embodiments, the cord blood for treating a subject is cryopreserved cord blood. Cord blood may be collected from a single cord or multiple cords.

In certain embodiments, HT cells, such as UCB cells, are unrelated to the subject and/or HPCs. In another embodiment, HT cells, such as UCB cells, are not partially matched to the subject and/or HPCs. In yet another embodiment, HT cells, such as UCB cells, are not matched to HPCs. In yet another embodiment, HT cells, such as UCB cells, are not HPC-associated and mismatched. In particular embodiments, the UCB matches the subject at 3/6, 4/6, or 5/6HLA loci. In particular embodiments, HT cells, e.g., from adult sources, are matched to a subject at 6/8, 7/8, or 8/8HLA loci.

In some embodiments, the cord blood is prepared from a premature umbilical cord. In other embodiments, the cord blood is prepared from umbilical cord. In certain embodiments, the cord blood is obtained from postpartum mammalian umbilical cord produced in term. In other embodiments, the umbilical cord blood is obtained from a post-partum mammalian umbilical cord that was born prematurely. In some embodiments, the umbilical cord is the umbilical cord of an infant born at about 23 to about 25 weeks of gestation. In some embodiments, the umbilical cord is the umbilical cord of an infant born at about 26 to about 29 weeks of gestation. In some embodiments, the umbilical cord is the umbilical cord of an infant born at about 30 to about 33 weeks of gestation. In some embodiments, the umbilical cord is the umbilical cord of an infant born at about 34 to about 37 weeks of gestation. In some embodiments, the umbilical cord is the umbilical cord of an infant born at about 37 to about 42 weeks of gestation.

The cord blood or cells obtained therefrom (e.g., total nucleated cells or stem cells from which it is derived) may be collected from a single individual (i.e., as a single unit) for administration, or may be mixed with other units. In certain embodiments, the cord blood or cells obtained therefrom (e.g., wherein the total nucleated cells or stem cells derived) are stored prior to use. When cord blood is mixed from multiple cords, the mixed cord blood may comprise cord blood from term production only, cord blood from combined term production, or cord blood from premature delivery only. For example, umbilical cord blood from a preterm infant may be mixed with, for example, umbilical cord blood from other preterm infants, umbilical cord blood from term birth only, or a mixture of umbilical cord blood from preterm and term born placentas. Cord blood, including autologous or allogeneic cord blood, may also be mixed with peripheral blood. In certain embodiments, cord blood from a preterm infant is used because such cord blood contains a relatively high number of CD34+ stem cells per unit volume as compared to cord blood from term birth. In certain embodiments, one unit of cord blood comprises a sufficient number of cells to administer at least about 1.0x10 per kilogram body weight of the subject⁶、1.5x10⁶、2.0x10⁶、1.5x10⁶、2.0x10⁶、2.5x10⁶、3.0x10⁶、3.5x10⁶、4.0x10⁶、4.5x10⁶、6.0x10⁶、6.5x10⁶、7.0x10⁶、7.5x10⁶、8.0x10⁶、8.5x10⁶、9.0x10⁶、9.5x10⁶、1.0x10⁷、1.5x10⁷、2.0x10⁷、2.5x10⁷、3.0x10⁷、3.5x10⁷、4.0x10⁷、4.5x10⁷、5.0x10⁷、5.5x10⁷Or 6.0x10⁷Cells obtained from the cord blood, e.g., total nucleated cells of the cord blood. In certain embodiments, one unit of cord blood or cells obtained therefrom is administered. In certain embodiments, less than one unit is administered. In certain embodiments, more than one unit is administered, e.g., 2 or more (e.g., 2, 3, 4, 5, 6, or more) units are administered.

Examples

6.1 example 1 human placental perfusate cell compositions

This example shows the determination of human placental perfusate compositions by cell type and associated phenotype.

Human Placental Perfusate (HPP) was obtained as described in section 5.2 above. The donor-matched HPP and human cord blood (HUCB) bags were thawed separately at 37 deg.C and then diluted with an equal volume of thawing solution (IMDM (Cat #30-2005, ATCC) + 2% FBS (Cat # SH30070.03, Hyclone) + P/S (Cat #15140-122, Gibco)). The diluted cell mixture was spun at 400g for 8min (using a 15ml conical tube) and if a 50ml conical tube was used for 10 min. Resuspend the cell pellet to 1X10 with FACS buffer (PBS (Cat #10010-023, Gibco) + 2% FBS + P/S)⁷And/ml. RBCs (red blood cells) were lysed by adding ammonium chloride solution (Cat #07850, Stem Cell) on ice at a ratio of ammonium chloride to cells 9:1 for 10 min. After RBC lysis, the samples were spun at 400g for 5min and then washed twice with FACS buffer. The cell pellet was then resuspended with a Cytofix/cytoperm solution (Cat #554722, BD Biosciences) at 4 ℃ for 20 minutes to 1ml per 1X10⁷A cell. The samples were washed twice with FACS buffer and then stained with fluorochrome-conjugated antibody for 20min at RT (room temperature) in the dark. The plaques (panel) are listed in tables 1 and 2. Antibody information is listed in table 3. For dyed samplesFACS buffer was washed twice and resuspended in 200 μ l FACS buffer for data collection: the 9 color plates were from FACS Aria (BD Biosciences) and the 6 color plates were from FACS Canto II (BD Biosciences) according to the instructions provided by the manufacturer. Data analysis was performed by flowjo (tree star). Statistical analysis was performed using paired student t-test.

Table 1: 9 color table board

Table 2: 6 color table board

Table 3: antibody information for phenotypic characterization.

Monocytes from HPPs were analyzed to determine the composition of various monocyte types. Table 4 details the cell types identified:

table 4: human placental perfusate composition

6.2 example 2 Total nucleated cell count in human placental perfusate

This example shows a complete nucleated cell count assay of human placental perfusate and cord blood units.

Total nucleated cell counts were determined by treatment of 43 pairs of donor-matched HPP and HUCB units. The average total nucleated cell count for a single unit of HPP was 135 million cells. The average total nucleated cell count for a single unit of HUCB was-666 million cells (fig. 1).

6.3 example 3 population of progenitor cells in human placental perfusate

This example shows CD34 in human placental perfusate and cord blood⁺CD45^-And CD34⁺CD45⁺Determination of cell population.

Subpopulations of human placental perfusate cells were determined using Fluorescence Activated Cell Sorting (FACS) (fig. 2A). CD34⁺A subset of cells is CD45^-And are therefore excluded from counting using the ISHAGE protocol (Barnett et al, 1999, Clin. Lab. Haem.21:301-308), a sequential gating strategy (FIG. 2B) with gating preferentially directed to CD45⁺A cell. A scheme was established using another sequential gate strategy, where the gate was preferentially directed to CD34⁺Cells to analyze CD34 in human placental perfusate⁺CD45^-And CD34⁺CD45⁺Both cells (fig. 2C). Using this protocol, a clear CD34⁺CD45^-The cell population is evident in human placental perfusate.

FACS cell sorting was performed as follows: the donor matched HPP and HUCB bags were thawed separately at 37 ℃ and then ammonium chloride RBC lysed. Then stained with FITC anti-human CD34(Cat #555821, BD Biosciences) and PE anti-human CD45(Cat #555483, BD Biosciences) for 15min at room temperature in the dark. After washing twice with FACS buffer, the samples were resuspended to 1x10⁷Per ml and manufactured by FACS Aria (BD Biosciences) usingThe sorting was performed according to the protocol provided by the supplier.

Human placental perfusate contained a greater proportion of CD34 than cord blood in donor-matched pairs as measured using FACS sorting protocols⁺Cells (FIGS. 3A-3B). Colony formation experiments using human placental perfusate cells showed CD34 after sorting⁺CD45⁺Cells and CD34⁺CD45^-And (4) growing the cells.

6.4 example 4 CD34 in human placental perfusate⁺Subgroup of

This example shows CD34 in human placental perfusate and cord blood⁺CD31⁺、CD34⁺KDR⁺And CD34⁺CXCR-4⁺And (4) determining the cell population.

Using the phenotypic characterization protocol as described in section 6.1, we determined HPP CD34⁺The cells contained specific HUCB CD34⁺Higher percentage of cells CD31⁺、KDR⁺And CXCR-4⁺Cells (fig. 4). These phenotypes are consistent with HPPs containing a population of angioblasts.

6.5 example 5: functional evaluation of human placental perfusate cells

This example shows the determination of the angiogenic properties of human placental perfusate cells compared to umbilical cord blood cells. As shown in figure 5, human placental perfusate showed higher angiogenic (vascularization) activity than umbilical cord blood in the experiments described herein.

HPP cells were obtained as described in section 5.5 above. HPP cells (FIG. 5, top left) were incubated with 10. mu.g/mL Dil-AC LDL (Cat # L3484, Life technology) for 4h at 37 ℃ and fluorescence photographs of lipoprotein uptake by endothelial cells from HPP were taken using Axiovert 200M (Zeiss) (FIG. 5, top right). In vitro functional assays were performed to assess the angiogenic properties of human placental perfusate cells. HPP cells obtained according to section 5.5 above were cultured in ECMATRIX^TMIncubate for 18-24 hours in 96-well plates to about 10⁶Cells per well, an in vitro angiogenesis assay kit (Chemicon cat # ECM625) was used, in which cells were cultured in the presence of TGF- β, FGF, plasminogen, tPA and matrix metalloproteinases. Microvessel formation was observed in human placental perfusate cell cultures (figure 5, bottom right). HUVECs (human umbilical vein endothelial cells) were used as positive controls (FIG. 5, bottom left). No significant tube formation was observed in cord blood cultures.

6.6 example 6 Primary progenitor cell population in human placental perfusate

This example shows various CD34 in human placental perfusate and cord blood⁺Determination of the progenitor cell population.

Using a phenotypic characterization protocol as described in section 6.1, we determined that human placental perfusate contains a substantially greater proportion of nestin than cord blood⁺/CD34⁺Cells (fig. 6). Nestin⁺CD34⁺Cells are suggested to be more primitive neuronal progenitors (Mii et al, j.cell biol., 2013).

Human placental perfusate contains a significantly greater number of immature hematopoietic stem cell populations (i.e., CD34) than umbilical cord blood⁺CD45^-，CD34⁺CD38^-) As shown in table 5. Similar to that shown in Table 2, a putative hemangioblast population (i.e., CD34) was found⁺C31⁺，CD34⁺KDR⁺And CD34⁺CXCR4⁺) More in human placental perfusate than in cord blood.

Table 5: human placental perfusate and primitive progenitor cells in umbilical cord blood.

6.7 example 7T-cell content in human placental perfusate

This example shows the determination of various T cell populations in human placental perfusate and cord blood.

Total HLA class I and II assessments and extensive immunophenotypic characterization of human placental perfusate and cord blood were performed using 9-color T cell FACS plates to describe T cell subsets, CD45RA, CD8, CD25, CD127, CD69, CD3, CCR7, HLADR and CD4, as shown in section 6.1 above.

As shown in table 6, the results show that human placental perfusate contains significantly lower T cell content than umbilical cord blood. Similarly, human placental perfusate cells had lower expression of class I and class II HLA (fig. 7). The relative proportions of specific T-cell populations expressing CD3, CD4, and/or CD8 were also determined for human placental perfusate and cord blood (fig. 8). The T cell content of human placental perfusate indicates, for example, that human placental perfusate cells are suitable for allogeneic mismatch transplantation.

Table 6: human placental perfusate and the T cell population in umbilical cord blood. "Hi" and "low" indicate the expression intensity of a particular phenotypic marker.

6.8 example 8T cell isolation, functional assessment and expansion

This example shows that T can be used for successful isolation, evaluation and amplification in human placental perfusate and cord blood_regMethods of cell populations. Similar methods can be used to isolate, evaluate and expand other populations or subpopulations of human placental perfusate cells.

Use can be made of a CD4 for a person⁺CD127^lowCD25⁺Complete kit for regulatory T cells (Cat #15861, Stem Cell) isolation of T from donor-matched HPP or HUCB, respectively_regA cell. From donor-matched HPP or HUCB, or T-free_regT isolated from donor-matched HPP or HUCB of cells, respectively_regCells can be evaluated by the in vitro magnetic bead T cell reaction (BTR) assay. Briefly, T cells from Peripheral Blood (PB) activated with anti-CD 3/CD28 magnetic beads can be co-cultured with the samples listed above for 5 days. Inhibition of CD4 and CD 8T cell proliferation can be detected by FACS.

Can use T_regAmplification kit (Cat #:130-Modulation of CD4⁺CD25⁺T cell reagentCassette (Cat #11363D, Life Technology) evaluation of T from two magnetic bead-based amplification kits of donor-matched HPP and HUCB, respectively_regAnd (4) expanding the cells. Members of the necrosis factor receptor family for enhancement may be used: OX40, 4-1BB to accomplish the improved capacity of expanding Treg cells for clinical use (Hippen et al, 2008)

Equivalence:

the scope of the invention should not be limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and such modifications are intended to fall within the scope of the appended claims.

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Scheme 1. composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 6 x10⁵CD34⁺A cell.

Scheme 2. the composition of scheme 1, wherein the composition further comprises a 2-fold greater amount of CD34⁺A cell.

Scheme 3. the composition of scheme 1, wherein the composition further comprises a 10-fold greater amount of CD34⁺A cell.

Scheme 4. the composition of scheme 1, wherein the composition further comprises a 50-fold greater amount of CD34⁺A cell.

Scheme 5. the composition of scheme 1, wherein the composition comprises substantially pure human placental perfusate CD34⁺A cell.

Scheme 6. composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 5x10⁵CD34⁺CD45^-A cell.

Scheme 7. the composition of scheme 6, wherein the composition further comprises a 2-fold greater amount of CD34⁺CD45^-A cell.

Scheme 8. the composition of scheme 6, wherein the composition further comprises a 10-fold greater amount of CD34⁺CD45^-A cell.

Scheme 9. the composition of scheme 6, wherein the composition further comprises a 50-fold greater amount of CD34⁺CD45^-A cell.

Scheme 10. the composition of scheme 6, wherein the composition comprises substantially pure human placental perfusate CD34⁺CD45^-A cell.

Scheme 11. composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 6 x10⁵CD34⁺CD31⁺A cell.

Scheme 12. the composition of scheme 11, wherein the composition further comprises a 2-fold greater amount of CD34⁺CD31⁺A cell.

Scheme 13. the composition of scheme 11, wherein the composition further comprises a 10-fold greater amount of CD34⁺CD31⁺A cell.

Scheme 14. the composition of scheme 11, wherein the composition further comprises a 50-fold greater amount of CD34⁺CD31⁺A cell.

Scheme 15. the composition of scheme 11, wherein the composition comprises substantially pure human placental perfusate CD34⁺CD31⁺A cell.

Scheme 16. composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 5x10⁵CD34⁺KDR⁺A cell.

Scheme 17. the composition of scheme 16, wherein the composition further comprises a 2-fold greater amount of CD34⁺KDR⁺A cell.

Scheme 18. the composition of scheme 16, wherein the composition further comprises a 10-fold greater amount of CD34⁺KDR⁺Cells。

Scheme 19. the composition of scheme 16, wherein the composition further comprises a 50-fold greater amount of CD34⁺KDR⁺A cell.

Scheme 20. the composition of scheme 16, wherein the composition comprises substantially pure human placental perfusate CD34⁺KDR⁺A cell.

Scheme 21. composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 5x10⁵CD34⁺CXCR4⁺A cell.

Scheme 22. the composition of scheme 21, wherein the composition further comprises a 2-fold greater amount of CD34⁺CXCR4⁺A cell.

Scheme 23. the composition of scheme 21, wherein the composition further comprises a 10-fold greater amount of CD34⁺CXCR4⁺A cell.

Scheme 24. the composition of scheme 21, wherein the composition further comprises a 50-fold greater amount of CD34⁺CXCR4⁺A cell.

Scheme 25. the composition of scheme 21, wherein the composition comprises substantially pure human placental perfusate CD34⁺CXCR4⁺A cell.

Scheme 26. composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 6 x10⁵CD34⁺CD38^-A cell.

Scheme 27. the composition of scheme 26, wherein the composition further comprises a 2-fold greater amount of CD34⁺CD38^-A cell.

Scheme 28. the composition of scheme 26, wherein the composition further comprises a 10-fold greater amount of CD34⁺CD38^-A cell.

Scheme 29. the composition of scheme 26, wherein the composition further comprises a 50-fold greater amount of CD34⁺CD38^-A cell.

Scheme 30. the composition of scheme 26, wherein the composition comprises substantially pure human placental perfusate CD34⁺CD38^-A cell.

Scheme 31. composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 7 x10⁵CD34⁺CD117^-A cell.

Scheme 32. the composition of scheme 31, wherein the composition further comprises a 2-fold greater amount of CD34⁺CD117^-A cell.

Scheme 33. the composition of scheme 31, wherein the composition further comprises a 10-fold greater amount of CD34⁺CD117^-A cell.

Scheme 34 the composition of scheme 31, wherein the composition further comprises a 50-fold greater amount of CD34⁺CD117^-A cell.

Scheme 35. the composition of scheme 31, wherein the composition comprises substantially pure human placental perfusate CD34⁺CD117^-A cell.

Scheme 36. composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 6 x10⁵CD34⁺CD140a⁺A cell.

Scheme 37. the composition of scheme 36, wherein the composition further comprises a 2-fold greater amount of CD34⁺CD140a⁺A cell.

Scheme 38. the composition of scheme 36, wherein the composition further comprises a 10-fold greater amount of CD34⁺CD140a⁺A cell.

Scheme 39. the composition of scheme 36, wherein the composition further comprises a 50-fold greater amount of CD34⁺CD140a⁺A cell.

Scheme 40. the composition of scheme 36, wherein the composition comprises substantially pure human placental perfusate CD34⁺CD140a⁺A cell.

Scheme 41. composition comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 3 x10⁵CD34⁺Nestin⁺A cell.

Scheme 42. the composition of scheme 40, wherein the composition further comprises a 2-fold greater amount of CD34⁺Nestin⁺A cell.

Scheme 43. the composition of scheme 40, wherein the composition further comprises a 10-fold greater amount of CD34⁺Nestin⁺A cell.

Scheme 44. the composition of scheme 40, wherein the composition further comprises a 50-fold greater amount of CD34⁺Nestin⁺A cell.

Scheme 45. the composition of scheme 40, wherein the composition is substantially pure human placental perfusate CD34⁺Nestin⁺A cell.

Scheme 46. compositions comprising isolated human placental perfusate, wherein the human placental perfusate comprises at least 3 x10⁴CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

Scheme 47. the composition of scheme 46, wherein the composition further comprises a 2-fold greater amount of CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

Scheme 48. the composition of scheme 46, wherein the composition further comprises a 10-fold greater amount of CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

Scheme 49. the composition of scheme 46, wherein the composition further comprises a 50-fold greater amount of CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

Scheme 50. the composition of scheme 46, wherein the composition is substantially pure human placental perfusate CD3⁺CD4⁺CD8^-CD25^hiCD127^lowA cell.

Scheme 51. the composition of any of schemes 1-50, wherein the human placental perfusate is isolated from a single placental perfusate.

Scheme 52. a method of treating a central nervous system injury, disease, or disorder in a subject, comprising administering to the subject a composition according to any of schemes 1-50 and hematopoietic cells from another source.

Scheme 53. the method of scheme 52, wherein said central nervous system injury, disease or disorder is hypoxic ischemic encephalopathy.

Scheme 54. a method of treating sarcopenia in a subject comprising administering to the subject a composition according to any one of schemes 1 to 50 and hematopoietic cells from another source.

A method of inducing chimerism in a subject comprising administering to the subject a composition according to any of claims 1-50 and hematopoietic cells from another source.

Scheme 56. a method for cell transplantation in a subject, comprising administering to the subject a composition according to any one of schemes 1-50 and hematopoietic cells from another source.

Scheme 57. A method of reducing the severity or duration of Graft Versus Host Disease (GVHD) in a subject comprising administering to the subject a composition according to any of schemes 1-50 and hematopoietic cells from another source.

Scheme 58. a method of treating a metabolic disorder in a subject comprising administering to the subject a composition according to any of schemes 1-50 and hematopoietic cells from another source.

Scheme 59. A method of treating a hematologic disease or hematologic malignancy in a subject, comprising administering to the subject a composition according to any one of schemes 1-50 and hematopoietic cells from another source.

Scheme 60. a composition as defined in any one of schemes 1-50 for use in a method comprising:

treating a central nervous system injury, disease, or disorder in a subject, preferably the central nervous system injury, disease, or disorder is hypoxic ischemic encephalopathy;

inducing a chimera in a subject;

for cell transplantation;

for reducing the severity or duration of Graft Versus Host Disease (GVHD) in a subject;

treating a metabolic disorder in a subject;

treating a hematologic or hematologic malignancy in a subject; or

Treating sarcopenia in a subject.

Scheme 61. the composition for use as in scheme 60, wherein the composition further comprises hematopoietic cells from another source.