阅读说明：本技术 用于治疗脑内出血的多潜能成体祖细胞 (Multipotent adult progenitor cells for the treatment of intracerebral hemorrhage ) 是由 R·梅斯 于 2020-01-30 设计创作，主要内容包括：本文所描述的发明的各方面涉及给予多潜能成体祖细胞用于治疗脑内出血。(Aspects of the invention described herein relate to the administration of multipotent adult progenitor cells for the treatment of intracerebral hemorrhage.)

1. A method of treating intracerebral hemorrhage in a subject, comprising administering to a subject in need thereof multipotent adult progenitor cells, which are not embryonic stem cells, embryonic germ cells, nor germ cells, can differentiate into at least one cell type of each of at least two of the endodermal, ectodermal and mesodermal embryonic lineages, and are allogeneic or xenogeneic with respect to the subject.

2. A method of treating intracerebral hemorrhage in a subject, comprising administering to a subject in need thereof multipotent adult progenitor cells, which are not embryonic stem cells, embryonic germ cells, or germ cells, that express telomerase and are allogeneic or xenogeneic to the subject.

3. A method of treating intracerebral hemorrhage in a subject, comprising administering to a subject in need thereof multipotent adult progenitor cells, which are not embryonic stem cells, embryonic germ cells, nor germ cells, that are positive for oct3/4 and are allogeneic or xenogeneic to the subject.

4. A method of treating intracerebral hemorrhage in a subject, comprising administering to a subject in need thereof multipotent adult progenitor cells that are not embryonic stem cells, embryonic germ cells, or germ cells, have undergone at least 40 cell doublings in culture prior to their use, and are allogeneic or xenogeneic to the subject.

5. A method according to any one of claims 2 to 4 wherein the cells can differentiate into at least one cell type of each of at least two of the endodermal, ectodermal and mesodermal embryonic lineages.

6. The method according to any one of claims 3-4, wherein the cells express telomerase.

7. The method according to 3, wherein the cell undergoes at least 40 doublings in culture prior to its use.

8. The method of claim 1, wherein said cells express telomerase and are positive for oct 3/4.

9. The method according to claim 1, wherein the cells express telomerase and have undergone at least 40 doublings in culture prior to their use.

10. The method according to claim 1, wherein said cells are positive for oct3/4 and undergo at least 40 doublings in culture prior to their use.

11. Use of a method according to claim 1 wherein the cells express telomerase, are positive for oct3/4, and undergo at least 40 doublings in culture prior to their use.

12. The method according to any one of claims 1-11, wherein the cell has a normal karyotype.

13. The method according to any one of claims 1-12, wherein the cell is non-tumorigenic.

14. The method according to any one of claims 1-13, wherein the cell is not immunogenic in the subject.

15. A method according to any one of claims 1 to 14 wherein the cells can differentiate into at least one cell type of each of the endodermal, ectodermal and mesodermal embryonic lineages.

16. The method according to any one of claims 1-15, wherein the cell is a mammalian cell.

17. The method according to any one of claims 1-16, wherein the cell is a human cell.

18. The method according to any one of claims 1 to 17, wherein the cells are derived from cells isolated from any one of placental tissue, umbilical cord blood, bone marrow, blood, spleen tissue, thymus tissue, spinal cord tissue, adipose tissue, and liver tissue.

19. The method according to any one of claims 1-18, wherein the cells are derived from bone marrow.

20. The method according to any one of claims 1-19, wherein the subject is a human.

21. The method according to any one of claims 1-20, wherein one or more doses of 10 are administered per kilogram body weight of the subject⁴To 10⁸And (c) isolating said cells.

22. The method according to any one of claims 1-21, wherein one or more doses of 10 are administered per kilogram body weight of the subject⁶To 5X 10⁷A main body composed ofA cell.

23. The method of any one of claims 1-22, wherein an antimicrobial agent, an antifungal agent, an antiviral agent, or a combination thereof is used concurrently.

24. The method according to any one of claims 1-23, wherein the cells are in a formulation comprising one or more additional pharmaceutically active agents.

25. The method according to any one of claims 1-24, wherein the cells are administered by a parenteral method, an intravenous method, or a stereotactic method.

26. A method according to any one of claims 1-25, wherein said cells are administered by an intravenous method, a method of treating intracerebral hemorrhage in a subject, comprising administering to a subject in need thereof multipotent adult progenitor cells, which are not embryonic stem cells, embryonic germ cells, nor germ cells, can differentiate into at least one cell type of each of at least two of the endodermal, ectodermal and mesodermal embryonic lineages, and are allogeneic or xenogeneic to the subject.

27. The method according to any one of claims 1-26, wherein immunosuppressive therapy is not administered adjunctively to the administration of the cells.

28. Use of a cell in the manufacture of a medicament for treating intracerebral hemorrhage in a subject, wherein the cell is a multipotent adult progenitor cell that is not a fetal stem cell, not an embryonic germ cell, nor a germ cell, that can differentiate into at least one cell type of each of at least two of the endodermal, ectodermal and mesodermal embryonic lineages, and is allogeneic or xenogeneic to the subject.

29. Use of a cell in the manufacture of a medicament for treating intracerebral hemorrhage in a subject, wherein the cell is a multipotent adult progenitor cell, that is not an embryonic stem cell, not an embryonic germ cell, nor a germ cell, that expresses telomerase and is allogeneic or xenogeneic to the subject.

30. Use of a cell in the manufacture of a medicament for treating intracerebral hemorrhage in a subject, wherein the cell is a multipotent adult progenitor cell that is not an embryonic stem cell, not an embryonic germ cell, nor a germ cell, that is positive for oct3/4 and is allogeneic or xenogeneic to the subject.

31. Use of cells in the manufacture of a medicament for treating intracerebral hemorrhage in a subject, wherein the cells are multipotent adult progenitor cells that are not embryonic stem cells, embryonic germ cells, or germ cells, undergo at least 40 cell doublings in culture prior to their use, and are allogeneic or xenogeneic to the subject.

32. Use according to any one of claims 29 to 31, wherein the cells can differentiate into at least one cell type of each of at least two of the endodermal, ectodermal and mesodermal embryonic lineages.

33. The use according to any of claims 30-31, wherein the cells express telomerase.

34. The use according to 30, wherein the cell has undergone at least 40 doublings in culture prior to its use.

35. The use according to claim 28, wherein said cells express telomerase and are positive for oct 3/4.

36. The use according to claim 28, wherein the cells express telomerase and have undergone at least 40 doublings in culture prior to their use.

37. The use according to claim 28, wherein said cells are positive for oct3/4 and undergo at least 40 doublings in culture prior to their use.

38. The use according to claim 28, wherein said cells express telomerase, are positive for oct3/4, and undergo at least 40 doublings in culture prior to their use.

39. The use according to any one of claims 28-38, wherein the cell has a normal karyotype.

40. The use according to any one of claims 28-39, wherein the cell is not tumorigenic.

41. The use according to any one of claims 28-40, wherein the cell is not immunogenic in the subject.

42. Use according to any one of claims 28 to 41, wherein the cells can differentiate into at least one cell type of each of the endodermal, ectodermal and mesodermal embryonic lineages.

43. The use according to any one of claims 28-42, wherein the cell is a mammalian cell.

44. The use according to any one of claims 28-43, wherein the cell is a human cell.

45. The use according to any one of claims 28 to 44, wherein the cells are derived from cells isolated from any one of placental tissue, umbilical cord blood, bone marrow, blood, spleen tissue, thymus tissue, spinal cord tissue, adipose tissue, and liver tissue.

46. The use according to any one of claims 28-45, wherein the cells are derived from bone marrow.

47. The use according to any one of claims 28-46, wherein the subject is a human.

48. Use according to any one of claims 28 to 47, wherein one or more doses of 10 are administered per kilogram body weight of the subject⁴To 10⁸And (c) isolating said cells.

49. Use according to any one of claims 28 to 48, wherein one or more doses of 10 are administered per kilogram body weight of the subject⁶To 5X 10⁷And (c) isolating said cells.

50. The use according to any one of claims 28 to 49, wherein an antimicrobial, antifungal, antiviral or a combination thereof is used simultaneously.

51. The use according to any one of claims 28 to 50, wherein the cells are in a formulation comprising one or more further pharmaceutically active agents.

52. The use according to any one of claims 28-51, wherein the cells are administered by a parenteral method, an intravenous method, or a stereotactic method.

53. The use according to any one of claims 28-52, wherein the cells are administered by intravenous methods.

54. The use according to any one of claims 28-53, wherein the medicament is for use without a co-immunosuppressive agent.

Technical Field

The present invention relates to intracerebral hemorrhage and pluripotent stem cells.

Capital of government

No government funds are used in making the invention disclosed herein.

RELATED APPLICATIONS

This application claims priority to U.S. patent application No. 16/265,373, filed on 1/2/2019, having the same name, the entire contents of which are incorporated herein by reference.

Ⅰ

Intracerebral hemorrhage ("ICH") refers to any hemorrhage within the intracranial fornix, including the parenchyma of the brain and the surrounding meningeal space. ICH is a devastating disease affecting a large population in the united states and throughout the world. (see, e.g., Caceres and Goldstein (2012): emery Med Clin North Am 30(3)771- & 794.) the incidence of spontaneous ICH worldwide is 24.6 cases per 100,000 persons-year, with approximately 40,000 to 67,000 cases per year in the United states. The 30-day mortality rate is 35% to 52%. About half of the total deaths occurred within the first 24 hours. Only 20% of survivors recovered full function at 6 months. Early effective treatment is considered to be of great importance. (see, e.g., van Asch et al (2010): The Lancet Neurology 9(2)167-

Intraparenchymal hemorrhage is usually caused by penetrating head trauma. They may also be caused by depressed skull fractures. Other causes include aneurysm rupture, arteriovenous malformations (AVM), intratumoral bleeding, and accelerated-decelerated trauma. Amyloid angiopathy is a common cause of intracerebral hemorrhage in patients over the age of 55. Cerebral sinus thrombosis accounts for a very small fraction of ICH.

Primary ICH is often a manifestation of underlying small vessel disease. First, long-term hypertension leads to hypertensive vascular lesions that result in microscopic degenerative changes (lipohyalinity) in the wall of small to medium sized penetrating blood vessels. Second, cerebral amyloid angiopathy develops, characterized by deposition of amyloid- β peptide (a β) in the walls of small leptomeningeal and cortical blood vessels. The mechanisms leading to amyloid accumulation are not clear; however, the consequences are well documented: degenerative changes in the vessel wall characterized by loss of smooth muscle cells, wall thickening, narrowing of the lumen, microaneurysms and microhemorrhages. (see, e.g., Fisher, CM. (1971): J Neuropathol Exp neuron 30(3) 536-50; Vinters H. (1987): Stroke 18(2)311-

After initial vascular rupture, hematomas cause direct mechanical damage to the brain parenchyma. Edema surrounding the hematoma develops within the first 3 hours after symptoms appear and peaks between 10 and 20 days. Next, the blood and plasma cause secondary injury processes including inflammatory reactions, activation of the coagulation cascade, and iron deposition from hemoglobin degradation. Finally, during the first 24 hours, hematomas continue to expand in up to 38% of patients. (see, e.g., Aronowski and ZHao (2011): Stroke 42 (6): 781-6 and Brott et al (1997): Stroke 28 (1): 1-5.)

In summary, ICH is a brain injury caused by blood infiltration into the brain and blood accumulation in the brain parenchyma. It may be caused by aneurysm rupture, cerebral artery injury (e.g., perforation), or arteriovenous malformations (AVM). It is a devastating nerve injury, accounting for approximately 20% of all stroke-related injuries worldwide, and nearly 30% in japan and asia. It has the highest mortality and worst long-term outcome of all stroke-related injuries, and ICH accounts for nearly 50% of all stroke-related deaths. Early clot regression is a major clinical goal, as hematoma volume is an independent determinant of ICH patient outcome. However, there is currently no FDA-approved therapy that improves the outcome of ICH.

Emergency surgical removal of blood clots (if possible) and rehabilitation is currently the only standard of care; however, surgical treatments have limited utility and rehabilitation is a problem of dealing with injury rather than preventing or repairing it. Indeed, the latest guidelines for AHA/ASA and the only recommended treatment for ICH patients are surgical removal of blood clots if surgery is available. There are no other recommended therapeutic interventions. (see, e.g., Hemphill III, J.C.et al. (2015): AHA/ASA Guideline, Stoke 46:2032-

Clearly, there is a great need to reduce and repair the brain damage that occurs in methods of treating ICH damage. It is therefore an object of embodiments of the invention disclosed herein to provide means and methods for improving the outcome of ICH cases.

To date, stem cells have been considered to be useless for treating ICH. As mentioned above, ICH is pathophysiologically thought to be driven by the appearance of red blood cells in the brain and subsequent breakdown, released hemoglobin and its neurotoxic heme breakdown products. Extravasation of red blood cells into the brain creates a space-occupying hematoma/clot which will be accompanied by mechanical tissue destruction, edema formation, increased intracranial pressure, increased microvascular compression, decreased blood flow and poor outcomes that do not appear to be easily repaired by cell therapy.

As described herein, surprisingly, it has been found that the administration of multipotent adult stem cells described herein to treat ICH has unexpected and unexpectedly efficacious therapeutic benefits for ICH results, as shown by measurements of hematoma volume, cerebral blood flow/perfusion results, and functional assessments.

Ⅱ

Some of the many embodiments covered by this specification are summarized in the following numbered paragraphs. The numbered paragraphs are self-referencing. In particular, the phrase "according to any of the preceding or following" as used in these paragraphs refers to other paragraphs. In the following paragraphs, this phrase means that the embodiments disclosed herein include subject matter described in the individual paragraphs and subject matter described in the combined paragraphs. In this regard, it is the applicant's intent to clarify the following paragraphs, particularly to describe various aspects and embodiments either individually or in any combination of paragraphs. That is, these paragraphs provide explicit written instructions for all embodiments they contain, individually or in combination with each other, in a compact manner. Applicants expressly reserve the following claims: any subject matter recited in any of the following paragraphs alone or in combination with any other subject matter of any one or more of the other paragraphs, is claimed whenever possible, including using any combination of any values listed therein alone or any combination thereof with any other values listed therein. The applicant specifically reserves the right to combine all of the combinations listed herein throughout the present application or in any subsequent application benefiting from the present application, if desired.

p1. A method of treating intracerebral hemorrhage in a subject, comprising administering to a subject in need thereof multipotent adult progenitor cells, which are not embryonic stem cells, embryonic germ cells, nor germ cells, that can differentiate into at least one cell type of each of at least two of the endodermal, ectodermal and mesodermal embryonic lineages, and are allogeneic or xenogeneic with respect to the subject.

p2. A method of treating intracerebral hemorrhage in a subject, comprising administering to a subject in need thereof multipotent adult progenitor cells, which are not embryonic stem cells, embryonic germ cells, or germ cells, that express telomerase and are allogeneic or xenogeneic to the subject.

p3. A method of treating intracerebral hemorrhage in a subject, comprising administering to a subject in need thereof multipotent adult progenitor cells, which are not embryonic stem cells, embryonic germ cells, or germ cells, that are positive for oct4 and are allogeneic or xenogeneic to the subject.

p4. A method of treating intracerebral hemorrhage in a subject, comprising administering to a subject in need thereof multipotent adult progenitor cells, which are not embryonic stem cells, embryonic germ cells, or germ cells, that have undergone at least 40 cell doublings in culture prior to use, and that are allogeneic or xenogeneic to the subject.

p5. A method according to any preceding and/or following, wherein at least one cell type can differentiate into each of at least two of the endodermal, ectodermal and mesodermal embryonic lineages.

p6. the method according to any preceding or following, wherein the cell expresses telomerase.

p7. the method according to any preceding or subsequent claim wherein the cell is positive for oct 4.

p8. for use according to the method of any preceding or subsequent claim, wherein the cells express telomerase and are positive for oct 4.

p9. A method according to any preceding or subsequent claim, wherein the cells express telomerase and have undergone at least 40 cell doublings prior to their use.

p10. a method according to any preceding or following, wherein said cell expresses oct4 and has undergone at least 40 cell doublings prior to its use.

p11. a method according to any preceding or following, wherein the cells express telomerase, are positive for oct4 and undergo at least 40 cell doublings prior to their use.

p12. a method according to any one of the preceding or following, wherein the cell expresses any one or more of rex-1, rox-1 or sox-2.

p13. the method according to any one of the preceding or following, wherein the cell has a normal karyotype.

p14. the method according to any one of the preceding or following, wherein the cell is non-tumorigenic.

P15. the method according to any one of the preceding or following, wherein said cells do not form teratomas.

P16. the method according to any one of the preceding or following, wherein said cell has not been genetically modified.

P17. the method according to any one of the preceding or following, wherein said cell is genetically engineered.

P18. the method according to any one of the preceding or following, wherein said cells are not immunogenic in said subject.

P19. the method according to any one of the preceding or following, wherein said cells can differentiate into at least one cell type of each of at least two of the endodermal, ectodermal and mesodermal embryonic lineages.

p20. the method according to any one of the preceding or following, wherein the cell is a mammalian cell.

p21. the method according to any one of the preceding or following, wherein the cell is a human cell.

p22. a method according to any one of the preceding or following, wherein the cells are derived from cells isolated from any one of: placental tissue, umbilical cord blood, bone marrow, blood, spleen tissue, thymus tissue, spinal cord tissue, adipose tissue, and liver tissue.

p23. the method according to any one of the preceding or following, wherein the cells are derived from bone marrow.

p24. the method according to any one of the preceding or following, wherein the subject is a human.

p25. the method according to any one of the preceding or following, wherein one or more doses of 10 are administered per kilogram body weight of the subject⁴To 10⁸And (c) isolating said cells.

p26. the method according to any one of the preceding or following, wherein one or more doses of 10 are administered per kilogram body weight of the subject⁶To 5X 10⁷And (c) isolating said cells.

p27. the method according to any of the preceding or following, wherein in addition to said cells one or more growth factors, differentiation factors, signalling factors and/or factors increasing homing are used simultaneously.

p28. the method according to any one of the preceding or following, wherein an antimicrobial, antifungal, antiviral or a combination thereof is used simultaneously.

p29. the method according to any one of the preceding or following, wherein the cells are in a preparation comprising one or more further pharmaceutically active agents.

p30. the method according to any one of the preceding or following, wherein the cells are administered parenterally.

p32. the method according to any one of the preceding or following, wherein the cells are administered intravenously.

p33. a method according to any of the preceding or following, wherein the cells are administered stereospecifically.

p34. a method according to any one of the preceding or following, wherein the cells are administered at any one or more of the following time points: 1, 5, 10, 15, 30, 45, or 60 minutes after ICH, or 60, 90, 120, 150, or 180 minutes after ICH, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after ICH, or 12, 18, 24, 30, 36, or 40 hours after ICH, or 1, 2, 3, 4, 5, 6, or 7 days after ICH, or 1, 2, 3, 4, 5, 6, 7, or 8 weeks after ICH, or any combination of the foregoing and/or any later time.

Ⅲ

As used herein, "a" or "an" means: one or more than one; at least one. Where plural is used herein, it typically includes the singular.

"cell bank" is the industry nomenclature for cells that have been cultured and stored for future use. The cells can be aliquotedIn partAnd (5) storing. They can be used directly when they are removed from the storage or they can be propagated after storage. This facilitates the provision of "ready-to-use" cells that can be used for drug administration. The cells may have been stored in a pharmaceutically acceptable excipient so they can be administered directly, or they can be mixed with a suitable excipient when they have been depalletized. The cells may be frozen or otherwise stored to maintain viability. In one embodiment of the invention, a cell bank is created in which cells are selected to increase the potential to achieve the effects described herein. After release from storage, and prior to administration, the cells may preferably be tested again for potency. This can be done using any direct or indirect test described in the present application or known in the art. Cells with the desired potential can then be administered. Autologous cells (from organ donors or recipients) can be used to make these libraries. Or these libraries may contain cells for allogeneic use.

As used herein, "co-administration" means co-administration with each other, together, in coordination, including administration of two or more agents simultaneously or sequentially.

As used herein, "comprising" means not limited to the mentioned things it must include, without any limiting conditions or exclusions to other things that may be included. For example, "a composition comprising x and y" encompasses any composition comprising x and y, regardless of what other components may be present in the composition. Likewise, "a method comprising x steps" encompasses any method in which x is performed, whether x is the only step or only one of the steps in the method, and regardless of how many other steps the method may have, and regardless of how simple or complex x is compared to the other steps. As used herein, the root "comprising" and similar phrases used herein as synonyms for "comprising" are used as if the root "comprises" and similar phrases.

As used herein, "comprising of" is synonymous with "comprising" (see above).

"conditioned cell culture medium" is a term well known in the art and refers to a medium in which cells have been grown. As used herein, the phrase means that the cells are grown in the medium for a sufficient time to secrete factors effective for growth of the particular type of cell in the conditioned medium.

As used herein, "reduce" and "reducing" and similar terms generally mean a reduction in an amount or value or effect when compared to another amount, value or effect. For example, reducing the severity of ICH may mean reducing hematoma volume and/or reducing functional damage, as compared to the volume or functional damage previously caused by ICH.

As used herein, "effective amount" generally means an amount that provides the desired local or systemic effect. For example, an effective amount is an amount sufficient to achieve a beneficial or desired clinical result. For example, an effective amount to treat ICH is an amount that reduces hematoma volume and/or improves and/or increases brain circulation and/or perfusion; and/or reducing the amount of nerve damage and/or functional damage and/or improving function (e.g., motor function, balance, etc.).

An effective amount may be provided all at once in a single administration or in divided amounts given in several administrations to provide an effective amount. The exact amount to be determined is considered an effective amount and will depend upon factors which are personal to each subject including their weight, age, condition of the injury and/or disease or injury being treated, and the length of time since the injury occurred or the disease began. One skilled in the art will be able to determine an effective amount for a given subject based on such considerations as are conventional in the art. As used herein, an "effective dose" is synonymous with an "effective amount".

As used herein, an "effective route" generally means a route to deliver an agent to a desired compartment (component), system or location. For example, an effective route is one by which an agent can be administered to provide an amount of the agent at a desired site of action sufficient to achieve a beneficial or desired clinical result.

As used herein, "ICH" is an acronym for "intracerebral hemorrhage" and has the same meaning as "intracerebral hemorrhage".

As used herein, "include" and "including" are non-limiting and have substantially the same meaning as "comprising" and "comprises".

As used herein, "increase" and "increasing" refer to making, for example, a biological event or property greater in size, quantity, intensity, or degree, including induction (e.g., from a zero or inactive state). For example, an effective amount to treat ICH is an amount that, for example, increases cerebral circulation and/or perfusion; and/or to improve functions, such as motor functions, balance, etc., such as by comparison to prior post-ICH functions.

"intracerebral hemorrhage" ("ICH") is the entry of intracranial hemorrhage into brain tissue, the ventricle, or both. Causes of ICH include, but are not limited to, bleeding from or due to any one or more of aneurysms, arteriovenous malformations, brain tumors, and brain trauma. ICH is also known as "cerebral hemorrhage".

As used herein, the term "isolated" refers to one or more cells that are not associated with one or more cells in vivo or one or more cellular components associated with one or more cells. By "enriched population" is meant a relative increase in the number of desired cells relative to one or more other cell types in vivo or in the original culture medium.

However, as used herein, the term "isolated" does not indicate the presence of only stem cells. In contrast, the term "isolated" means that cells are removed from their natural tissue environment and are present in higher concentrations than in the normal tissue environment. Thus, an "isolated" cell population may further include cell types other than stem cells and may include additional tissue components. This can also be expressed, for example, in terms of cell doubling. A cell may undergo 10, 20, 30, 40 or more doublings in vitro (vitro) or ex vivo (ex vivo), and is therefore enriched compared to its original number in vivo or within its original tissue environment (e.g., bone marrow, peripheral blood, adipose tissue, etc.).

"MAPC" is an acronym for "multipotent adult progenitor cell". It refers to cells that are not embryonic stem cells or germ cells. MAPCs can be characterized in a number of alternative descriptions, each of which confers novelty to cells in their discovery. Thus, they may be characterized by one or more of these descriptions. First, they expand replication capacity in culture without genetic engineering, transformation (tumorigenic), and under conditions with normal karyotype. This means that these cells express telomerase (i.e., have telomerase activity). Second, they can, upon differentiation, produce cell progeny of more than one embryonic layer, e.g., two or all three embryonic layers (i.e., endoderm, mesoderm, and ectoderm). Third, although they are not embryonic stem or germ cells, they may express markers for these primitive cell types so that MAPCs may express one or more of oct4, rex-1, and rox-1. They may also express sox-2. Rex-1 is controlled by oct4, which activates expression downstream of Rex-1. Rox-1 and sox-2 are expressed in non-ES cells. Thus, a cell type designated as "MAPC" can be characterized by alternative essential features that describe the cell by some novel property thereof.

The term "adult" in MAPC is non-limiting. It refers to non-embryonic somatic cells, such as postnatal somatic cells. MAPCs do not form teratomas in vivo. This acronym is first used in U.S. patent No. 7,015,037 to describe pluripotent cells isolated from bone marrow. However, cells with pluripotency markers and/or differentiation potential were subsequently discovered and, for the purposes of the present invention, they could be equated with those originally designated as "MAPCs". A basic description of MAPC type cells is provided in the summary of the invention above.

MAPC represents a more primitive population of progenitor cells than MSC (Verfailie, CM, Trends Cell Biol 12:502-8 (2002); Jahagirdar, BN, et al, Exp Hematol,29:543-56 (2001); Reyes, M. and CM Verfailie, Ann NY Acad Sci,938: 231-.

MAPCs may not be immunogenic. MAPCs may be immunosuppressive. MAPCs may or may not be genetically modified to improve their characteristics. MAPCs may be used with or without adjuvant immunosuppressive therapy. Other aspects of MAPC are described herein.

"may" as used herein, the term "may" is synonymous with "optionally," and "may" as used herein includes "may not even if not stated. That is, a statement that something may be also means that it may or may not be. That is, "may" as used herein expressly includes "may not," and applicants reserve the right to claim subject matter consistent therewith. For example, as used herein, the statement that MAPC may be administered with other agents also means that MAPC may be administered without any other agent. For another example, as used herein, the statement that MAPC may be genetically engineered also means that MAPC may not be genetically engineered.

As used herein, "pluripotent" with respect to MAPCs refers to the ability of MAPCs, when differentiated, to give rise to a cell lineage with more than one of the three primitive germ layers (endoderm, mesoderm and ectoderm), e.g., two or all three layers.

Is the trade name for a cell preparation based on MAPC of U.S. patent No. 7,015,037, namely non-embryonic stem cells, non-germ cells as described above.Prepared according to the cell culture method disclosed in this patent application, in particular lower oxygen and higher serum.Is highly scalable, karyotype is normal, and does not form teratomas in vivo. It can differentiate into cell lineages with more than one germ layer and can express one or more of telomerase, oct4, rex-1, rox-1, and sox-2.

"Oct-4" is a member of the POU family of transcription factors (1). Mouse proteins were first identified and classified as Oct-3. Human homologues were initially classified as Oct-3 based on their 87% homology at the amino acid level to the mouse Oct-3 protein. Subsequently, two human Oct-3 transcripts were identified (Takeda, et al, Nucleic Acids Research 20(17)4613-20(1992)), Oct-3A and 3B, which are alternative splice products of the same gene. Oct-3A has been identified and renamed Oct-4 by some communities and Oct-3/4 by others. There are also groups that divide oct4 into oct4A and oct4B as alternative transcripts. The A transcripts from which the nucleoprotein was produced (i.e., oct4, oct3A, oct3/4) were associated with pluripotency and were limited to a portion of exon 1. B transcripts are present as cytoplasmic proteins in many cell types and encompass the remainder of exon 1 as well as exons 2-5.

As used herein, "optionally" has substantially the same meaning as "may". As used herein, the statement that X optionally comprises a includes that X comprises a and that X does not comprise a.

A "pharmaceutically acceptable carrier" is any pharmaceutically acceptable medium for the cells used in the present invention. Such media can maintain isotonicity, cellular metabolism, pH, and the like. It is compatible with in vivo administration to a subject and thus useful for cell delivery and therapy.

A "progenitor cell" is a cell that arises during the differentiation of a stem cell, and which has some (but not all) of the characteristics of its terminally differentiated progeny. A defined progenitor cell, such as a "cardiac progenitor cell", belongs to a lineage, but not to a specific or terminally differentiated cell type. The term "progenitor cells" as used in the acronym "MAPC" does not limit these cells to a particular lineage.

As used herein, the term "reduce" means prevent as well as reduce. Within the context of treatment, "reducing" is preventing or ameliorating one or more clinical symptoms. A clinical symptom is a symptom(s) that, if left untreated, negatively affects the quality of life (health) of the subject.

"selecting" a cell having a desired level of potency can mean identifying (e.g., by assay), isolating, and expanding the cell. This can create a population with higher potential than the parental cell population from which the cell was isolated. A "parental" cell population refers to the parental cell from which the selected cell is split. "parent" refers to the actual P1 → F1 relationship (i.e., daughter cells). Thus, if cell X is isolated from a mixed population of cells X and Y, where X is an expressor and Y is not, then the X isolate alone will not be classified as having enhanced expression. However, if the progeny cells of X are higher expressors, the progeny cells will be classified as having enhanced expression.

As used herein, "self-renewal" refers to the ability to generate replicon-substituted stem cells that have the same differentiation potential as the cells that generated them. A similar term is used in this context to be "proliferation".

As used herein, "stem cell" means a cell that is self-renewing (i.e., progeny having the same differentiation potential) and that also produces progeny cells that are more limited in differentiation potential.

As used herein, "subject" refers to a vertebrate, e.g., a mammal, e.g., a human. Mammals include, but are not limited to, humans, dogs, cats, horses, cattle, and pigs.

The term "therapeutically effective amount" as used herein refers to an amount determined to produce any beneficial therapeutic response in a subject. For example, an effective amount of a therapeutic cell or cell-associated agent may prolong the survival of a patient and/or inhibit overt clinical symptoms. Within the meaning of the terms used herein, therapeutically effective treatments include those that improve the quality of life of a subject, even though they do not themselves improve the outcome of the disease. For example, a therapeutic effect may indicate, for example, a reduction in bleeding volume, an improvement in cerebral blood flow, and/or an improvement in neurological and/or behavioral functions after ICH. Such therapeutically effective amounts are readily determined by one of ordinary skill in the art.

"treatment", "treating" or "treatment" are used broadly in the present invention, and each such term essentially means administration to a cell as described herein, in particularly advantageous embodiments having some or all of the following effects, but not necessarily: prevent, ameliorate, inhibit or cure defects, dysfunctions, diseases or other deleterious processes, including those that interfere with and/or result from treatment. For example, treatment may mean reducing bleeding volume, improving cerebral blood flow, and/or improving neurological and/or behavioral functions, e.g., after ICH. Treatment of these aspects is readily determined by one of ordinary skill in the art.

As used herein, "verify" means confirm. In the context of the present invention, cells are identified as expressors with the desired potential. In this way, one can use the cell (in therapy, banking, drug screening, etc.) with reasonable expectation of efficacy. Thus, validation means confirming that cells originally found/established to have the desired activity actually retained that activity. Thus, authentication is an authentication event in a two-event process involving an original confirmation and a subsequent confirmation. The second event is referred to herein as "verification".

III

Drawings

The various features and advantages of the embodiments described herein may be more fully understood as the same becomes better understood when considered with reference to the accompanying drawings in which:

fig. 1A and 1B: following the collagenase-induced ICH, the collagen protein is, cell reduction of hematoma volume

Placebo (PBS, n-10) or(n-11) was administered to mice intravenously. Hematoma volume was assessed by MRI (T2W) using 7T small animal MRI. Representative coronal brain images at day 3 and day 7 are provided

FIG. 1AShow thatSignificant benefit of cells on hematoma volume.

FIG. 1BData from all mice over the 21 day evaluation period are depicted.

Data are presented as mean +/-SEM and analyzed by Student's t-test (Student's t-test) at each time point. P <0.01 vs placebo treated ICH mice.

Detailed information is provided in example 3.

Fig. 2A and 2B: after the induction of the ICH by the collagenase, cell-improved brain perfusion

Placebo (PBS, n-10) or(n-11) was administered to mice intravenously. Brain perfusion was assessed by MRI (ASL; FAIR-RARE) using 7T small animal MRI.

FIG. 2A-Representative coronary brain mapLike this.

FIG. 2B-Quantitative perfusion data.

Data showBrain perfusion was improved within the first week after ICH. Data are presented as mean +/-SEM and analyzed by student t-test at each time point. P<0.05、**p<0.01 versus placebo treated ICH mice.

Detailed information is provided in example 4.

Fig. 3A, 3B and 3C: after the induction of the ICH by the collagenase, cellular improvement of motor function

Placebo (PBS, n-10) or(n-1) was administered to mice by intravenous injection. Neurological assessment of motor function was assessed on day 7 post injury (or in sham operated mice; n-8).

FIG. 3A-And (5) testing the grip strength.

FIG. 3B-Narrow Beam Task (Narrow Beam Task) test results.

FIG. 3CElevation Body Swing Task (Elevated Body Swing Task) test results.

Data are mean +/-SEM and compared using One-Way analysis of variance (One Way ANOVA) followed by Tukey post hoc test. P <0.5, p <0.01, p <0.001, ns is not significant.

Detailed information is provided in example 5.

Ⅴ

As described herein, some aspects of the invention relate to administering MAPCs (as defined herein) to a subject experiencing intracerebral haemorrhagic bleeding (also known as hemorrhagic stroke). If the size and location of the blood clot in the brain is suitable for surgery, these patients have no therapeutic intervention other than surgical removal of the blood clot.

As described herein, some aspects of the invention provide methods of administering cells to a patient suffering from or in need of treatment for intracerebral hemorrhage in order to have one or more, but not necessarily some or all, of the following beneficial effects: prevent, ameliorate, inhibit or cure intracerebral hemorrhage. Cells and methods according to this are described below.

Embodiments of the invention provide for administration of cells by intravenous route after the onset of ICH, e.g., within a sub-acute time frame (hours). Can be administered by a variety of routes and times that may be found to be effective.

Without being limited to any particular mechanism of action, it is noted thatCells can modulate the acute inflammatory response in other preclinical and clinical injuries. Can make it possible toThe effect in treating ICH is in some aspects by analogy toTo the acute inflammatory response that occurs after ICH.

The cells may achieve these effects naturally (i.e., without genetic or pharmaceutical modification). However, the cells may also be genetically or pharmaceutically modified to increase effectiveness and/or improve their properties.

In one embodiment, the cells undergo a desired number of cell doublings in culture. For example, the cells undergo at least 10-40 cell doublings, e.g., 30-35 cell doublings, in culture, and wherein the cells are untransformed and have a normal karyotype. If the cells are transformed or tumorigenic and it is desired to use them for infusion, these cells cannot be used and therefore they cannot form tumors in vivo, such as by treatment that prevents the cells from proliferating into tumors. Such treatments are well known in the art.

Oct4, specific for ES, EG and germ cells, was considered a marker for undifferentiated cells with broad spectrum differentiation capacity. Oct4 is also generally believed to play a role in maintaining cells in an undifferentiated state. Oct4 belongs to the POU (pit Oct Unc) family of transcription factors and is a DNA binding protein capable of activating transcription of genes and contains an octamer sequence called "octamer motif" in the promoter or enhancer region. Oct4 was expressed during the cleavage phase of fertilized eggs until oocysts (eg cylinder) were formed. Oct4 functions to inhibit differentiation-inducing genes (i.e., FoxaD3, hCG) and to activate genes to promote pluripotency (FGF4, Utfl, Rexl). Sox2 is a member of the High Mobility Group (HMG) box transcription factor, and it activates transcription of genes expressed in the inner cell mass in cooperation with Oct 4. Oct4 expression in embryonic stem cells must be maintained within a certain level range. Over-or down-regulation of > 50% of Oct4 expression levels will alter embryonic stem cell fate, forming primitive endoderm/mesoderm or trophectoderm, respectively. In vivo, Oct 4-deficient embryos developed to the blastocyst stage, but the inner cell mass cells were not pluripotent. Instead, they differentiate along the extraembryonic trophoblast lineage.

Sall4 is a mammalian Spalt transcription factor, an upstream regulator of Oct4, and is therefore important for maintaining adequate Oct4 levels during the early stages of embryology. When the Sall4 level is below a certain threshold, the trophectoderm cells will ectopically expand into the inner cell mass.

Such cells include, but are not limited to, the features in the numbered embodiments below:

pb1. isolated expanded non-embryonic stem, non-germ cells that have undergone at least 10-40 cell doublings in culture, wherein the cells express oct4, are untransformed, and have a normal karyotype.

pb2. the non-embryonic stem cell, non-germ cell of 1 above, further expressing one or more of telomerase, rex-1, rox-1, or sox-2.

pb3. the non-embryonic stem, non-germ cell of above 1, which can differentiate into at least one cell type of at least two of the endodermal, ectodermal and mesodermal embryonic lineages.

pb4. the non-embryonic stem cell, non-germ cell of 3 above, further expressing one or more of telomerase, rex-1, rox-1, or sox-2.

pb5. the non-embryonic stem, non-germ cell of 3 above that can differentiate into at least one cell type of each of the endodermal, ectodermal and mesodermal embryonic lineages.

pb6. the non-embryonic stem cell, non-germ cell of 5 above, further expressing one or more of telomerase, rex-1, rox-1, or sox-2.

pb7. isolated expanded non-embryonic stem, non-germ cells obtained by culturing non-embryonic, non-germ tissue, said cells having undergone at least 40 cell doublings in culture, wherein said cells are untransformed and have a normal karyotype.

pb8. the non-embryonic stem cell, non-germ cell of claim 7, which expresses one or more of oct4, telomerase, rex-1, rox-1, or sox-2.

pb9. the non-embryonic stem cell, non-germ cell of above 7, which can differentiate into at least one cell type of at least two of the endodermal, ectodermal and mesodermal embryonic lineages.

pb10. the non-embryonic stem cell, non-germ cell of above 9, which expresses one or more of oct4, telomerase, rex-1, rox-1, or sox-2.

pb11. the non-embryonic stem, non-germ cell of above 9 that can differentiate into at least one cell type of each of the endodermal, ectodermal and mesodermal embryonic lineages.

pb12. the non-embryonic stem cell, non-germ cell described above, which expresses one or more of oct4, telomerase, rex-1, rox-1, or sox-2.

Isolated expanded non-embryonic stem, non-germ cells that undergo at least 10-40 cell doublings in culture, wherein the cells express telomerase, are untransformed, and have a normal karyotype.

pb14. the non-embryonic stem cell, non-germ cell of above 13, further expressing one or more of oct4, rex-1, rox-1, or sox-2.

pb15. the non-embryonic stem, non-germ cell of above 13, which can differentiate into at least one cell type of at least two of the endodermal, ectodermal and mesodermal embryonic lineages.

pb16. the non-embryonic stem cell, non-germ cell of 15 above, further expressing one or more of oct4, rex-1, rox-1, or sox-2.

pb17. the non-embryonic stem cell, non-germ cell of 15 above that can differentiate into at least one cell type of each of the endodermal, ectodermal and mesodermal embryonic lineages.

pb18. the non-embryonic stem cell, non-germ cell of 17 above, further expressing one or more of oct4, rex-1, rox-1, or sox-2.

pb19. isolated expanded non-embryonic stem, non-germ cells that can differentiate into at least one cell type of at least two of the endodermal, ectodermal and mesodermal embryonic lineages, said cells having undergone at least 10-40 cell doublings in culture.

pb20. the non-embryonic stem cell, non-germ cell of 19 above, which expresses one or more of oct4, telomerase, rex-1, rox-1, or sox-2.

pb21. the non-embryonic stem cell, non-germ cell of above 19, which can differentiate into at least one cell type of each of the endodermal, ectodermal and mesodermal embryonic lineages.

pb22. the non-embryonic stem cell, non-germ cell of claim 21, which expresses one or more of oct4, telomerase, rex-1, rox-1, or sox-2.

Selection of cells

As described herein, MAPCs can be used in isolated and amplified form. MAPCs can also be selected for specific characteristics prior to use, with or without the use of genetic engineering techniques.

Selecting cells with a desired level of potency can mean identifying (e.g., by assay), isolating, and expanding the cells. This can create a population with higher potential than the parental cell population from which the cells were isolated. A "parental" cell population refers to the parental cell from which the selected cell is split. "parent" refers to the actual P1 → F1 relationship (i.e., daughter cells). Thus, if cell X is isolated from a mixed population of cells X and Y, where X is an expressor and Y is not, then the X isolate alone will not be classified as having enhanced expression. However, if the progeny cells of X are higher expressors, the progeny cells will be classified as having enhanced expression.

Selecting cells that achieve the desired effect will include determining whether the cells achieve the desired effect and obtaining such cells. The cell can naturally achieve the desired effect because the effect is not achieved by the exogenous transgene/DNA. However, effective cells may be improved by incubation with or exposure to agents that enhance their effect. Prior to performing the assay, the cell population from which the effective cells were selected may not be known to have potential. It may also be unknown that the cells will achieve the desired effect prior to performing the assay. Since the effect may depend on gene expression and/or secretion, it may also be selected based on the gene or genes producing the effect.

Cells in the tissue may be selected. For example, in such a case, cells will be isolated from the desired tissue, expanded in culture, selected for the desired effect, and then the selected cells further expanded.

Ex vivo cells, e.g., cells in culture, may also be selected. In this case, to achieve the desired effect, one or more cells in culture are assayed, and the cells obtained to achieve the desired effect can be further expanded.

Cells may also be selected that have an enhanced ability to achieve the desired effect. In such a case, the cell population from which such enhanced cells are obtained already has the desired effect. The enhanced effect means that the average number of each cell is higher than the parent population.

The parent population from which the enhanced cells are selected may be substantially homogeneous (same cell type). One way to obtain such enhanced cells from the population is to create single cells or cell banks and assay these cells or cell banks to obtain clonal cells that naturally have an enhanced (greater) effect (as opposed to cells treated with a modulator that induces or increases an effect), and then expand those cells that naturally have an enhanced effect.

However, the cells may be treated with one or more agents that induce or increase the effect. Thus, a substantially homogenous population may be processed to enhance the effect.

If the population is not substantially homogeneous, it is preferred that the parental cell population to be treated comprises at least 100 desired cell types for which enhanced effect is sought, more preferably at least 1,000 cells, still more preferably at least 10,000 cells. After treatment, the subpopulation can be recovered from the heterogeneous population by known cell selection techniques and further expanded if desired.

Thus, the desired level of effect may be higher than in a given previous population. For example, cells that are primary cultured from tissue and expanded and isolated by culture conditions that are not specifically designed to produce this effect may provide a parental population. Such a parental population may be treated to enhance the average effect per cell, or to screen the population for cells or cells expressing a greater degree of effect without deliberate treatment. Such cells can then be expanded to provide populations with higher (desired) expression.

Use and administration of drugs

In some embodiments, the cell is used as the sole active agent for therapy. In some embodiments of the invention, MAPC is used as the primary treatment modality, together with one or more other agents and/or treatment modalities. In some embodiments of the invention, the cells are used as an adjunct therapy modality, i.e., as an adjunct to another primary therapy modality. In some embodiments, the cells are used as the sole active agent in an adjunctive therapeutic modality. In other embodiments, the cells are used as an adjunct therapy modality with one or more other agents or therapies. In some embodiments, the cells are used as both a primary and a secondary therapeutic agent and/or treatment modality. In both aspects, the cells can be used alone in a primary and/or secondary manner. They may also be used with other therapeutic agents or treatments (either in primary or secondary or both).

As noted above, the primary treatment (e.g., therapeutic agent, therapy, and/or treatment modality) targets (i.e., is intended to act upon) the primary dysfunction, e.g., disease, to be treated. Adjunctive therapy (e.g., therapy and/or treatment modality) can be administered in combination with the primary therapy (e.g., therapeutic agent, therapy and/or treatment modality) to affect the primary dysfunction (e.g., disease) and complement the effects of the primary therapy, thereby improving the overall efficacy of the treatment regimen. Adjunctive therapies (e.g., agents, therapies and/or treatment modalities) may also be administered to act on complications and/or side effects of the primary dysfunction (e.g., disease) and/or those resulting from the treatment (e.g., therapeutic agents, therapies and/or treatment modalities). For any of these uses, one, two, three or more primary treatments may be used with one, two, three or more secondary treatments.

In some embodiments, the MAPC is administered to the subject prior to the onset of ICH. In some embodiments, the cells are administered at a time when ICH and/or the resulting dysfunction is developing. In some embodiments, the cells are administered after having suffered an ICH and/or resulting dysfunction. MAPC may be administered at any stage of development, persistence and/or spread (suppression) of ICH or related dysfunction or following regression thereof.

The cells may be administered at any one or more of the following time points: 1, 5, 10, 15, 30, 45, or 60 minutes before or after the ICH, or 60, 90, 120, 150, or 180 minutes before or after the ICH, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours before or after the ICH, or 12, 18, 24, 30, 36, or 40 hours after the ICH, or 1, 2, 3, 4, 5, 6, or 7 days after the ICH, or 1, 2, 3, 4, 5, 6, 7, or 8 weeks after the ICH, or any combination and/or any later time of the above.

Cells can be administered immediately at the following time points: 60 minutes after ICH, 30 to 90 minutes after ICH, 1 to 6 hours after ICH, 5 to 15 hours after ICH, 10 to 20 hours after ICH, 15 to 25 hours after ICH, 20 to 40 hours after ICH, 1 to 5 days after ICH, 1 to 1 week after ICH, 1 to 2 weeks after ICH, 1 to several weeks after ICH, several weeks to 1 month after ICH, 1 or several months after ICH, any time after ICH.

The cells may also be administered prior to the ICH, at any time prior to the ICH, or at any time or interval prior to the ICH as mentioned in the preceding two paragraphs.

As noted above, embodiments of the invention provide cells and methods for primary or adjunct therapy. In certain embodiments of the invention, the cells are administered to an allogeneic subject (i.e., allogeneic to the subject). In some embodiments, they are autologous to the subject. In some embodiments, they are syngeneic with the subject. In some embodiments, the cell is xenogeneic to the subject. Whether allogeneic, autologous, homologous, or xenogeneic, in various embodiments of the invention, MAPCs are only weakly immunogenic or non-immunogenic in a subject. In some embodiments, MAPCs have sufficiently low immunogenicity and non-immunogenicity such that they do not typically elicit an adverse immune response when administered to allogeneic and/or xenogeneic subjects, and thus they can be used as "universal" donor cells without the need for tissue typing and matching.

Further, in this regard, the MAPCs in various embodiments can be administered without adjunctive immunosuppressive therapy. MAPCs may also be stored and preserved in cell banks, according to various embodiments of the present invention, and thus may be maintained in a useable state when desired.

In all of these and other aspects, embodiments of the invention provide MAPCs from mammals, including humans in one embodiment, and non-human primates, rats and mice, as well as dogs, pigs, goats, sheep, horses and cattle in other embodiments. MAPCs prepared from mammals as described above may be used in all of the methods and other aspects of the invention described herein.

According to various embodiments of the invention, MAPCs may be isolated from the various compartments and tissues of such mammals in which they are present, including, but not limited to, bone marrow, peripheral blood, cord blood, spleen, liver, muscle, brain, adipose tissue, placenta, and other tissues described below. In some embodiments, MAPCs are cultured prior to use.

In some embodiments, MAPCs are isolated from bone marrow. In some embodiments of this aspect, the MAPCs are isolated from human bone marrow.

In many embodiments, MAPCs are not genetically engineered.

In some embodiments, the MAPCs are genetically engineered. MAPCs can be genetically engineered for a variety of purposes, such as those well known in the art. For example, they may be engineered to have improved growth characteristics, to enhance their therapeutic efficacy, to express one or more exogenous genes to produce beneficial substances, and to alter their immunological profile.

In some embodiments, the genetically engineered MAPCs are produced by in vitro culture. In some embodiments, the genetically engineered MAPCs are produced by transgenic organisms.

Preparation

MAPCs can be prepared from a variety of tissues, such as bone marrow cells, as discussed in more detail elsewhere herein.

In many embodiments, the purity of the MAPC administered to the subject is about 100%.

In other embodiments, the purity is 95% to 100%. In some embodiments, the purity is 85% to 95%. Particularly in the case of mixing with other cells, the percentage of MAPC may be 2% -5%, 3% -7%, 5% -10%, 7% -15%, 10% -20%, 15% -20%, 20% -25%, 25% -30%, 30% -35%, 35% -40%, 40% -45%, 45% -50%, 60% -70%, 70% -80%, 80% -90% or 90% -95%.

In some embodiments, the purity of the cells used for administration is about 100% (substantially homogeneous). In other embodiments, the purity is 95% to 100%. In some embodiments, the purity is from 85% to 95%. In particular, the percentage may be about 10% -15%, 15% -20%, 20% -25%, 25% -30%, 30% -35%, 35% -40%, 40% -45%, 45% -50%, 60% -70%, 70% -80%, 80% -90%, or 90% -95% in the case of mixing with other cells. Or isolation/purity can be expressed in terms of the cell doubling that the cell undergoes (e.g., 10-20, 20-30, 30-40, 40-50, or more cell doublings).

Treatment of disorders or diseases with MAPCs and the like may use undifferentiated MAPCs. It is also possible to treat MAPCs with already treated MAPCs in order to focus them on differentiation pathways. Treatment may also involve MAPCs that have been treated to differentiate into less potent stem cells with limited differentiation potential. It may also involve MAPCs that have been treated to differentiate into terminally differentiated cell types. The optimal type or mixture of MAPCs will depend on the particular environment in which they are used, and determining an effective type or combination of MAPCs in this regard is a matter of routine design for one skilled in the art.

The choice of formulation to administer MAPC for a given application will depend on a variety of factors. Prominent among these are the species of the subject, the nature of the intracerebral hemorrhage being treated and its status and distribution in the subject, the nature of the other therapies and agents being administered, the optimal route of administration of the MAPCs, the viability of the MAPCs by that route, the dosing regimen, and other factors that will be apparent to those skilled in the art. In particular, the selection of suitable carriers and other additives will depend, for example, on the exact route of administration and the nature of the particular dosage form.

Cell survival may be an important determinant of the efficacy of treatment with MAPC. This is true for both primary and adjuvant treatments. Another problem arises when the target site is not suitable for cell seeding and cell growth. This may hinder the entry of the therapeutic MAPC into the site and/or implant there. In embodiments, the invention includes the use of measures to increase cell survival and/or overcome problems caused by seeding and/or growth barriers.

Various additives are typically included to enhance the stability, sterility and isotonicity of the composition, such as antimicrobial preservatives, antioxidants, chelating agents, and buffering agents, and the like.

For example, prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents.

Pharmaceutically acceptable preservatives or cell stabilizers can be used to increase the longevity of MAPC compositions.

It is well within the ability of the skilled artisan to select compositions that do not affect the viability or efficacy of MAPC, if such additives are included.

Reference may be made to standard texts, such as "REMINGTON's pharmaceutical science", 17 th edition, 1985, incorporated herein by reference, to prepare suitable formulations without undue experimentation.

In preferred embodiments are solutions for injection, including those for topical, intravenous infusion, and stereotactic injection.

In some embodiments, the MAPCs are formulated in unit dose injectable form.

The skilled person can easily determine the amount of cells and optional additives, excipients and/or carriers in the composition to be administered in the method of the invention.

Other active agents

MAPCs may be administered with other pharmaceutically active agents. In some embodiments, one or more such agents are formulated with MAPCs for administration. In some embodiments, the MAPC and the one or more agents are in separate formulations. In some embodiments, compositions comprising MAPCs and/or one or more agents are formulated for adjunctive use with one another.

MAPCs may be administered in a formulation comprising an immunosuppressive agent, e.g., any number of any combination of corticosteroids, cyclosporin a, cyclosporin-like immunosuppressants, cyclophosphamide, antithymocytocin, azathioprine, FK-506, and macrolide immunosuppressants.

Such agents also include antibiotic, antifungal and antiviral agents, to name but a few other pharmacologically active substances and compositions that may be used in accordance with embodiments of the present invention.

Typical antibiotic and antifungal compounds include, but are not limited to, penicillin, streptomycin, amphotericin, ampicillin (ampicilin), gentamicin, kanamycin (kanamycin), mycophenolic acid (mycophenolic acid), nalidixic acid (nalidixic acid), neomycin (neomycin), nystatin (nystatin), paromomycin (paromomycin), polymyxin (polymyxin), puromycin (puromycin), rifampin (rifampicin), spectinomycin (spectinomycin), tetracycline (tetracycline), tylosin (tylosin), zeocin and cephalosporins (cephalosporins), aminoglycosides (aminoglycosides), and echinocandins (echinodins).

MAPCs may also be administered in combination with agents that enhance cellular homing to the site of injury (i.e., the site of injury caused by ICH). For example, MAPC may be administered with growth factors and trophic signaling agents, such as cytokines, e.g., stromal cell derived factor-1 (SDF-1), Stem Cell Factor (SCF), angiopoietin-1, placenta derived growth factor (PIGF), granulocyte colony stimulating factor (G-CSF), and those that stimulate the expression of endothelial adhesion molecules.

They may be administered to a subject as a pretreatment with MAPC, or following administration of MAPC, to promote homing to the desired site and achieve improved therapeutic effect by improved homing or by other mechanisms. These factors may be combined with MAPCs in a formulation suitable for their administration together. Alternatively, these factors may be formulated and administered separately.

The order of administration, formulation, dose, frequency of administration, and route of administration of the other active agents and MAPCs will generally vary with the ICH being treated, its severity, the subject, the other therapy being administered, the stage of the disorder or disease, and prognostic factors, among other factors. A general protocol that has been established for other treatments provides a framework for determining the appropriate dose in MAPC-mediated direct or adjuvant therapy. These, along with the additional information provided herein, will enable a skilled artisan to determine an appropriate dosing procedure according to embodiments of the invention without undue experimentation.

In embodiments, the cells are suitably formulated for use in the treatment of brain injury, including brain injury and/or dysfunction and/or disorder and/or disease as described herein. In some embodiments, the formulation is effective for parenteral administration. In some embodiments, the formulation is effective for intravenous infusion. In some embodiments, the formulation is effective for stereotactic injection.

Route of administration

MAPCs can be administered to a subject by any of a variety of routes known to those of skill in the art to be potentially useful for administering cells to a subject.

In various embodiments, MAPCs are administered to a subject by any effective route for delivering a cellular therapeutic agent. In some embodiments, the cells are administered by injection, including local and/or systemic injection. In certain embodiments, the cells are administered within and/or near the ICH site where the cells are to be treated. In some embodiments, the cells are administered by injection at a location that is not proximal to the site of the dysfunction. In some embodiments, the cells are administered by systemic injection, such as intravenous injection.

A method that may be used in this aspect of embodiments of the invention is a method of administering MAPC by systemic injection. Systemic injections, such as intravenous injections, provide the simplest and minimally invasive route of MAPC administration. In some cases, these routes may require high MAPC doses to achieve optimal efficacy and/or homing of MAPCs to the target. In various embodiments, MAPCs may be administered by targeted and/or local injection to ensure optimal efficacy at the target site.

In some embodiments of the invention, the MAPC may be administered to the subject via a syringe through a hypodermic needle. In various embodiments, the MAPC is administered to the subject via a catheter. In various embodiments, MAPC is administered by surgical implantation. Further in this aspect, in various embodiments of the invention, the MAPC is administered to the subject by implantation using arthroscopic methods. In some embodiments, the MAPC is administered to the subject by stereotactic injection.

Dosage form

The compositions are administered by dosages and techniques known to those of skill in the medical and veterinary arts, taking into account such factors as the age, sex, weight and condition of the particular patient, and the formulation (e.g., solid versus liquid) to be administered. The skilled artisan can determine dosages for humans or other mammals without undue experimentation, based on the present disclosure, the references cited herein, and the knowledge in the art.

The dosage of MAPC suitable for use in various embodiments of the invention will depend on a variety of factors. It may vary greatly from case to case. Parameters for determining the optimal dose of MAPC to be administered for primary and adjuvant therapy typically include some or all of the following: treated ICH and its staging; the species of the subject, their health, sex, age, weight, and metabolic rate; the immunological competence of the subject; other therapies administered; and potential complications as expected from the subject's medical history or genotype. The parameters may further include: whether the MAPCs are syngeneic, autologous, allogeneic or xenogeneic; their potency (specific activity); the site or distribution that must be targeted for MAPC to be effective; and such characteristics of the site such as accessibility of MAPCs and/or implantation of MAPCs. Other parameters include co-administration of other factors (such as growth factors and cytokines) with MAPCs. The optimal dosage in a given situation will also take into account the manner in which the cells are formulated, the manner in which they are administered, and the extent to which the cells localize to the target site after administration. Finally, determination of the optimal dose will necessarily provide an effective dose that is neither below the threshold of maximal beneficial effect nor above the threshold where the detrimental effects associated with MAPC doses outweigh the advantages of increasing the dose.

For some embodiments, the optimal dose of MAPC will be within the dose range for autologous mononuclear bone marrow transplantation. This can be estimated by extrapolation, from animal studies taking into account size (mass) differences and metabolic factors, and from the dose required for the establishment of other cell therapies (e.g. transplantation therapies).

In some embodimentsThe optimal dosage range for each administration is 10⁴To 10⁹Individual MAPC cells/kg recipient mass. In some embodiments, the optimal dose for each administration is 10⁵To 10⁸One MAPC cell/kg. In some embodiments, the optimal dose for each administration is 5 × 10⁵To 5X 10⁷One MAPC cell/kg. In some embodiments, the optimal dose for each administration is 1, 2, 3, 4, 5, 6, 7, 8, or 9 x 10⁶To 1, 2, 3, 4, 5, 6, 7, 8 or 9 × 10⁷Any dosage of (a).

For reference, some of the high and medium doses above are similar to the dose of nucleated cells used in autologous mononuclear bone marrow transplantation. Some of the mid-low doses were similar to the number of CD3+ cells/kg used in autologous mononuclear bone marrow transplantation.

It is understood that a single dose may be delivered all at once, in several fractions, or continuously over a period of time. It is also possible to deliver the entire dose to a single location or spread it in several portions over a plurality of locations.

In various embodiments, MAPCs may be administered at an initial dose, and then maintained by further administration of MAPCs. MAPC may be administered initially by one method and then by the same method or one or more different methods. MAPC levels in a subject can be maintained by ongoing cellular administration. Various embodiments administer MAPC initially by intravenous injection or to maintain its level in the subject, or both. In various embodiments, other forms of administration are used, depending on the condition of the patient and other factors discussed elsewhere herein.

Notably, the treatment time of human subjects is generally longer than that of experimental animals; however, the duration of treatment is generally proportional to the duration of the disease process and the effectiveness of the treatment. One skilled in the art will take this into account when using the results of other procedures performed in humans and/or animals (e.g., rats, mice, non-human primates, etc.) to determine an appropriate dosage for a human. Such determinations, based on such considerations and in view of the guidance provided by the present disclosure and prior art, will enable the skilled artisan to do so without undue experimentation.

Suitable regimens for initial administration and further or sequential administration may all be the same or may vary. The skilled person can determine suitable protocols based on the present disclosure, the documents cited herein and the knowledge in the art.

MAPCs may be administered at various frequencies over a wide time frame, for example, until the desired therapeutic effect is achieved. In some embodiments, the MAPC is administered over a period of less than one day. In other embodiments, they are administered within two, three, four, five or six days. In some embodiments, MAPCs are administered once or more times per week over weeks. In other embodiments, they are administered over a period of weeks for one to several months. In various embodiments, they may be administered over a period of months. In other cases, they may be administered over a period of one or more years. Generally, the length of treatment will be proportional to the length of the disease process, the effectiveness of the therapy applied, and the condition and response of the subject being treated.

In some embodiments, the MAPCs are administered once, twice, three times, or more than three times until the desired therapeutic effect is achieved or administration does not appear to be any more likely to provide a benefit to the subject. In some embodiments, MAPCs are administered continuously for a period of time, e.g., by intravenous drip. MAPC administration may be for a short time, days, weeks, months, years, or longer.

In some embodiments, administration is by a single bolus. In some embodiments, two or more administrations in a single bolus are administered one or more days apart in time. In some embodiments, each dose is administered by intravenous infusion over any period of time from a few minutes to a few hours. In some embodiments, a single dose of cells is administered by stereotactic injection. In some embodiments, two or more doses are administered to the same or different regions of the brain by stereotactic injection. In some embodiments, this aspect relates to bolus, intravenous, and stereotactic injections for the treatment of brain injuryFor injection, the cell dose per administration was 10⁴To 10⁹Individual MAPC cells/kg recipient mass. In some embodiments, the dose is 10⁵To 10⁸One MAPC cell/kg. In some embodiments, the dose is 5 x 10⁵To 5X 10⁷One MAPC cell/kg. In some embodiments, the dose is 1, 2, 3, 4, 5, 6, 7, 8, or 9 x 10⁶To 1, 2, 3, 4, 5, 6, 7, 8 or 9 × 10⁷Any one of them.

Isolation and growth of MAPC

Methods for MAPC isolation are known in the art. See, for example, U.S. patent 7,015,037, which methods and characterization (phenotype) of MAPCs are incorporated herein by reference. MAPCs can be isolated from a variety of sources, including but not limited to bone marrow, placenta, umbilical cord and cord blood, muscle, brain, liver, spinal cord, blood or skin. Thus, it is possible to obtain bone marrow aspirates, brain or liver biopsies and other organs, and isolate cells using positive or negative selection techniques available to those skilled in the art, depending on the genes expressed (or not) in these cells (e.g., by functional or morphological assays, such as those disclosed in the above-referenced applications, which are incorporated herein by reference).

MAPC has also been obtained by modification Methods described in Breyer et al, Experimental Hematology,34:1596-1601(2006) and Subramanian et al, Cellular Programming and reproduction: Methods and Protocols; ding (ed.), Methods in Molecular Biology,636:55-78(2010), which Methods are incorporated by reference.

Isolation and growth of MAPC as described in U.S. Pat. No. 7,015,037

Methods for isolating MAPCs from, e.g., humans, rats, mice, dogs, and pigs are known in the art. Exemplary methods are described, for example, in U.S. patent No. 7,015,037 and PCT/US02/04652 (published as WO 02/064748), and these methods, along with the features of the MAPCs disclosed therein, are incorporated herein by reference, by way of example and not limitation.

MAPCs were initially isolated from bone marrow and subsequently established from other tissues, including brain and muscle (Jiang, Y. et al (2002): Nature 418: 41-49). MAPCs can be isolated from many sources, including but not limited to bone marrow, placenta, umbilical cord and cord blood, muscle, brain, liver, spinal cord, blood, adipose tissue, and skin. For example, MAPCs can be derived from Bone Marrow aspirates, which can be obtained by standard methods available to those skilled in the art (see, e.g., Muschler, G.F. et al, (1997) J Bone Joint Surg am.79(11):1699-709 and Batinic, D. et al, (1990): Bone Marrow transfer 6(2): 103-7).

The human MAPC phenotype under certain conditions is proposed in U.S. Pat. No. 7,015,037

FACS immunophenotypic analysis of human MAPC obtained after 22-25 cell doublings showed that the cells did not express CD31, CD34, CD36, CD38, CD45, CD50, CD62E and-P, HLA-DR, Mucl8, STRO-1, cKit, Tie/Tek; and express low levels of CD44, HLA class I and beta.2-microglobulin, but express CD10, CD13, CD49b, CD49e, CDw90, Flkl (N > 10).

Once the cells are at a temperature of about 2X 10³/cm²(ii) experience in re-inoculated cultures>At 40 doublings, the phenotype became more uniform and no cells expressed HLA class I or CD44 (n-6). When cells were grown with higher fusions (confluences), they expressed high levels of Muc 18, CD44, HLA class I and β.2-microglobulin, similar to the described MSC (N ═ 8) (Pittenger,1999) phenotype.

Immunohistochemistry showed that at about 2X 10³3/cm²Human MAPCs grown at inoculation density express EGF-R, TGF-R1 and TGF-2, BMP-R1A, PDGF-Rla and PDGF-B, and a small subset (1% to 10%) of MAPCs were stained with anti-SSEA 4 antibody (Kannagi, R, 1983).

Determined at about 2X 10 using Clontech cDNA array³Cells/cm²The gene profile of expression of human MAPCs cultured over 22 and 26 cell doublings:

mapc does not express CD31, CD36, CD62E, CD62P, CD44-H, cKit, Tie; receptors for IL1, IL3, IL6, IL11, G CSF, GM-CSF, Epo, Flt3-L, or CNTF; and low levels of HLA class I, CD44-E and Muc-18 mRNA.

Mapc express mRNA to: cytokines BMP1, BMP5, VEGF, HGF, KGF, MCP 1; cytokine receptors Flk1, EGF-R, PDGF-R1. alpha., gp130, LIF-R, activin-R1 and activin-R2, TGFR-2, BMP-R1A; adhesion receptors CD49c, CD49d, CD 29; and CD 10.

mRNA of mapc expressing hTRT and TRF 1; POU domain transcription factors oct-4, sox-2 (oct-4 is required to maintain the undifferentiated state of ES/EC (Uwanogho, D. (1995): Mech Dev 49 (1-2): 23-36)); sox 11 (neurodevelopment), sox 9 (chondrogenesis) (Lefebvre V. et al (1998): Matrix Biol 16(9): 529-40); homeodomain transcription factors Hox-a4 and-a 5 (cervical and sternal norms; airway organogenesis) (Packer AI (2000): Dev Dyn 217 (1): 62-74); hox-a9 (myelopoiesis) (Lawrence H. (1997): Blood 89 (6): 1922-30); dlx4 (head forebrain and peripheral structural norms) (Akimenko MA (1994): J Neurosci (6):3475-86), MSX1 (embryonic mesoderm, adult heart and muscle, cartilage and osteogenesis) (Foerst-Potts L. (1997) Dev Dyn209(1): 70-84); PDX1 (pancreas) (Offield MF et al (1996): development.122(3): 983-95).

The presence of oct-4, LIF-R and hTRT mRNAs was confirmed by RT-PCR.

E. In addition, RT-PCR showed that rex-1 mRNA and rox-1 mRNA were expressed in MAPC.

Oct-4, rex-1 and rox-1 are expressed in MAPCs derived from human and murine bone marrow and murine liver and brain. Human MAPCs express LIF-R and SSEA-4 stains positive. Finally, oct-4, LIF-R, rex-1, and rox-1 mRNA levels were found to increase in human MAPCs cultured for more than 30 cell doublings, resulting in phenotypically more uniform cells. In contrast, MAPCs cultured at high densities lost the expression of these markers. This was associated with senescence and loss of differentiation into cells other than chondroblasts, osteoblasts and adipocytes prior to 40 cell doublings. Thus, the presence of oct-4 and rex-1, rox-1 and sox-2 correlates with the presence of the most primitive cells in MAPC cultures.

Culturing MAPC

Methods of culturing MAPCs are well known in the art. (see, e.g., U.S. patent forNo. 7,015,037, incorporated herein by reference for a method of culturing MAPCs. ) The density for culturing MAPCs can be about 100 cells/cm²Or about 150 cells/cm²To about 10,000 cells/cm²Including about 200 cells/cm²To about 1500 cells/cm²To about 2000 cells/cm²An internal variation. The density may vary from species to species. In addition, the optimum density may vary depending on the culture conditions and the cell source. Determining the optimal density of cells for a given set of culture conditions is within the skill of the ordinary artisan.

In addition, effective ambient oxygen concentrations of less than about 10%, including about 3% to 5%, may be used at any time during isolation, growth and differentiation of MAPCs in culture.

Other methods of cultivation

In other experiments, the density used to culture MAPCs can be about 100 cells/cm²Or about 150 cells/cm²To about 10,000 cells/cm²Including about 200 cells/cm²To about 1500 cells/cm²To about 2000 cells/cm²An internal variation. The density may vary from species to species. In addition, the optimum density may vary depending on the culture conditions and the cell source. Determining the optimal density of cells for a given set of culture conditions is within the skill of the ordinary artisan.

In addition, effective ambient oxygen concentrations of less than about 10%, including about 1-5%, especially 3-5%, can be used at any time during isolation, growth and differentiation of MAPCs in culture.

Cells can be cultured at different serum concentrations, e.g., about 2-20%. Fetal bovine serum may be used. Higher serum may be used in combination with lower oxygen tension, e.g., about 15-20%. Cells need not be selected prior to adhering to the culture dish. For example, after a Ficoll gradient, cells can be plated directly, e.g., 250,000-². Adherent clusters can be picked, possibly pooled and expanded.

In one embodiment, for the experimental procedures in the examples, high serum (about 15-20%) and low oxygen (about 3-5%) conditions were used for cell culture. In particular, adherence from clustersCells were grown at about 1700-2300 cells/cm²Density of (d) was plated and passaged in 18% serum and 3% oxygen (with PDGF and EGF).

Detailed Description

The following examples are offered by way of illustration only and are in no way limiting, exclusive or exhaustive of the many aspects and embodiments of the invention disclosed herein.

Example 1 preparation of MAPC

Those who use Athersys Inc. Cleveland in the examples described belowMAPC. These are MAPCs derived from human bone marrow, isolated from bone marrow aspirates, obtained with consent from healthy donors, and processed according to the methods described previously, essentially as described in Penn, MS et al, Circ Res 2012; 110(2) 304-11; maziarz, RT et al, Biology of Blood and Marrow transfer 2012; 18(2 Sup): S264-S265; and clinicaltirials. gov # NCT01436487, # NCT01240915 and # NCT 01841632). Briefly, in fibronectin-coated plastic tissue culture flasks at low oxygen tension at 5% CO₂In a humid atmosphere, MAPCs were cultured. Cells were maintained in MAPC medium (Low glucose DMEM [ Life Technologies Invitrogen)]Medium supplemented with the following supplements: FBS (Atlas Biologicals, Fort Collins, CO), ITS liquid Medium supplement [ Sigma]、MCDB[Sigma]Platelet derived growth factor (R)&D Systems, Minneapolis, MN), epidermal growth factor (R)&D Systems), dexamethasone ([ Sigma)]Penicillin/streptomycin [ Life Technologies Invitrogen]2-phospho-L-ascorbic acid [ Sigma, St. Louis, Mo.) and linoleic acid albumin (Sigma). Cells were passaged every 3-4 days and harvested using trypsin/EDTA (Life Technologies Invitrogen, Carlsbad, Calif.). Cells were positive for CD49c and CD90, and negative for MHC class II and CD 45. The cells were then multiplied 30-35 times in population, 1-10X 10 in 1ml in the gas phase of liquid nitrogen⁶Was frozen in cryovials at the concentrations of (PlasmaLyte, 5% HSA and 10% DMSO). MAPCs were thawed immediately prior to use and then used directly.

Example 2 collagenase ICH induction in rats

The mouse collagenase model of ICH was used for the study as described previously (Sukumura-Ramesh et al, J Neurotrauma 29(18): 2798-. Briefly, adult male C57B1/6J mice (8-10 weeks old) were placed in a stereotactic frame and a 0.5mm diameter drill hole was drilled in the apical cortex 2.2mm outside the bregma. A26-GHamilton syringe containing 0.04. mu.I bacterial type IV collagenase in 0.5. mu.I saline was lowered 3mm deep from the surface of the skull directly into the left striatum. The syringe was pressed at a rate of 450nl/min and left in place for a few minutes after this step to prevent reflux and excessive diffusion of the solution. The hole was closed with bone wax after the syringe was removed, the incision was surgically sutured, and the mouse was kept warm until the orthotropic reflex was restored. Littermates (littermates) were used for all studies to reduce the source of experimental variability.

Animals received Intravenous (IV) saline (control; n-10) orCells (n-11).

Cells or saline were administered intravenously 2 hours after the start of bleeding (2 hours after collagenase injection). All cell treated animals received 100 million cells.

Hematoma volume and brain perfusion were assessed by Magnetic Resonance Imaging (MRI) within three weeks post-injury as described below.

Neurobehavioral results, including grip strength test, narrow beam test, and elevated body swing test, were evaluated on day 7 post injury as described below.

Example 3 MAPC reduction of hematoma volume after ICH

Intravenous injection as early as 1 day after injuryThe cells significantly reduce the volume of the hematoma. This effect persists the first week after ICH. Consistent with accelerating the resolution of the hematoma. Mice were anesthetized with isoflurane (3% for induction, 1.5% for maintenance in N₂/O₂In a 2:1 mixture) and useImaging on a horizontal 7 Tesla BioSpec MRI spectrometer (Bruker Instruments) equipped with a 12-cm self-shielding gradient device (maximum 45 Gauss/cm). The radio frequency pulses were applied using a standard transmit/receive volume coil (72-mm id) that was actively decoupled from a two-channel Bruker quadrature receiver coil located on the centerline of the animal's skull. Stereotactic ear rods (stereotaxic ear bars) were used to minimize motion during imaging. The temperature of the mice was maintained at 37 ± 0.5 ℃ using a pad heated by a circulating water bath. After localization using a three-plane fast low-angle acquisition sequence, MR was performed using a T2' -weighted MRI scan. MRI was acquired using the following parameters: t2 mapping sequence (two-dimensional gradient echo sequence with multiple echoes; TE.5, 10, 15, 20, 25 and 30 ms; TR.3,000 ms; FOV: 32 mm; slice thickness of 1-mm (IS slice)](ii) a A 256x256 matrix; NEX ═ 2). The obtained image was volume segmented using ImageJ software and hematoma volume was calculated. The T2 × W images were further processed using Bruker software to generate magnetosensitive weighted images (Sehgaletak, 2006), providing an alternative approach to segmentation and quality control referencing of blood clot volumes. Hematoma and ventricle volumes were determined by drawing an irregular region of interest (ROI) on all MRI slices containing the lesion/ventricle, and the sum (area) was multiplied by the slice thickness to calculate the volume. Analysis was performed using ImageJ software.

Cell treated animals showed a statistically significant reduction in hematoma volume (about 4-fold reduction) within one day of treatment. At least on the first 7 days, the reduction in cell treated animals relative to saline treated animals was statistically significant.

The results are shown in FIG. 1. Mice were given intravenous placebo (PBS, n-10) or 2 hours after collagenase induction of ICH(n-11). Hematoma volume was assessed by MRI (T2W) using 7T small animal MRI. Representative coronal brain images at day 3 and day 7 are provided, and panel A showsSignificant benefit to hematoma volume. FIG. B depicts allData for mice over the 21 day evaluation period. Data are presented as mean +/-SEM and analyzed by student t-test at each time point. P<0.01 vs placebo treated ICH mice.

Example 4 improvement of brain perfusion following ICH by MAPC

Within more than 1 week after the ICH,improving brain perfusion in and around the injured striatum. Mice were anesthetized with isoflurane (3% for induction, 1.5% for N maintenance at 2: 1)₂/O₂And imaged (maximum 45 gauss/cm) using a horizontal 7 Tesla BioSpec MRI spectrometer (Bruker Instruments) equipped with a 12-cm self-shielding gradient device. The radio frequency pulses were applied using a standard transmit/receive volume coil (72 mm id) that was actively decoupled from a two-channel Bruker quadrature receiver coil located on the centerline of the animal's skull. The use of a stereotactic ear rod minimizes movement during imaging. The temperature of the mice was maintained at 37 ± 0.5 ℃ using a pad heated by a circulating water bath. After localization using a three-plane fast low-angle acquisition sequence, MR studies were performed using a T2-weighted MRI scan. MRI was acquired using the following parameters: t2-fluid decay inversion recovery sequence (RARE-IR, T1 · 2000; TR · 10,000 ms; TE · 36 ms; RARE factor ═ 8; FOV ═ 32 mm; 256 × 256 matrix; l-mm slice thickness; JS slice analysis using ImageJ software.

Blood flow in cell treated animals was statistically significantly improved in the first 7 days after treatment compared to injured animals treated with physiological saline. This indicates intravenous infusionThe cellular products have a dramatic improvement in cerebral blood flow in the brain following the onset of a hemorrhagic stroke, possibly resulting in edema, tissue damage and a reduction in nerve circuit interruption.

The results are shown in fig. 2. Mice were given intravenous placebo (PBS, n-10) or 2 hours after collagenase induction of ICH(n-11). Brain perfusion was assessed by MRI (ASL; FAIR-RARE) using 7T small animal MRI. A representative coronary brain image is provided in panel a and the quantized data is shown in panel B. The data indicate that within the first week after the ICH,improves the cerebral perfusion. Data are presented as mean +/-SEM and analyzed by student t-test at each time point. P<0.05，**p<0.01 versus placebo treated ICH mice.

Example 5 MAPC reduces functional deficits and improves behavior after ICH

The changes in observed infarct volume reduction and brain perfusion improvement reflect functional improvement in motor behavior in grip testing, reduction in time to pass through the beam, and normalized left/right swing ratio.

Behavioral testing

Hanging wire test (Hanging wire test)

The grip evaluation was performed by placing the mouse on a device consisting of a 50-cm rope pulled between two vertical supports. Mice were evaluated as follows: 0: dropping; 1: hanging the rope by two front claws; 2: same as 1, but trying to climb on a rope; 3: hanging the two front claws and one or two hind limbs on a rope; 4: the front claw is hung on the rope, and the tail is wound on the rope; and 5: escape. The highest reading of three consecutive trials was taken per animal at each time point.

Narrow beam walking (Narrow beamwalk)

The motor coordination was assessed on a fixed narrow beam (6mm wide, 1m long) for three consecutive days. The first two days included training and the third day quantified the behavior on the beam by measuring the time required to traverse the beam. Each mouse was tested 3 times by an blinded investigator and the average was recorded.

Elevated body swing test

Holding the animal 1cm from the tail root and hanging 1-5cm above the plane. One oscillation is recorded per suspension. Swing is defined as being >10 degrees off the body midline or rotated about a vertical axis. The mice were placed on the surface between the two suspensions, allowed to significantly reposition so that no lateral preference was observed, and then resuspended. The evaluator changes the hand and standing position, the test area has no visual cues to avoid deviating from the swing direction. Each test recorded 20 oscillations and the lateral preference was calculated as the oscillation/total oscillation of one side.

These data show that in three tests carried out on animals, it is demonstrated that the hemorrhagic stroke is followed by its onset, in comparison with animals treated with saline onlyAcute treatment can significantly improve the benefits of the motor and nervous system.

The results are shown in fig. 3. Placebo (PBS, n-10) or(n-11) was intravenously administered to mice. Neurological assessment of motor function was assessed on day 7 post injury (or in sham operated mice; n-8). (A) And (6) testing the grip strength. (B) The narrow beam task. (C) Raising the body swing task. Data are mean +/-SEM and compared using factorial analysis of variance and subsequent Tukey post hoc test. P<.05，**p<0.01，***p<0.001, ns is not significant.

The above results are surprisingly good, especially considering that there is currently no approved treatment for patients with hemorrhagic stroke other than surgical clearance in patients when the location and size of the blood clot is suitable for surgery. No approved drugs or therapies exist.

The Dhandapani group published the latest preclinical paper for the evaluation of experimental therapies in 2018, 9 months, focusing on the reduction of hematoma volume by inhibiting adenosine monophosphate kinase α -1(AMPKal) (Vaibhav, 2018). In this paper, the same type of MRI results as in the present application, as well as motor and neurological endpoints, were evaluated.

Compared to the results provided in the present application, administrationCells consistently gave better results in reducing hematoma and blood flow, and to the same extent, if not better, in motor outcomes. These are the best results seen in ICH treatment studies.

As will be understood by those of skill in the art, the foregoing description and examples are illustrative, but are not exhaustive of the many aspects and implementations encompassed by the invention disclosed herein.

All publications mentioned in the foregoing disclosure are incorporated in their entirety into the present disclosure by reference, particularly in the section thereof most relevant to the subject matter to which they have been specifically referenced.