Multipotent adult progenitor cells for use in treating intracerebral hemorrhage - Patent Application 20070122999

Abstract

Aspects of the invention described herein relate to the administration of multipotent adult progenitor cells to treat intracerebral hemorrhage.Stem cells have not previously been considered useful for treating ICH.As described herein, it has surprisingly been found that administering multipotent adult stem cells as described herein to treat ICH has an unexpected and surprisingly effective therapeutic benefit on the outcome of ICH, as shown by hematoma volume, cerebral blood flow / perfusion results and functional neurological assessment measures.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to intracerebral hemorrhage and multipotent stem cells.

[0002] government funding No government funds were used in making the invention disclosed herein.

[0003] Related Applications This application claims priority to U.S. Patent Application No. 16 / 265,373, filed February 1, 2019, and having the same title, the entirety of which is incorporated herein by reference. [Background technology]

[0004] I Intracerebral hemorrhage ("ICH") refers to any bleeding within the intracranial vault, including the brain parenchyma and surrounding medullary cavity. ICH is a devastating disease that affects substantial populations in the United States and worldwide (see, e.g., Caceres and Goldstein (2012): Emerg Med Clin North Am 30(3) 771-794). The worldwide incidence of spontaneous ICH is 24.6 per 100,000 observed person-years, with approximately 40,000 to 67,000 cases per year in the United States. The 30-day mortality rate ranges from 35% to 52%. Approximately half of all mortality occurs within the first 24 hours. Only 20% of survivors have a complete functional recovery by 6 months. Early and effective treatment is believed to be crucial (see, e.g., van Asch et al. (2010): The Lancet Neurology 9(2) 167-176; Aguilar and Freeman (2010): Semin Neurol 30(5) 555-64; Broderick et al. (2007): Stroke 38(6) 2001-2023; Elliott and Smith (2010): Anesthesia & Analgesia 110(5) 1419-1427; and Feigin et al. (2009): The Lancet Neurology 8(4) 355-369).

[0005] Intraparenchymal hemorrhages often result from penetrating head trauma. They can also result from depressed skull fractures. Additional causes include ruptured aneurysms, arteriovenous malformations (AVMs), bleeding within tumors, and acceleration-deceleration trauma. In patients over 55 years of age, amyloid angiopathy is a frequent cause of intracerebral hemorrhage. Cerebral venous sinus thrombosis accounts for a very small percentage of ICH cases.

[0006] Primary ICH is often a manifestation of underlying small vessel disease. First, longstanding hypertension leads to hypertensive vasculopathy, which causes microscopic degenerative changes (lipohyaline degeneration) in the walls of small to medium-sized penetrating vessels. Second, cerebral amyloid angiopathy develops, which is characterized by the deposition of amyloid-β peptide (Aβ) in the walls of small leptomeningeal and cortical vessels. The mechanisms leading to amyloid deposition are unknown; however, the conclusions are well documented: degenerative changes in the vascular wall characterized by loss of smooth muscle cells, wall thickening, luminal narrowing, microaneurysm formation, and microbleeding (see, e.g., Fisher, C.M. (1971): J Neuropathol Exp Neurol 30(3) 536-50; Vinters H. (1987): Stroke 18(2) 311-324; and Viswanathan et al. (2011): Annals of Neurology 70(6) 871-880).

[0007] After initial vascular rupture, the hematoma causes direct mechanical injury to the brain parenchyma. Perihematomal edema develops within the first 3 hours of symptom onset and peaks between 10 and 20 days. Blood and plasma products then mediate secondary injury processes, including inflammatory responses, activation of the coagulation cascade, and iron deposition from hemoglobin degradation. Finally, the hematoma may continue to expand in up to 38% of patients during the first 24 hours (see, e.g., Aronowski and Zhao (2011): Stroke 42(6): 781-6 and Brott et al. (1997): Stroke 28(1) 1-5).

[0008] Briefly, ICH is a type of brain injury resulting from leakage of blood into the brain and accumulation of blood in the brain parenchyma. ICH can result from aneurysm rupture, damage (e.g., perforation) of a cerebral artery, or arteriovenous malformation (AVM). ICH is a devastating neurological injury, accounting for approximately 20% of all stroke-related injuries worldwide and nearly 30% in Japan and Asia. It has the highest mortality rate and the worst long-term outcomes of all stroke-related injuries, and ICH is responsible for nearly 50% of all stroke-related deaths. Because hematoma volume is an independent determinant of ICH patient outcome, early clot resolution is a primary clinical objective. However, there are no FDA-approved treatments that improve ICH outcomes. Emergency surgery to remove the clot (if possible) and rehabilitation are the only current standard of care; however, surgical management is of limited utility, and rehabilitation addresses damage rather than preventing or repairing it. In fact, the current guidance from the AHA / ASA and the only recommended treatment for patients with ICH is surgical removal of the clot, if surgery is feasible. No other therapeutic interventions are recommended (see, e.g., Hemphill III, JC et al. (2015): AHA / ASA Guideline, Stoke 46: 2032-2060). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Caceres and Goldstein (2012): Emerg Med Clin North Am 30(3) 771-794 [Non-patent document 2] van Asch et al. (2010): The Lancet Neurology 9(2) 167-176 [Non-patent document 3] Aguilar and Freeman (2010): Semin Neurol 30(5) 555-64 Summary of the Invention [Problem to be solved by the invention]

[0010] Clearly, there is a great need for methods to address ICH injury and reduce and repair the resulting brain damage. Accordingly, among the goals of the embodiments of the invention disclosed herein are to provide means and methods for improving outcomes in cases of ICH.

[0011] Stem cells have not traditionally been considered useful for treating ICH. The pathophysiology of ICH, as mentioned above, is thought to be driven by the presence of red blood cells in the brain, which then degrade and release hemoglobin and its neurotoxic heme degradation products. Extravasation of red blood cells into the brain results in space-occupying hematomas / clots that are associated with mechanical tissue destruction, edema formation, elevated intracranial pressure, increased microvascular pressure, reduced blood flow, and poor outcomes, and do not appear to be amenable to treatment with cell therapy. [Means for solving the problem]

[0012] As described herein, it has surprisingly been found that administering multipotent adult stem cells as described herein to treat ICH has an unexpected and surprisingly effective therapeutic benefit on ICH outcome, as shown by measures of hematoma volume, cerebral blood flow / perfusion results, and functional neurological assessment.

[0013] II Some of the many embodiments encompassed by the present description are summarized in the following numbered paragraphs. The numbered paragraphs are self-referential. In particular, the phrase "in accordance with any of the foregoing or the following," as used in these paragraphs, also refers to the other paragraphs. The phrase means that the embodiments disclosed herein include both the subject matter described in each paragraph taken alone and the subject matter described by the paragraphs taken in combination in the following paragraphs. In this regard, in setting forth the following paragraphs, it is expressly Applicant's intent to describe various aspects and embodiments, particularly in terms of the paragraphs taken alone or in any combination. That is, the paragraphs are a concise way of setting forth and providing an explicit written description of all embodiments encompassed by them individually and in any combination with one another. Applicant specifically reserves the right to claim any subject matter set forth in any of the following paragraphs at any time, alone or together with any one or more other subject matter of the other paragraphs, whether employed alone or in any combination with any other value set forth therein, including any combination of any value set forth therein. If desired, applicants specifically reserve the right to disclose in full all of the combinations set forth herein in this application or in any subsequent application having the benefit of this application.

[0014] p1. A method for treating intracerebral hemorrhage in a subject, said method comprising administering to a subject in need thereof multipotent adult progenitor cells which are not embryonic stem cells, embryonic germ cells or germ cells, and which are capable of differentiating into at least one cell type of each of at least two of the endodermal, ectodermal and mesodermal lineages, and which are allogeneic or xenogeneic to said subject.

[0015] p2. A method of treating intracerebral hemorrhage in a subject, said method comprising administering to a subject in need thereof multipotent adult progenitor cells which are not embryonic stem cells, embryonic germ cells or germ cells, which express telomerase and which are allogeneic or xenogeneic to said subject.

[0016] p3. A method for treating intracerebral hemorrhage in a subject, said method comprising administering to a subject in need thereof multipotent adult progenitor cells which are not embryonic stem cells, embryonic germ cells or germ cells, which are positive for Oct4, and which are allogeneic or xenogeneic to said subject.

[0017] p4. A method of treating intracerebral hemorrhage in a subject, said method comprising administering to a subject in need thereof multipotent adult progenitor cells which are not embryonic stem cells, embryonic germ cells or germ cells, which have undergone at least 40 cell doublings in culture prior to their use, and which are allogeneic or xenogeneic to said subject.

[0018] p5. A method according to any of the foregoing and / or the following, wherein said cells are capable of differentiating into at least one cell type of each of at least two of the endodermal, ectodermal, and mesodermal lineages.

[0019] p6. The method according to any of the foregoing or following, wherein said cells express telomerase.

[0020] p7. A method according to any of the foregoing or the following, wherein said cells are positive for oct4.

[0021] p8. A method according to any of the foregoing or the following, wherein said cells express telomerase and are positive for oct4.

[0022] p9. A method according to any of the foregoing or the following, wherein said cells express telomerase and have undergone at least 40 cell doublings prior to their use.

[0023] p10. A method according to any of the foregoing or the following, wherein said cells express oct4 and have undergone at least 40 cell doublings prior to their use.

[0024] p11. A method according to any of the foregoing or the following, wherein said cells express telomerase, are positive for oct4 and have undergone at least 40 cell doublings prior to their use.

[0025] p12. A method according to any of the foregoing or the following, wherein said cells express any one or more of rex-1, rox-1, or sox-2.

[0026] p13. The method according to any one of the foregoing or the following, wherein said cells have a normal karyotype.

[0027] p14. The method according to any one of the foregoing or the following, wherein said cells are not tumorigenic.

[0028] p15. A method according to any of the foregoing or the following, wherein said cells do not form teratomas.

[0029] p16. A method according to any of the foregoing or following, wherein said cells are not genetically altered.

[0030] p17. A method according to any of the foregoing or the following, wherein said cells are genetically altered.

[0031] p18. A method according to any of the foregoing or the following, wherein said cells are not immunogenic in said subject.

[0032] p19. A method according to any of the foregoing or the following, wherein said cells are capable of differentiating into at least one cell type of each of the endodermal, ectodermal and mesodermal lineages.

[0033] p20. A method according to any of the foregoing or the following, wherein said cell is a mammalian cell.

[0034] p21. A method according to any of the foregoing or the following, wherein said cells are human cells.

[0035] p22. The method according to any of the foregoing or following, wherein said cells are derived from cells isolated from any one of placental tissue, umbilical cord tissue, umbilical cord blood, bone marrow, blood, spleen tissue, thymus tissue, spinal cord tissue, adipose tissue, and liver tissue.

[0036] p23. A method according to any of the foregoing or the following, wherein said cells are derived from bone marrow.

[0037] p24. The method according to any of the foregoing or following, wherein said subject is a human.

[0038] p25. 10 per kg of the subject's mass 4 ~10 8 one or more doses of said cells are used, or by one of the following methods:

[0039] p26. 10 per kg of the subject's mass 6 ~5×10 7 A method according to any of the foregoing or the following, wherein one or more doses of said cells of

[0040] p27. A method according to any of the foregoing or the following, wherein in addition to said cells one or more growth factors, differentiation factors, signaling factors, and / or factors that enhance homing are simultaneously used.

[0041] p28. A method according to any of the foregoing or following wherein an antimicrobial agent, an antifungal agent, an antiviral agent or a combination thereof is used concomitantly.

[0042] p29. A method according to any of the foregoing or the following, wherein said cells are in a formulation comprising one or more other pharmaceutically active agents.

[0043] p30. A method according to any of the foregoing or the following, wherein said cells are administered parenterally.

[0044] p32. The method according to any of the foregoing or the following, wherein said cells are administered intravenously.

[0045] p33. A method according to any of the foregoing or the following, wherein said cells are administered stereotactically.

[0046] p34. A method according to any of the foregoing or below, wherein the cells are administered at any one or more of 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes or 60 minutes after ICH, or 60 minutes, 90 minutes, 120 minutes, 150 minutes or 180 minutes after ICH, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours after ICH, or 12 hours, 18 hours, 24 hours, 30 hours, 36 hours or 40 hours after ICH, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after ICH, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks after ICH, or any combination of the foregoing and / or any later time.

[0047] III "A" or "an," as used herein, means one and more than one; at least Both refer to 1. When the plural is used herein, it generally includes the singular as well.

[0048] A "cell bank" is the industry term for cells that have been propagated and stored for future use. Cells may be stored in aliquots. They may be used directly off the shelf or expanded after storage. This facilitates the presence of "off-the-shelf" cells available for administration. The cells may already be stored in a pharmaceutically acceptable vehicle so that they can be administered directly or mixed with an appropriate vehicle when released from storage. Cells may be frozen or otherwise stored in a form that preserves viability. In one embodiment of the present invention, a cell bank is created in which the cells have been selected for enhanced potency to achieve the effects described herein. After release from storage and prior to administration, it may be preferable to re-assay the cells for potency. This can be done using any of the assays described herein (direct or indirect) or other methods known in the art. Cells with the desired potency can then be administered. The bank may be created using autologous cells (derived from an organ donor or recipient). Alternatively, the bank may contain cells for allogeneic use.

[0049] "Co-administer," as used herein, means administering two or more agents in conjunction with, together, or in a coordinated manner, including simultaneous or sequential administration.

[0050] "Comprising," as used herein, means including, without other limitation, what is recited, without any limitation or exclusion as to what else may necessarily be included. For example, "a composition comprising x and y" includes any composition comprising x and y, no matter what other components may be present in the composition. Similarly, "a method comprising the step of x" includes any method in which x is performed, whether x is the only step or only one of the steps, no matter how many other steps may be present, and no matter how simple or complex x is in comparison thereto. "Comprised of" and similar phrases using the root word "comprise" are used herein as synonyms of "comprising" and have the same meaning.

[0051] "Comprised of" as used herein is synonymous with "comprising" (see above).

[0052] "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 have been grown in that medium for a sufficient period of time to secrete factors that are effective against the growth of the particular type of cells to which the medium is being conditioned.

[0053] "Decrease" and "decreasing" and similar terms are used herein to generally mean a lessening in the amount, value, or effect of one thing as compared to another. For example, a decrease in the severity of ICH can mean a decrease in hematoma volume and / or functional impairment, respectively, as compared to the previous volume or functional impairment resulting from the ICH.

[0054] As used herein, "effective amount" generally refers to an amount that provides a 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 for treating ICH is an amount that reduces hematoma volume and / or improves and / or increases cerebral circulation and / or perfusion; and / or reduces neurological and / or functional impairments and / or improves functions such as motor function, balance, etc.

[0055] The effective amount can be provided all at once in a single administration, or can be provided in divided amounts that provide an effective amount in several administrations. The exact determination of what is considered to be an effective amount can be based on factors specific to each subject, including their size, age, injury, and / or the disease or injury being treated, and the amount of time since the injury occurred or the disease began. Those skilled in the art can determine the effective amount for a given subject based on these considerations, which are conventional in the art. As used herein, "effective dose" means the same as "effective amount."

[0056] "Effective route," as used herein, generally refers to a route that provides for delivery of a drug to a desired compartment, system, or location. For example, an effective route is one by which a drug can be administered to provide a sufficient amount of drug at a desired site of action to achieve a beneficial or desired clinical result.

[0057] "ICH" as used herein is an acronym for "intracerebral hemorrhage" and has the same meaning.

[0058] The terms "includes" and "including," as used herein, are not limiting and mean roughly the same as "comprises" and "comprising."

[0059] "Increase" and "increasing," as used herein, mean making (including inducing) a biological event or characteristic, such as in size, amount, intensity, or degree (as from a zero or inactive state), greater. For example, an effective amount for treating ICH is one that increases, for example, cerebral circulation and / or perfusion; and / or improves function such as motor function, balance, etc. (e.g., by comparison with previous post-ICH function).

[0060] "Intracerebral hemorrhage" ("ICH") is intracranial bleeding into brain tissue, the ventricles, or both. Causes of ICH include, but are not limited to, bleeding from, or as a result of, any one or more of the following: aneurysm, arteriovenous malformation, brain tumor, and brain trauma. ICH is also referred to as "brain bleed."

[0061] The term "isolated," as used herein, refers to a cell that is not associated with one or more cells or one or more cellular components associated with said cell in vivo. An "enriched population" refers to an increase in the number of a desired cell relative to one or more other cell types in vivo or in primary culture.

[0062] However, as used herein, the term "isolated" does not refer to the presence of stem cells alone. Rather, the term "isolated" indicates that the cells have been removed from their natural tissue environment and are present in a higher concentration than in their normal tissue environment. Thus, an "isolated" cell population may contain additional cell types and tissue components in addition to stem cells. This may also be expressed, for example, in terms of cell doublings. Cells may have undergone 10, 20, 30, 40, or more doublings in vivo or ex vivo so as to be enriched compared to their original number in vivo or in their original tissue environment (e.g., bone marrow, peripheral blood, adipose tissue, etc.).

[0063] "MAPC" is an acronym for "multipotent adult progenitor cell." It refers to cells that are neither embryonic stem cells nor germ cells. MAPCs can be characterized by many alternative descriptions, each of which conferred novelty to the cells when they were discovered. Thus, they can be characterized by one or more of these descriptions. First, they are not genetically engineered or transformed (tumorigenic), have a normal karyotype, and have the capacity for long-term replication in culture. This means that these cells express telomerase (i.e., have telomerase activity). Second, upon differentiation, they can give rise to cell progeny of more than one germ layer, for example, two or all three germ layers (i.e., endoderm, mesoderm, and ectoderm). Third, although they are not embryonic stem cells or germ cells, MAPCs can express markers of these primitive cell types, such that they can express one or more of oct4, rex-1, and rox-1. They can also express sox-2. Rex-1 is regulated by oct4, which activates downstream expression of rex-1. Rox-1 and sox-2 are expressed in non-ES cells. Thus, the cell type designated "MAPC" can be characterized by alternative cardinal features that describe these cells through some of its novel properties.

[0064] The term "adult" in MAPC is non-limiting. It refers to non-embryonic somatic cells (e.g., postnatal). MAPCs do not form teratomas in vivo. This acronym was first used in U.S. Pat. No. 7,015,037 to describe pluripotent cells isolated from bone marrow. However, cells with pluripotency markers and / or differentiation potential have been subsequently discovered and may be equivalent, for purposes of the present invention, to those cells originally designated "MAPC." An essential description of MAPC types of cells is provided in the Summary of the Invention above.

[0065] MAPCs represent a more primitive progenitor population than MSCs (Verfaillie, CM, Trends Cell Biol 12:502-8 (2002); Jahagirdar, BN et al., Exp Hematol, 29:543-56 (2001); Reyes, M. and CM Verfaillie, Ann NY Acad Sci, 938:231-233 (2001); Jiang, Y. et al., Exp Hematol, 30896-904 (2002); and Jiang, Y. et al., Nature, 418:41-9. (2002)).

[0066] MAPCs may not be immunogenic. MAPCs may be immunosuppressive. MAPCs may or may not be genetically altered to improve their characteristics. MAPCs may be used with or without concomitant immunosuppressive treatment. Further aspects of MAPCs are described herein.

[0067] As used herein, the phrase "may" means the same as "optionally," and even if not stated otherwise, when used herein, "may" also includes "may not." That is, a statement that something may be present also means that it may not be present. That is, when used herein, "may" expressly includes "may not," and applicants reserve the right to claim subject matter accordingly. For example, when used herein, a statement that MAPC may be administered with other agents also means that MAPC may be administered without any other agents. For another example, when used herein, a statement that MAPC may be genetically engineered also means that MAPC may not be genetically engineered.

[0068] "Multipotent," as used herein with respect to MAPCs, refers to the ability of MAPCs, upon differentiation, to give rise to cell lineages of more than one of the three primitive germ layers: endoderm, mesoderm, and ectoderm (e.g., two of all three).

[0069] "MultiStem®" is the trade name for the MAPC-based cell preparation (i.e., non-embryonic stem, non-germ cells as described above) of U.S. Patent No. 7,015,037. MultiStem® is prepared according to the cell culture methods disclosed in this patent application, particularly the lower oxygen and higher serum conditions. MultiStem® is highly expandable, karyotypically normal, and does not form teratomas in vivo. It can differentiate into cell lineages of more than one germ layer and express one or more of telomerase, oct4, rex-1, rox-1, and sox-2.

[0070] "Oct-4" is a member of the POU family of transcription factors (1). The mouse protein was first identified and classified as Oct-3. The human homolog was initially classified as Oct-3 based on its 87% amino acid identity with the mouse Oct-3 protein. Two human Oct-3 transcripts were subsequently identified (Takeda et al., Nucleic Acids Research 20(17) 4613-20 (1992)). Oct-3A and Oct-3B are alternative splice products of the same gene. Oct-3A has been identified and renamed Oct-4 by some groups and Oct-3 / 4 by others. Still other groups have subdivided Oct-4 into Oct-4A and Oct-4B as alternative transcripts. The A transcript (i.e., Oct-4, Oct-3A, Oct-3 / 4) produces a nuclear protein, is associated with pluripotency, and is restricted to a portion of exon 1. The B transcript is found in many cell types as a cytoplasmic protein and encompasses the remainder of exon 1 and exons 2-5.

[0071] "Optionally," as used herein, means roughly the same as "may," and a statement that X optionally includes A, as used herein, encompasses both X including A and X not including A.

[0072] A "pharmaceutically acceptable carrier" is any pharmaceutically acceptable medium for the cells used in the present invention. Such a medium can maintain isotonicity, cellular metabolism, pH, etc. It is compatible with administration to a subject in vivo and therefore can be used for cell delivery and treatment.

[0073] "Progenitor cells" are cells generated during the differentiation of stem cells that have some (but not all) of the characteristics of their terminally differentiated progeny. Defined progenitor cells (e.g., "cardiac progenitor cells") are committed to one lineage, but not to a specific or terminally differentiated cell type. The term "progenitor" as used in the acronym "MAPC" does not restrict these cells to a specific lineage.

[0074] The term "reduce," as used herein, means to prevent and to decrease. In the context of treatment, "reducing" is both preventing or ameliorating one or more clinical symptoms. A clinical symptom is one (or more) symptoms that, if left untreated, have or will have a negative impact on a subject's quality of life (well-being).

[0075] "Selecting" cells with a desired level of potency can mean identifying (by assay), isolating, and expanding the cells. This can create a population with higher potency than the parent cell population from which the cells were isolated. The "parent" cell population refers to the parent cell from which the selected cell divided. "Parent" refers to the actual P1→F1 relationship (i.e., progeny cell). Thus, if cell X is isolated from a mixed population of cells X and Y (where X is an expressor but Y is not), the mere isolate of X would not be classified as having enhanced expression. However, if a progeny cell of X is a higher expressor, the progeny cell would be classified as having enhanced expression.

[0076] "Self-renewal," as used herein, refers to the ability of a replicating daughter hepatocyte to generate its replicating daughter stem cells that have differentiation potential identical to the cell from which it arose. A similar term used in this context is "proliferation."

[0077] "Stem cell," as used herein, means a cell that can undergo self-renewal (i.e., progeny with the same differentiation potential) and also generate progeny cells with more restricted differentiation potential.

[0078] "Subject," as used herein, means a vertebrate, such as a mammal (e.g., a human). Mammals include, but are not limited to, humans, dogs, cats, horses, cows, and pigs.

[0079] 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 therapeutic cells or cell-related agents can prolong a patient's chances of survival and / or inhibit overt clinical symptoms. A treatment that is therapeutically effective within the meaning of the term, as used herein, includes a treatment that improves a subject's quality of life, even if the subject does not improve the disease outcome itself. For example, therapeutically effective can mean reducing the volume of hemorrhage, improving cerebral blood flow, and / or improving neurological and / or behavioral function (e.g., after ICH). Such therapeutically effective amounts are easily ascertained by those skilled in the art.

[0080] "Treat," "treating," or "treatment" are used broadly in the context of the present invention, and each such term essentially refers to the administration of cells as described herein, particularly with, in advantageous embodiments, the beneficial effect of one or more (but not necessarily any or all) of preventing, ameliorating, inhibiting, or curing a defect, dysfunction, disease, or other deleterious process (including those that interfere with and / or result from the treatment). For example, treating can mean reducing the volume of hemorrhage, improving cerebral blood flow, and / or improving neurological and / or behavioral function (e.g., after ICH). Such aspects of treatment are readily ascertained by those skilled in the art.

[0081] "Validate," as used herein, means to confirm. In the context of the present invention, cells are confirmed to be manifesters of the desired efficacy. This allows the cells to be used later (in treatments, banking, drug screening, etc.) with a reasonable expectation of effectiveness. Thus, validating means confirming that cells originally found / established to have the desired activity actually retain that activity. Validation is therefore the confirmatory event in a two-event process involving its original determination and a follow-up determination. The second event is referred to herein as "validation."

[0082] III The various features and advantages of the embodiments described herein may be better appreciated as the same becomes better understood when considered in light of the accompanying drawings. [Brief explanation of the drawings]

[0083] [Figure 1] Figures 1A and 1B: MULTISTEM® cells reduce hematoma volume after collagenase-induced ICH. Placebo (PBS, n=10) or MultiStem® (n=11) was administered intravenously to mice 2 hours after collagenase-induced ICH. Hematoma volume was assessed by MRI (T2W) using a 7T small animal MRI. Representative coronal brain images on days 3 and 7 are provided. Figure 1A shows the dramatic benefit of MultiStem® cells on hematoma volume. Figure 1B shows data from all mice over the 21-day evaluation period. Data are presented as mean ± SEM and analyzed by Student's t-test within each time point. **p<0.01 vs. placebo-treated ICH mice. Details are provided in Example 3.

[0084] [Figure 2]Figures 2A and 2B: MULTISTEM® cells improve cerebral perfusion after collagenase-induced ICH. Placebo (PBS, n=10) or MultiStem® (n=11) was administered intravenously to mice 2 hours after collagenase-induced ICH. Cerebral perfusion was assessed by MRI (ASL; FAIR-RARE) using a 7T small animal MRI. Figure 2A - Representative coronal brain images. Figure 2B - Quantified perfusion data. The data show that MultiStem® improves cerebral perfusion over the first week after ICH. Data are presented as mean ± SEM and analyzed by Student's t-test within each time point. *p<0.05, **p<0.01 vs. placebo-treated ICH mice. Details are provided in Example 4.

[0085] [Figure 3] Figures 3A, 3B, and 3C: MULTISTEM® cells improve motor function after collagenase-induced ICH. Placebo (PBS, n=10) or MultiStem® (n=1) was administered intravenously to mice 2 hours after collagenase-induced ICH. Neurological assessment of motor function was assessed 7 days after injury (or in sham-operated mice; n=8). Figure 3A - Grip strength test results. Figure 3B - Beam task test results. Figure 3C - Elevated body swing task test results. Data are mean ± SEM and compared using one-way ANOVA followed by Tukey's post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ns=not significant. Details are provided in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0086] V As described herein, aspects of the invention relate to administering MAPC (as defined herein) to subjects who have experienced an intracerebral hemorrhage (otherwise known as hemorrhagic stroke). These patients will not receive therapeutic intervention other than surgical evacuation of the clot, provided the size and location of the clot in the brain is amenable to surgery.

[0087] Aspects of the present invention, as described herein, provide methods of administering the cells to a subject suffering from and / or in need of treatment for intracerebral hemorrhage, to have one or more (but not necessarily any or all) beneficial effects of preventing, ameliorating, inhibiting, or curing intracerebral hemorrhage. Cells and methods therefor are described below.

[0088] Embodiments of the present invention provide for the administration of MultiStem® cells via an intravenous route, for example, in a subacute time frame (hours) after an ICH event. Administration can be by various routes and times as may be found to be effective.

[0089] Without being limited to any particular mechanism of action, it is noted that MultiStem® cells modulate acute inflammatory responses in other preclinical and clinical injuries. The effect of MultiStem® in treating ICH may be mediated in some aspects by a similar effect of MultiStem® on the acute inflammatory response that occurs after ICH.

[0090] The cells may achieve these effects naturally (i.e., not genetically or pharmaceutically modified), but the cells may also be genetically or pharmaceutically modified to increase efficacy and / or improve their properties.

[0091] In one embodiment, the cells have undergone a desired number of cell doublings in culture. For example, the cells have undergone at least 10-40 cell doublings (e.g., 30-35 cell doublings) in culture, wherein the cells are not transformed and have a normal karyotype. If the cells are transformed or tumorigenic and it is desired to use them for injection, such cells can be rendered incompetent, such as by treatment to prevent cell proliferation from becoming tumorous, so that they cannot form tumors in vivo. Such treatments are well known in the art.

[0092] Oct4, which is otherwise specific to ES, EG, and germ cells, is thought to be a marker of undifferentiated cells with broad differentiation potential. Oct4 is also thought to play a role in maintaining cells in a general undifferentiated state. Oct4 belongs to the POU (Pit Oct Unc) family of transcription factors and is a DNA-binding protein that can activate the transcription of genes containing an octamer sequence, termed an "octamer motif," within their promoter or enhancer regions. Oct4 is expressed during the cleavage stage of fertilized zygotes until the formation of the egg cylinder. Oct4 functions to repress differentiation-inducing genes (i.e., FoxaD3, hCG) and activate pluripotency-promoting genes (FGF4, Utf1, Rex1). Sox2, a member of the high mobility group (HMG) box transcription factors, cooperates with Oct4 to activate the transcription of genes expressed in the inner cell mass. It is essential that Oct4 expression in embryonic stem cells be maintained at a specific level. Overexpression or downregulation of Oct4 expression levels by >50% alters embryonic stem cell fate, with formation of primitive endoderm / mesoderm or trophectoderm, respectively. In vivo, Oct4-deficient embryos develop to the blastocyst stage, but the cells of the inner cell mass are not pluripotent; instead, they differentiate along the extraembryonic trophoblast lineage.

[0093] Sall4 (the mammalian Spalt transcription factor) is an upstream regulator of Oct4 and is therefore important for maintaining appropriate levels of Oct4 during early embryology. When Sall4 levels fall below a certain threshold, trophectoderm cells ectopic expand into the inner cell mass.

[0094] The cells include, but are not limited to, the features in the following numbered embodiments:

[0095] pb1. Isolated and expanded non-embryonic stem, non-germ cells, which have undergone at least 10-40 cell doublings in culture, wherein the cells express Oct4, are not transformed, and have a normal karyotype.

[0096] pb2. The non-embryonic stem, non-germ cell of 1 above, further expressing one or more of telomerase, rex-1, rox-1, or sox-2.

[0097] pb3. A non-embryonic stem, non-germ cell of 1 above, capable of differentiating into at least one cell type of at least two of the endodermal, ectodermal, and mesodermal lineages.

[0098] pb4. The non-embryonic stem, non-germ cells of 3 above, further expressing one or more of telomerase, rex-1, rox-1, or sox-2.

[0099] pb5. A 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 lineages.

[0100] pb6. A non-embryonic stem, non-germ cell of 5 above, further expressing one or more of telomerase, rex-1, rox-1, or sox-2.

[0101] pb7. Isolated and 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 not transformed and have a normal karyotype.

[0102] pb8. A non-embryonic stem, non-germ cell as described in 7 above, which expresses one or more of oct4, telomerase, rex-1, rox-1, or sox-2.

[0103] pb9. A non-embryonic stem, non-germ cell according to item 7 above, which can differentiate into at least one cell type of at least two of the endodermal, ectodermal, and mesodermal lineages.

[0104] pb10. A non-embryonic stem, non-germ cell as described in 9 above, which expresses one or more of oct4, telomerase, rex-1, rox-1, or sox-2.

[0105] pb11. A non-embryonic stem, non-germ cell according to claim 9, which can differentiate into at least one cell type of each of the endodermal, ectodermal, and mesodermal lineages.

[0106] pb12. A non-embryonic stem, non-germ cell as described in claim 11, which expresses one or more of oct4, telomerase, rex-1, rox-1, or sox-2.

[0107] pb13. Isolated and expanded non-embryonic stem, non-germ cells, which have undergone at least 10-40 cell doublings in culture, and which express telomerase, are not transformed, and have a normal karyotype.

[0108] pb14. A non-embryonic stem, non-germ cell as described in claim 13, which further expresses one or more of oct4, rex-1, rox-1, or sox-2.

[0109] pb15. A non-embryonic stem, non-germ cell according to claim 13, which is capable of differentiating into at least one cell type of at least two of the endodermal, ectodermal, and mesodermal lineages.

[0110] pb16. A non-embryonic stem, non-germ cell as described in claim 15, further expressing one or more of oct4, rex-1, rox-1, or sox-2.

[0111] pb17. A non-embryonic stem, non-germ cell according to claim 15, which is capable of differentiating into at least one cell type of each of the endodermal, ectodermal, and mesodermal lineages.

[0112] pb18. A non-embryonic stem, non-germ cell as described in 17 above, which further expresses one or more of oct4, rex-1, rox-1, or sox-2.

[0113] pb19. Isolated and expanded non-embryonic stem, non-germ cells capable of differentiating into at least one cell type of at least two of the endodermal, ectodermal, and mesodermal lineages, said cells having undergone at least 10 to 40 cell doublings in culture.

[0114] pb20. A non-embryonic stem, non-germ cell as described in claim 19, which expresses one or more of oct4, telomerase, rex-1, rox-1, or sox-2.

[0115] pb21. A non-embryonic stem, non-germ cell according to claim 19, which can differentiate into at least one cell type of each of the endodermal, ectodermal, and mesodermal lineages.

[0116] pb22. A non-embryonic stem, non-germ cell as described in claim 21, which expresses one or more of oct4, telomerase, rex-1, rox-1, or sox-2.

[0117] Cell selection MAPCs can be used when isolated and expanded as described herein. MAPCs can also be selected for particular properties prior to use, with or without the use of genetic engineering techniques.

[0118] Selecting cells with a desired level of potency can mean identifying (as by assay), isolating, and expanding the cells. This can create a population with higher potency than the parent cell population from which the cells were isolated. The "parent" cell population refers to the parent cell from which the selected cell divided. "Parent" refers to the actual P1→F1 relationship (i.e., progeny cell). Thus, if cell X is isolated from a mixed population of cells X and Y (where X is an expressor but Y is not), the mere isolate of X would not be classified as having enhanced expression. However, if a progeny cell of X is a higher expressor, the progeny cell would be classified as having enhanced expression.

[0119] Selecting cells that achieve a desired effect includes assaying to determine whether the cells achieve the desired effect, and also includes obtaining the cells. The cells may naturally achieve the desired effect, in that the desired effect is not achieved by an exogenous transgene / DNA. However, effective cells may be improved by incubation with or exposure to an agent that enhances the effect. The cell population from which the effective cells are selected may not be known to have that effect before performing the assay. The cells may not be known to achieve the desired effect before performing the assay. Because the effect may depend on gene expression and / or secretion, selection may also be based on one or more of the genes that cause the effect.

[0120] Selection can be from cells in a tissue, for example, where cells are isolated from the desired tissue, expanded in culture, selected to achieve the desired effect, and the selected cells are further expanded.

[0121] Selection can also be from cells ex vivo (e.g., cells in culture), in which case one or more of the cells in the culture are assayed for achieving the desired effect, and those cells that achieve the desired effect can be further expanded.

[0122] Cells can also be selected for their enhanced ability to achieve a desired effect, in which case the cell population from which the enhanced cells are derived already possesses the desired effect, where enhanced effect means a higher average amount per cell than in the parent population.

[0123] The parent population from which the enhanced cells are selected can be substantially homogeneous (same cell type). One way to obtain such enhanced cells from this population is to create single cells or pools of cells, assay these cells or pools of cells to obtain clones that naturally have the enhanced (greater) effect (as opposed to treating the cells with a modulator that induces or increases the effect), and then expand those naturally enhanced cells.

[0124] However, the cells may be treated with one or more agents that induce or enhance the effect. Thus, the substantially homogenous population may be treated to enhance the effect.

[0125] If the population is not substantially homogeneous, it is preferred that the parent spore population to be treated contain at least 100 of the desired cell type for which an enhanced effect is sought, more preferably at least 1,000 such cells, and even more preferably at least 10,000 such cells. After treatment, this subpopulation can be recovered from the heterogeneous population by known cell selection techniques and further expanded, if desired.

[0126] Thus, the desired level of effect can be higher than that in a given previous population. For example, cells isolated from tissue into primary culture, expanded, and cultured under conditions not specifically designed to produce the effect can provide a parent population. Such parent populations can be treated to enhance the average effect per cell, or can be screened for cells within the population that exhibit a greater degree of effect without deliberate treatment. Such cells can then be expanded to provide a population with higher (desired) expression.

[0127] Use and Administration In some embodiments, the cells are used as the sole active agent of a treatment. In some embodiments of the invention, MAPCs are used as a primary therapeutic modality together with one or more other agents and / or therapeutic modalities. In some embodiments of the invention, the cells are used as an adjunctive therapeutic modality, i.e., as an adjunct to another primary therapeutic modality. In some embodiments, the cells are used as the sole active agent of an adjunctive therapeutic modality. In others, the cells are used as an adjunctive therapeutic modality together with one or more other agents or therapeutic modalities. In some embodiments, the cells are used as both a primary and an adjunctive therapeutic agent and / or modality. In both respects, the cells can be used alone in the primary and / or adjunctive modality. They can also be used together with other therapeutic agents or modalities, either as a primary modality or an adjunctive modality, or both.

[0128] As discussed above, a primary treatment (e.g., a therapeutic agent, treatment, and / or therapeutic modality) targets (i.e., is intended to act against) the primary dysfunction (e.g., disease) to be treated. An adjunctive treatment (e.g., a treatment and / or therapeutic modality) may be administered in combination with a primary treatment (e.g., a therapeutic agent, treatment, and / or therapeutic modality) to act against the primary dysfunction (e.g., disease) and supplement the effects of the primary treatment, thereby increasing the overall effectiveness of the treatment regimen. An adjunctive treatment (e.g., an agent, treatment, and / or therapeutic modality) may also be administered to act against complications and / or side effects of the primary dysfunction (e.g., disease) and / or those caused by the treatment (e.g., a therapeutic agent, treatment, and / or therapeutic modality). For any of these uses, one, two, three, or more primary treatments may be used in conjunction with one, two, three, or more adjunctive treatments.

[0129] In some embodiments, MAPCs are administered to a subject before the onset of ICH. In some embodiments, the cells are administered during the development of ICH and / or resulting dysfunction. In some embodiments, the cells are administered after ICH and / or after resulting dysfunction has been established. MAPCs can be administered at any stage in the development, persistence, and / or propagation of ICH or associated dysfunction, or after it has regressed.

[0130] The cells may be administered at any one or more of 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes or 60 minutes before or after ICH, or 60 minutes, 90 minutes, 120 minutes, 150 minutes or 180 minutes before or after ICH, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours before or after ICH, or 12 hours, 18 hours, 24 hours, 30 hours, 36 hours or 40 hours after ICH, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after ICH, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks after ICH, or any combination of the foregoing and / or any later times.

[0131] The cells can be administered any time after ICH, including immediately after up to 60 minutes after ICH, 30-90 minutes after ICH, 1-6 hours after ICH, 5-15 hours after ICH, 10-20 hours after ICH, 15-25 hours after ICH, 20-40 hours after ICH, 1-5 days after ICH, 1 day to 1 week after ICH, 1-2 weeks after ICH, 1 to several weeks after ICH, several weeks to 1 month after ICH, or 1 or several months after ICH.

[0132] The cells may also be administered before the ICH, at any time preceding the ICH, or at any of the times or intervals before the ICH noted in the previous two paragraphs.

[0133] As discussed above, embodiments of the present invention provide cells and methods for primary or adjunctive therapy. In certain embodiments of the present 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 isogeneic to the subject. In some embodiments, the cells are xenogeneic to the subject. Whether allogeneic, autologous, isogeneic, or xenogeneic, in various embodiments of the present invention, the MAPCs are only weakly immunogenic or non-immunogenic in the subject. In embodiments, the MAPCs are sufficiently low immunogenic or non-immunogenic so that they generally do not induce a harmful immune response when administered to allogeneic and / or xenogeneic subjects and can be used as "universal" donor cells without tissue typing and matching.

[0134] Furthermore, in this regard, MAPCs in various embodiments may be administered without concomitant immunosuppressive treatment. According to various embodiments of the present invention, the MAPCs may also be stored and maintained in cell banks, and thus maintained available for use when needed.

[0135] In these and all other respects, embodiments of the present invention provide MAPCs derived from mammals (including, in one embodiment, humans, and in other embodiments, non-human primates, rats and mice, as well as dogs, pigs, goats, sheep, horses and cows). MAPCs prepared from mammals as described above can be used in all of the above methods and other aspects of the invention described herein.

[0136] MAPCs according to various embodiments of the present invention can be isolated from the various compartments and tissues of such mammals in which they are found, including, but not limited to, bone marrow, peripheral blood, umbilical cord blood, blood, spleen, liver, muscle, brain, adipose tissue, placenta, and others discussed below. MAPCs in some embodiments are cultured prior to use.

[0137] In some embodiments, the MAPCs are isolated from bone marrow. In some specific embodiments, the MAPCs may be isolated from human bone marrow in this regard.

[0138] In many embodiments, the MAPCs are not genetically engineered.

[0139] In some embodiments, MAPCs are genetically engineered. MAPCs can be genetically engineered for a wide variety of purposes, well known in the art. For example, they can be engineered to have improved growth characteristics, to improve their therapeutic efficacy, to express one or more heterologous genes to produce beneficial substances, and to alter their immunological profile.

[0140] In some embodiments, the genetically engineered MAPCs are produced by in vitro culture. In some embodiments, the genetically engineered MAPCs are produced from transgenic organisms.

[0141] formulation MAPCs can be prepared from a variety of tissues (eg, bone marrow cells), as discussed in more detail elsewhere herein.

[0142] In many embodiments, the purity of MAPCs for administration to a subject is about 100%. In other embodiments, it is 95%-100%. In some embodiments, it is 85%-95%. Particularly when mixed with other cells, the percentage of MAPCs can be 2%-5%, 3%-7%, 5%-10%, 7%-15%, 10%-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%.

[0143] In some embodiments, the purity of cells for administration is about 100% (substantially homogeneous). In other embodiments, it is 95%-100%. In some embodiments, it is 85%-95%. Particularly when mixed with other cells, 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%. Alternatively, isolation / purity may be expressed in terms of cell doublings, where the cells have undergone, for example, 10-20, 20-30, 30-40, 40-50, or more cell doublings.

[0144] Treatment with MAPCs of disorders or diseases may involve undifferentiated MAPCs. Treatment may also be performed on MAPCs that have been treated to commit them to a differentiation pathway. 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 best type or mixture of MAPCs will be determined by the specific environment in which they will be used, and determining effective types or combinations of MAPCs in this regard is a routine design matter for those skilled in the art.

[0145] The choice of MAPC administration formulation for a given application depends on various factors.Notably among these are the species of the subject, the nature of intracerebral hemorrhage during treatment, and the state and distribution of intracerebral hemorrhage in the subject, the nature of other treatments and drugs being administered, the optimal route of administration of MAPC, the viability of MAPC via that route, the administration regimen, and other factors that are obvious to those skilled in the art.In particular, for example, the selection of suitable carriers and other additives depends on the exact route of administration and the nature of the specific administration form.

[0146] Cell survival can be an important determinant of the effectiveness of treatments using MAPCs. This applies to both primary and secondary treatments. Another concern arises when the target site is inadequate for cell seeding and cell proliferation. This can hinder access to the site and / or engraftment of therapeutic MAPCs there. In embodiments, the present invention involves the use of means to increase cell survival and / or overcome the problems posed by barriers to seeding and / or proliferation.

[0147] Various additives are often included to enhance the stability, sterility, and isotonicity of the composition (eg, antimicrobial preservatives, antioxidants, chelating agents, and buffers, among others).

[0148] Prevention of microbial activity can be ensured, for example, by various antibacterial and antifungal agents.

[0149] Pharmaceutically acceptable preservatives or cell stabilizers may be used to increase the shelf life of the MAPC compositions.

[0150] If such additives are included, it is well within the skill of the art to select compositions that do not affect the viability or effectiveness of the MAPCs.

[0151] Standard texts (eg, "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th ed., 1985, incorporated herein by reference) can be consulted to prepare suitable preparations without undue experimentation.

[0152] Among the preferred embodiments are injectable solutions, including those for topical, IV infusion and stereotactic injection.

[0153] In some embodiments, the MAPC is formulated in a unit dosage injectable form.

[0154] One of ordinary skill in the art can readily determine the amount of cells and optional additives, vehicles, and / or carriers in the composition to be administered in the methods of the present invention.

[0155] Further active agents MAPCs may be administered with other pharmaceutically active agents. In some embodiments, one or more of such agents may be formulated with the MAPC for administration. In some embodiments, the MAPC and the one or more agents are in separate formulations. In some embodiments, compositions comprising the MAPC and / or the one or more agents are formulated for adjunctive use with each other.

[0156] MAPCs may be administered in a formulation containing immunosuppressants (e.g., any combination of an appropriate number of corticosteroids, cyclosporine A, cyclosporine-like immunosuppressants, cyclophosphamide, antithymocyte globulin, azathioprine, FK-506, and macrolide-like immunosuppressants).

[0157] Such agents also include antibiotics, antifungals, and antivirals, to name just a few other pharmacologically active substances and compositions that may be used in accordance with embodiments of the present invention.

[0158] Representative antibiotic and antifungal compounds include, but are not limited to, penicillin, streptomycin, amphotericin, ampicillin, gentamicin, kanamycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, zeocin, and cephalosporins, aminoglycosides, and echinocandins.

[0159] MAPCs can also be administered in combination with agents that enhance their homing to the site of injury (i.e., the site of injury resulting from ICH). For example, MAPCs can be administered with growth factors and trophic signaling factors (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 cytokines that stimulate the expression of endothelial adhesion molecules).

[0160] They can be administered to a subject as a pretreatment, together with MAPC, or after MAPC has been administered, to promote homing to the desired site and achieve improved therapeutic effects, either through improved homing or other mechanisms. Such factors can be combined with MAPC in a formulation suitable for their co-administration. Alternatively, such factors can be formulated and administered separately.

[0161] The order, formulation, dose, frequency, and route of administration of other active agents and MAPC generally vary depending on, among other factors, the ICH being treated, its severity, the subject, other treatments being administered, the stage of the disorder or disease, and prognostic factors. Comprehensive regimens established for other treatments provide a framework for determining appropriate dosing in MAPC-mediated direct or concomitant treatments. These, together with the additional information provided herein, will enable one of ordinary skill in the art to determine appropriate dosing procedures in accordance with embodiments of the present invention without undue experimentation.

[0162] In embodiments, the cells are formulated appropriately for treating brain injury (including brain injury and / or dysfunction and / or disorders and / or diseases as set forth herein). In embodiments, the formulation is effective for parenteral administration. In embodiments, the formulation is effective for IV infusion. In embodiments, the formulation is effective for stereotactic injection.

[0163] Route of administration MAPCs can be administered to a subject by any of a variety of administration routes known to those of skill in the art that can be used to administer cells to a subject.

[0164] In various embodiments, the MAPCs are administered to a subject by any route for effective delivery of 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 site of ICH where they are intended to be treated. In some embodiments, the cells are administered by injection at a location that is not near the site of dysfunction. In some embodiments, the cells are administered by systemic injection (e.g., intravenous injection).

[0165] Among the methods used in this regard in embodiments of the present invention are methods for administering MAPC by systemic injection. Systemic injection (e.g., intravenous injection) provides one of the simplest and least invasive routes of MAPC administration. In some cases, these routes may require high MAPC doses for optimal efficacy and / or homing by MAPC to the target site. In various embodiments, MAPC may be administered by targeted and / or local injection to ensure optimal effect at the target site.

[0166] In some embodiments of the invention, MAPC may be administered to the subject via a syringe through a hypodermic needle. In various embodiments, MAPC is administered to the subject via a catheter. In various embodiments, MAPC is administered by surgical implantation. Further in this regard, in various embodiments of the invention, MAPC is administered to the subject by implantation using an arthroscopic procedure. In some embodiments, MAPC is administered to the subject by stereotactic injection.

[0167] Administration The compositions can be administered in dosages and by techniques well known to those skilled in the medical and veterinary fields, taking into account factors such as the age, sex, weight, and condition of the particular patient, and the formulation to be administered (e.g., solid vs. liquid). Dosages for humans or other mammals can be determined by those skilled in the art from this disclosure, the documents incorporated herein, and knowledge in the art without undue experimentation.

[0168] The dosage of MAPC suitable for use in accordance with various embodiments of the present invention depends on many factors, which can vary considerably under different circumstances. Parameters determining the optimal dosage of MAPC to be administered for primary and adjunctive therapy generally include some or all of the following: the ICH being treated and its stage; the subject's species, body weight, sex, age, weight, and metabolic rate; the subject's immunocompetence; other treatments being administered; and potential complications predicted by the subject's history or genotype. These parameters may also include whether the MAPC are autologous, autologous, allogeneic, or xenogeneic; their potency (specific activity); the site and / or distribution to which the MAPC must be targeted to be effective; and characteristics of the site, such as accessibility and / or engraftment of the MAPC. Additional parameters include coadministration of MAPC with other factors (e.g., growth factors and cytokines). The optimal dosage for a given situation also takes into account how the cells are formulated, how they are administered, and the extent to which the cells are localized to the target site after administration. Ultimately, optimal dosing determination will necessarily provide an effective dose that is neither below the threshold for maximal beneficial effect nor above the threshold at which adverse effects associated with that dose of MAPC outweigh the benefits of the increased dose.

[0169] The optimal dose of MAPC for some embodiments is in the range of doses used for autologous mononuclear bone marrow transplantation, which can be predicted by extrapolation from animal studies, taking into account differences in size (mass) and metabolic factors, and from established dosing requirements for other cell therapies (e.g., transplantation therapy).

[0170] In an embodiment, the optimal dose is 10 4 ~10 9 MAPC cells / kg recipient mass. In embodiments, the optimal dose per administration ranges from 10 5 ~10 8In an embodiment, the optimal dose per administration is between 5 x 10 MAPC cells / kg. 5 ~5.×10 7 In embodiments, the optimal dose per administration is 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, or 9×10 MAPC cells / kg. 6 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, or 9×10 7 The limit is one of the following.

[0171] For reference, some of the above mid- to high-dose regimens are similar to the doses of nucleated cells used in autologous mononuclear bone marrow transplantation. Some of the above mid- to high-dose regimens are similar to the number of CD3+ cells / kg used in autologous mononuclear bone marrow transplantation.

[0172] It should be appreciated that a single dose can be delivered all at once, in portions, or continuously over a period of time. The total dose can also be delivered to a single location or divided and spread over several locations.

[0173] In various embodiments, MAPC can be administered at an initial dose and then maintained by further administrations of MAPC. MAPC can be initially administered by one method, and then administered by the same method or by one or more different methods. A subject's MAPC levels can be maintained by ongoing administration of the cells. Various embodiments administer the MAPC by intravenous injection either initially, or to maintain those levels in the subject, or both. In various embodiments, other forms of administration are used, depending on the patient's condition and other factors (discussed elsewhere herein).

[0174] It should be noted that human subjects are generally treated longer than experimental animals; however, the treatment duration is generally proportional to the length of disease process and the effectiveness of the treatment.Those skilled in the art will take this into consideration when using the results of other procedures carried out on humans and / or animals (for example, rats, mice, non-human primates, etc.) to determine the dosage that is appropriate for humans.Based on these considerations and taking into account the guidance provided by the present disclosure and the prior art, those skilled in the art can determine this without undue experimentation.

[0175] Suitable regimens for the initial administration and further doses or for sequential administration may all be the same or may vary, and suitable regimens can be ascertained by one skilled in the art from this disclosure, the documents cited herein, and knowledge in the art.

[0176] MAPCs can be administered many times over a wide range of time periods (e.g., until the desired therapeutic effect is achieved). In some embodiments, MAPCs are administered for less than one day. In other embodiments, they are administered for 2, 3, 4, 5, or 6 days. In some embodiments, MAPCs are administered once or more times per week for several weeks. In other embodiments, they are administered for several weeks, ranging from one to several months. In various embodiments, they can be administered for a period of several months. In other embodiments, they can be administered for a period of one year or longer. Generally, the length of treatment will be proportional to the length of the disease process, the effectiveness of the therapy being administered, and the condition and response of the subject being treated.

[0177] In some embodiments, MAPCs are administered once, twice, three times, or more than three times until the desired therapeutic effect is achieved or until administration no longer provides benefit to the subject. In some embodiments, MAPCs are administered continuously (e.g., by intravenous infusion) over a period of time. Administration of MAPCs can be for a short period of time, days, weeks, months, years, or longer.

[0178] In embodiments, a single bolus is administered. In embodiments, two or more doses of a single bolus are administered, separated by a time period of one day or more. In embodiments, each dose is administered by IV infusion over any period ranging from minutes to hours. In embodiments, a single dose of cells is administered by stereotactic injection. In embodiments, two or more doses are administered by stereotactic injection into the same or different regions of the brain. In embodiments involving bolus, IV, and stereotactic injection to treat brain injury in this regard, the dose of cells per administration is 10 or more per administration. 4 ~10 9 MAPC cells / kg recipient mass. In embodiments, the dose is 10 5 ~10 8 In an embodiment, the dose is 5.x10 MAPC cells / kg. 5 ~5.×10 7 In embodiments, the dose is 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, or 9×10 MAPC cells / kg. 6 to 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, or 9x10 7 The limit is one of the following.

[0179] Isolation and expansion of MAPCs Methods for isolating MAPCs are known in the art. See, for example, U.S. Patent No. 7,015,037. These methods, along with the characterization (phenotype) of MAPCs, are incorporated herein by reference. MAPCs can be isolated from a number 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 based on the genes expressed (or not expressed) 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).

[0180] MAPCs were also obtained by modified methods described in Breyer et al., Experimental Hematology, 34:1596-1601 (2006) and Subramanian et al., Cellular Programming and Reprogramming: Methods and Protocols; S. Ding (ed.), Methods in Molecular Biology, 636:55-78 (2010) (incorporated by reference for these methods).

[0181] Isolation and expansion of MAPCs as described in U.S. Patent No. 7,015,037 Methods for isolating MAPCs, e.g., from 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), which methods, together with the characterization of MAPCs disclosed therein, are incorporated herein by reference, by way of illustrative and non-limiting example only.

[0182] MAPCs were first isolated from bone marrow and then 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 obtained from bone marrow aspirates, which can be obtained by standard means available to those skilled in the art (see, e.g., Muschler, GF et al. (1997) J Bone Joint Surg Am.; 79(11): 1699-709 and Batinic, D. et al. (1990): Bone Marrow Transplant 6(2): 103-7).

[0183] Human MAPC phenotype under conditions set forth in U.S. Patent No. 7,015,037 Immunophenotypic analysis by FACS of human MAPCs obtained after 22–25 cell doublings showed that these cells did not express CD31, CD34, CD36, CD38, CD45, CD50, CD62E and -P, HLA-DR, Muc18, STRO-1, cKit, or Tie / Tek; they expressed low levels of CD44, HLA-class I, and β2-microglobulin, but did express CD10, CD13, CD49b, CD49e, CDw90, and Flk1 (N>10).

[0184] Once the cells reach approximately 2 x 10 3 / cm 2 After undergoing >40 doublings in cultures reseeded in PBS, the phenotype became more homogeneous, with no cells expressing HLA class I or CD44 (n=6). When cells were grown to higher confluence, they expressed high levels of Muc18, CD44, HLA class I, and β2-microglobulin (n=8), a phenotype similar to that described for MSCs (Pittenger, 1999).

[0185] By immunohistochemistry, approximately 2 × 10 3 3 / cm 2Human MAPCs grown at seeding density have been shown to express EGF-R, TGF-R1 and -2, BMP-R1A, PDGF-R1a and -B, and a small subpopulation of MAPCs (between 1 and 10%) stains with anti-SSEA4 antibodies (Kannagi, R, 1983).

[0186] Using Clontech cDNA arrays, approximately 2 × 10 for 22 and 26 cell doublings 3 cells / cm 2 The expressed gene profile of human MAPCs cultured at a seeding density of 1000 was determined.

[0187] A. MAPCs did 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 expressed low levels of HLA-class-I, CD44-E, and Muc-18 mRNA.

[0188] B. MAPCs expressed mRNA for the cytokines BMP1, BMP5, VEGF, HGF, KGF, and MCP1; the cytokine receptors Flk1, EGF-R, PDGF-R1α, gp130, LIF-R, activin-R1 and -R2, TGFR-2, and BMP-R1A; the adhesion receptors CD49c, CD49d, and CD29; and CD10.

[0189] C. MAPCs expressed mRNA for hTRT and TRF1; POU domain transcription factors oct-4 and sox-2 (required together with oct-4 to maintain the undifferentiated state of ES / ECs (Uwanogho, D. (1995): Mech Dev 49(1-2): 23-36); sox 11 (neurogenesis), sox 9 (chondrogenesis) (Lefebvre V. et al. (1998): Matrix Biol 16(9): 529-40); homeodomain transcription factors Hox-a4 and -a5 (cervical and thoracic skeleton specification; respiratory organogenesis) (Packer AI (2000): Dev Dyn 217(1): 62-74); Hox-a9 (myelopoiesis) (Lawrence H. (1997): Blood 89(6): 1922-30); Dlx4 (specification of peripheral structures in the forebrain and head) (Akimenko MA (1994): J Neurosci (6): 3475-86), MSX1 (mesoderm, adult heart and muscle, chondrogenesis and osteogenesis) (Foerst-Potts L. (1997) Dev Dyn 209(1): 70-84); PDX1 (pancreas) (Offield MF et al. (1996): Development. 122(3): 983-95).

[0190] D. The presence of oct-4, LIF-R, and hTRT mRNA was confirmed by RT-PCR.

[0191] E. Furthermore, RT-PCR showed that rex-1 mRNA and rox-1 mRNA were expressed in MAPCs.

[0192] Oct-4, rex-1, and rox-1 were expressed in MAPCs derived from human and mouse bone marrow, as well as from mouse liver and brain. Human MAPCs expressed LIF-R and stained positively with SSEA-4. Finally, we found that Oct-4, LIF-R, rex-1, and rox-1 mRNA levels increased in human MAPCs cultured beyond 30 cell doublings, resulting in phenotypically more homogeneous cells. In contrast, MAPCs cultured at high density lost expression of these markers. This is associated with senescence before 40 cell doublings and loss of differentiation into cells other than chondroblasts, osteoblasts, and adipocytes. Thus, the presence of Oct-4, along with rex-1, rox-1, and sox-2, correlates with the presence of the most primitive cells in MAPC cultures.

[0193] Culturing MAPCs Methods for culturing MAPCs are well known in the art (see, e.g., U.S. Pat. No. 7,015,037, which is incorporated herein by reference for methods for culturing MAPCs). The density for culturing MAPCs is approximately 100 cells / cm. 2 or approximately 150 cells / cm 2 ~about 10,000 cells / cm 2 (approximately 200 cells / cm 2 ~about 1500 cells / cm 2 ~about 2000 cells / cm 2 The density may vary from species to species (up to 1000 sq. m). The density may vary between species. Furthermore, the optimal density may vary depending on the culture conditions and the source of the cells. It is within the skill of one of ordinary skill in the art to determine the optimal density for a given set of culture conditions and cells.

[0194] Additionally, effective atmospheric oxygen concentrations of less than about 10% (including about 3-5%) may be used at any time during the isolation, expansion, and differentiation of MAPCs in culture.

[0195] Further culture methods In further experiments, the density at which MAPCs were cultured was approximately 100 cells / cm 2or approximately 150 cells / cm 2 ~about 10,000 cells / cm 2 (approximately 200 cells / cm 2 ~about 1500 cells / cm 2 ~about 2000 cells / cm 2 The density may vary from species to species (up to 1000 sq. m). The density may vary between species. Furthermore, the optimal density may vary depending on the culture conditions and the source of the cells. It is within the skill of one of ordinary skill in the art to determine the optimal density for a given set of culture conditions and cells.

[0196] Additionally, effective atmospheric oxygen concentrations of less than about 10% (including about 1-5% and especially 3-5%) may be used at any time during isolation, expansion, and differentiation of MAPCs in culture.

[0197] Cells can be cultured under various serum concentrations (e.g., about 2-20%). Fetal bovine serum can be used. Higher concentrations of serum can be used in combination with lower oxygen tension (e.g., about 15-20%). Cells do not need to be selected before attachment to the culture dish. For example, after a Ficoll gradient, cells can be plated directly (e.g., 250,000-500,000 / cm). 2 ). Adherent colonies can be picked, possibly pooled, and expanded.

[0198] In one embodiment used in the experimental procedures in the Examples, high serum (approximately 15-20%) and low oxygen (approximately 3-5%) conditions were used for cell culture. Specifically, adherent cells from colonies were plated at approximately 1700-2300 cells / cm in 18% serum and 3% oxygen (with PDGF and EGF). 2 The cells were passaged at a density of . [Example]

[0199] The following examples are offered by way of illustration only and in no way limit, exclude, or exhaust the many aspects and embodiments of the invention disclosed herein.

[0200] Example 1 – Preparation of MAPCs Human MultiStem® MAPCs from Athersys Inc. (Cleveland) were used in the experiments described below. These are human bone marrow-derived MAPCs isolated from bone marrow aspirates from consenting healthy donors and processed according to previously described methods, essentially as described in Penn, MS et al., Circ Res 2012;110(2):304-11; Maziarz, RT et al., Biology of Blood and Marrow Transplantation 2012;18(2 Sup):S264-S265; and clinicaltrials.gov #NCT01436487, #NCT01240915, and #NCT01841632. Briefly, MAPCs were cultured in fibronectin-coated plastic tissue culture flasks in a humidified atmosphere of 5% CO2 under low oxygen tension. Cells were cultured in MAPC culture medium (low-glucose DMEM [Life Technologies Invitrogen] supplemented with 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, CA). The cells were positive for CD49c and CD90 and negative for MHC class II and CD45. The cells were then cultured in 1 ml (PlasmaLyte, 5% HSA and 10% DMSO) medium 1~10×10 6MAPCs were frozen at population doubling 30-35 in cryovials in the vapor phase of liquid nitrogen at a concentration of 0.01%. MAPCs were thawed immediately before their use and then used directly.

[0201] Example 2 – Collagenase ICH induction in rats The murine collagenase model of ICH was used for the study as previously described (Sukumari-Ramesh et al., J Neurotrauma 29(18):2798-804 (2012). Briefly, adult male C57Bl / 6J mice (8-10 weeks old) were placed in a stereotactic frame and a 0.5 mm diameter burr hole was drilled over the parietal cortex, 2.2 mm lateral to bregma. A 26-G Hamilton syringe (0.5 μI) was used to insert the collagenase into the ICH. A syringe (loaded with 0.04 μL of bacterial type IV collagenase in saline) was lowered from the skull surface directly into the left striatum to a depth of 3 mm. The syringe was depressed at a rate of 450 nl / min and allowed to rest for several minutes after the procedure to prevent backflow and excessive diffusion of the solution. After removing the syringe, bone wax was used to close the burr hole, the incision was surgically stapled, and the mouse was kept warm until the righting reflex was restored. For all studies, littermates were used to reduce sources of experimental variability.

[0202] Animals were randomized to receive either intravenous (IV) saline (control; n=10) or MultiStem® cells (n=11) after injury.

[0203] IV administration of cells or saline was given 2 hours after the onset of bleeding (2 hours after collagenase injection). All cell-treated animals received 1 million cells.

[0204] Hematoma volume and cerebral perfusion were assessed by magnetic resonance imaging (MRI) for 3 weeks after injury, as described below.

[0205] Neurobehavioral outcomes (including grip strength testing, beam testing, and elevated plus body sway task) were assessed 7 days after injury as described below.

[0206] Example 3 - MAPC reduces hematoma volume after ICH IV administration of MultiStem® cells significantly reduced hematoma volume as early as 1 day after injury. This effect persisted throughout the first week after ICH, consistent with accelerated hematoma dissolution. Mice were anesthetized with isoflurane (3% for induction and 1.5% in a 2:1 N2 / O2 mixture for maintenance) and imaged using a horizontal 7 Tesla BioSpec MRI spectrometer (Bruker Instruments) equipped with a 12 cm self-shielded gradient set (45 Gauss / cm maximum). Radiofrequency pulses were applied using a standard transmit / receive volume coil (72 mm inner diameter) actively separated from a two-channel Bruker quadrature receive coil placed on the midline of the animal's skull. Stereotaxic ear bars were used to minimize movement during the imaging procedure. Mouse body temperature was maintained at 37 ± 0.5°C using a pad heated by a recirculating water bath. After positioning using a triplanar fast low angle shot sequence, MR examination was performed using a T2'-weighted MRI scan. MRIs were acquired using the following parameters: T2* mapping sequence (a multi-echo 2D gradient-echo sequence; TE 5, 10, 15, 20, 25, and 30 ms; TR 3,000 ms; FOV = 32 mm; 1-mm slice thickness (IS slices); 256 × 256 matrix; NEX = 2). Acquired images were volumetrically segmented using ImageJ software, and hematoma volumes were calculated. T2*W images were further processed using Bruker software to obtain susceptibility-weighted images (Sehgal et al., 2006), providing an alternative segmentation method and a quality control reference for clot volume. Both hematoma and ventricular volumes were determined by drawing regions of interest (ROIs) on all MRI sections containing the lesion / ventricle, and the sum (area) was multiplied by the slice thickness to calculate volume. Analysis was performed using ImageJ software.

[0207] Cell-treated animals showed a statistically significant reduction in hematoma volume (approximately a four-fold reduction) within one day of treatment, and the reduction was statistically significant in cell-treated animals versus saline-treated animals over at least the first seven days.

[0208] The results are shown in Figure 1. Placebo (PBS, n=10) or MultiStem® (n=11) was administered intravenously to mice 2 hours after collagenase-induced ICH. Hematoma volume was assessed by MRI (T2W) using a 7T small animal MRI. Representative coronal brain images on days 3 and 7 are provided in Panel A, demonstrating the dramatic benefit of MultiStem® on hematoma volume. Panel B shows data from all mice over the 21-day evaluation period. Data are presented as mean ± SEM and were analyzed by Student's t-test within each time point. **p<0.01 vs. placebo-treated ICH mice.

[0209] Example 4 - MAPC improves cerebral perfusion after ICH. MultiStem® improved cerebral perfusion in and around the injured striatum for 1 week after ICH. Mice were anesthetized with isoflurane (3% for induction and 1.5% in a 2:1 N2 / O2 mixture for maintenance) and imaged using a horizontal 7 Tesla BioSpec MRI spectrometer (Bruker Instruments) equipped with a 12 cm self-shielded gradient set (45 Gauss / cm maximum). Radiofrequency pulses were applied using a standard transmit / receive volume coil (72 mm inner diameter) actively separated from a two-channel Bruker quadrature receive coil placed on the midline of the animal's skull. Stereotaxic ear bars were used to minimize movement during the imaging procedure. Mouse body temperature was maintained at 37 ± 0.5°C using a pad heated by a recirculating water bath. After positioning using a three-plane fast-low-angle shot sequence, MR examinations were performed using T2'-weighted MRI scans. MRI was acquired using the following parameters: T2-fluid-attenuated inversion recovery sequence (RARE-IR, Tl 2,000; TR 10,000 ms; TE 36 ms; RARE factor = 8; FOV = 32 mm; 256 x 256 matrix; 1 mm slice thickness; JS slices). Analysis was performed using ImageJ software.

[0210] Blood flow in cell-treated animals was statistically significantly improved over the first 7 days after treatment when compared to saline-treated injured animals, indicating that IV infusion of the MultiStem® cell product resulted in a rapid restoration of cerebral blood flow after the occurrence of hemorrhagic stroke, likely resulting in less edema, tissue damage, and disruption of neural circuits.

[0211] The results are shown in Figure 2. Placebo (PBS, n=10) or MultiStem® (n=11) was administered intravenously to mice 2 hours after collagenase-induced ICH. Cerebral perfusion was assessed by MRI (ASL; FAIR-RARE) using a 7T small animal MRI. Representative coronal brain images are provided in Panel A, and quantified data are shown in Panel B. The data demonstrate that MultiStem® improves cerebral perfusion over the first week after ICH. Data are presented as mean ± SEM and analyzed by Student's t-test within each time point. *p<0.05, **p<0.01 vs. placebo-treated ICH mice.

[0212] Example 5 - MAPC reduces functional deficits and improves outcome after ICH The observed changes in infarct volume reduction and improved cerebral perfusion were reflected by functional improvements in motor performance in the grip strength test, reduced latency to traverse the trabecula, and normalized left / right sway ratio.

[0213] Behavioral testing Hanging Wire Test Grip strength was assessed by placing mice on an apparatus consisting of a 50 cm string stretched between two vertical posts. Mice were scored as follows: 0: fall; 1: hanging from the string by two front legs; 2: same as 1, but attempting to climb; 3: hanging from the string by two front legs and one or two hind legs; 4: hanging from the string by front legs and wrapping the tail around the string; and 5: escape. For each animal at each time point, the best performance of three consecutive trials was recorded.

[0214] Beam walking Motor coordination was assessed on a stationary thin beam (6 mm wide, 1 m long) for three consecutive days. The first two days consisted of training, and performance on the beam was quantified on the third day by measuring the time required to traverse the beam. Each mouse was tested three times by a blinded investigator, and the average was recorded.

[0215] Elevated body sway test Animals were suspended 1 cm from the base of the tail, 1–5 cm above a flat surface. One flinch was recorded per suspension. Flips were defined as a deviation of >10° from the body midline or a rotation around the vertical axis. During suspension, the mouse was placed on a surface and visually adjusted to avoid any observed lateral deviations, after which it was resuspended. The assessor changed hand and standing positions, and the testing area was clear of landmarks to avoid bias in the direction of flinches. Twenty flinches were recorded per trial, and lateral deviations were calculated as flinches to one side / total flinches.

[0216] These data suggest that acute treatment with MultiStem® following the occurrence of hemorrhagic stroke results in a markedly significant improvement in locomotion and neurological benefit as evidenced across the three studies in which animals were administered MultiStem® compared to animals treated with saline alone.

[0217] The results are shown in Figure 3. Placebo (PBS, n=10) or MultiStem® (n=11) was administered intravenously to mice 2 hours after collagenase-induced ICH. Neurological assessment of motor function was assessed 7 days after injury (or in sham-operated mice; n=8). (A) Grip strength test. (B) Beam task. (C) Elevated plus sway task. Data are mean ± SEM and were compared using one-way ANOVA followed by Tukey's post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ns=not significant.

[0218] The outcomes described above are surprisingly good in light of the fact that there are currently no approved treatments for patients who suffer hemorrhagic stroke other than surgical evacuation, particularly in patients whose clot location and size are amenable to surgery.

[0219] The most recent preclinical paper evaluating an experimental treatment was published in September 2018 by Dhandapani's group and focused on reducing hematoma volume via inhibition of adenosine monophosphate kinase alpha-1 (AMPKa1) ( Vaibhav, 2018 ), in which the same types of MRI outcomes and gait and neurological endpoints were evaluated as in the present application.

[0220] Administration of MultiStem® cells consistently resulted in better hematoma reduction and perfusion outcomes, and similar, if not better, locomotor outcomes when compared to the results presented in this application, which are the best results seen in studies of the treatment of ICH.

[0221] The foregoing description and examples are illustrative, but not exhaustive, of the many aspects and embodiments encompassed by the invention disclosed herein, as will be recognized by those skilled in the art to which they pertain.

[0222] All publications mentioned in the foregoing disclosure are incorporated by reference into the present disclosure in their entirety, particularly in the portions most relevant to the subject matter for which they are specifically cited. The present invention provides, for example, the following items. (Item 1) 1. A method of treating intracerebral hemorrhage in a subject, the method comprising administering to a subject in need thereof a multipotent adult progenitor cell that is not an embryonic stem cell, an embryonic germ cell, or a germ cell, and that is capable of differentiating into at least one cell type of each of at least two of the endodermal, ectodermal, and mesodermal lineages, and that is allogeneic or xenogeneic to the subject. (Item 2) 1. A method of treating intracerebral hemorrhage in a subject, the method comprising administering to a subject in need thereof multipotent adult progenitor cells which are not embryonic stem cells, embryonic germ cells or germ cells, which express telomerase, and which are allogeneic or xenogeneic to the subject. (Item 3) A method for treating intracerebral hemorrhage in a subject, said method comprising administering to a subject in need thereof multipotent adult progenitor cells which are not embryonic stem cells, embryonic germ cells or germ cells, which are positive for oct3 / 4, and which are allogeneic or xenogeneic to said subject. (Item 4) A method of treating intracerebral hemorrhage in a subject, said method comprising administering to a subject in need thereof multipotent adult progenitor cells that are not embryonic stem cells, embryonic germ cells or germ cells, that have undergone at least 40 cell doublings in culture prior to their use, and that are allogeneic or xenogeneic to said subject. (Item 5) 5. The method of any one of items 2 to 4, wherein the cells are capable of differentiating into at least one cell type of each of at least two of the endodermal lineage, the ectodermal lineage, and the mesodermal lineage. (Item 6) 5. The method according to any one of items 3 to 4, wherein the cells express telomerase. (Item 7) 4. The method of claim 3, wherein the cells have undergone at least 40 cell doublings in culture prior to their use. (Item 8) 2. The method of claim 1, wherein the cells express telomerase and are positive for oct3 / 4. (Item 9) 2. The method of claim 1, wherein the cells express telomerase and have undergone at least 40 cell doublings in culture prior to their use. (Item 10) 2. The method of item 1, wherein the cells are positive for oct3 / 4 and have undergone at least 40 cell doublings in culture prior to their use. (Item 11) 2. The method of claim 1, wherein the cells express telomerase, are positive for oct3 / 4, and have undergone at least 40 cell doublings in culture prior to their use. (Item 12) 12. The method according to any one of items 1 to 11, wherein the cells have a normal karyotype. (Item 13) 13. The method of any one of items 1 to 12, wherein the cells are not tumorigenic. (Item 14) 14. The method of any one of items 1 to 13, wherein the cells are not immunogenic in the subject. (Item 15) 15. The method of any one of items 1 to 14, wherein the cells are capable of differentiating into at least one cell type of each of the endodermal, ectodermal, and mesodermal lineages. (Item 16) 16. The method according to any one of items 1 to 15, wherein the cells are mammalian cells. (Item 17) 17. The method according to any one of items 1 to 16, wherein the cells are human cells. (Item 18) 18. The method of any one of items 1 to 17, wherein the cells are derived from cells isolated from any one of placental tissue, umbilical cord tissue, umbilical cord blood, bone marrow, blood, spleen tissue, thymus tissue, spinal cord tissue, adipose tissue, and liver tissue. (Item 19) 19. The method according to any one of items 1 to 18, wherein the cells are derived from bone marrow. (Item 20) 20. The method according to any one of items 1 to 19, wherein the subject is a human. (Item 21) 10 per kg of mass of the subject 4 ~10 8 21. The method of any one of items 1 to 20, wherein one or more doses of said cells are used. (Item 22) 10 per kg of mass of the subject 6 ~5×10 7 22. The method of any one of items 1 to 21, wherein one or more doses of said cells are used. (Item 23) 23. The method according to any one of items 1 to 22, wherein an antimicrobial agent, an antifungal agent, an antiviral agent or a combination thereof is used simultaneously. (Item 24) 24. The method according to any one of items 1 to 23, wherein the cells are in a formulation comprising one or more other pharmaceutically active agents. (Item 25) 25. The method of any one of items 1 to 24, wherein the cells are administered parenterally, intravenously or stereotaxically. (Item 26) 26. The method of any one of items 1 to 25, wherein the cells are administered by intravenous means. 27. 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, and that are capable of differentiating into at least one cell type of each of at least two of the endodermal, ectodermal, and mesodermal lineages, and that are allogeneic or xenogeneic to the subject. (Item 27) 27. The method of any one of items 1 to 26, wherein an immunosuppressive treatment is not administered concomitantly with the administration of said cells. (Item 28) 1. Use of a cell in the manufacture of a medicament for the treatment of intracerebral hemorrhage in a subject, wherein the cell is not an embryonic stem cell, an embryonic germ cell, or a germ cell, but is a multipotent adult progenitor cell that is capable of differentiating into at least one cell type of each of at least two of the endodermal, ectodermal, and mesodermal lineages, and that is allogeneic or xenogeneic to the subject. (Item 29) 1. Use of a cell in the manufacture of a medicament for the treatment of intracerebral hemorrhage in a subject, wherein the cell is not an embryonic stem cell, an embryonic germ cell, or a germ cell, but is a multipotent adult progenitor cell that expresses telomerase and is allogeneic or xenogeneic to the subject. use. (Item 30) Use of cells in the manufacture of a medicament for the treatment of 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, that are positive for oct3 / 4, and that are allogeneic or xenogeneic to the subject. (Item 31) 1. Use of cells in the manufacture of a medicament for the treatment of intracerebral hemorrhage in a subject, wherein the cells are not embryonic stem cells, embryonic germ cells or germ cells, but are multipotent adult progenitor cells that have undergone at least 40 cell doublings in culture prior to their use, and that are allogeneic or xenogeneic to the subject. (Item 32) 32. The use of any one of items 29 to 31, wherein the cells are capable of differentiating into at least one cell type of each of at least two of the endodermal, ectodermal, and mesodermal lineages. (Item 33) 32. The use according to any one of items 30 to 31, wherein the cells express telomerase. (Item 34) 31. The use of item 30, wherein the cells have undergone at least 40 cell doublings in culture prior to their use. (Item 35) 29. The use according to item 28, wherein the cells express telomerase and are positive for oct3 / 4. (Item 36) 29. The use of item 28, wherein the cells express telomerase and have undergone at least 40 cell doublings in culture prior to their use. (Item 37) 29. The use of item 28, wherein the cells are positive for oct3 / 4 and have undergone at least 40 cell doublings in culture prior to their use. (Item 38) 29. The use of item 28, wherein the cells express telomerase, are positive for oct3 / 4, and have undergone at least 40 cell doublings in culture prior to their use. (Item 39) 39. The use according to any one of items 28 to 38, wherein the cells have a normal karyotype. (Item 40) 40. The use of any one of items 28 to 39, wherein the cells are not tumorigenic. (Item 41) 41. The use of any one of items 28 to 40, wherein the cells are not immunogenic in the subject. (Item 42) 42. The use of any one of items 28 to 41, wherein the cells are capable of differentiating into at least one cell type of each of the endodermal, ectodermal, and mesodermal lineages. (Item 43) 43. The use according to any one of items 28 to 42, wherein the cells are mammalian cells. (Item 44) 44. The use according to any one of items 28 to 43, wherein the cells are human cells. (Item 45) 45. The use of any of items 28 to 44, wherein the cells are derived from cells isolated from any one of placental tissue, umbilical cord tissue, umbilical cord blood, bone marrow, blood, spleen tissue, thymus tissue, spinal cord tissue, adipose tissue, and liver tissue. (Item 46) 46. ​​The use according to any of items 28 to 45, wherein the cells are derived from bone marrow. (Item 47) 47. The use according to any one of items 28 to 46, wherein the subject is a human. (Item 48) 10 per kg of mass of the subject 4 ~10 8 48. The use according to any one of items 28 to 47, wherein one or more doses of said cells are used. (Item 49) 10 per kg of mass of the subject 6 ~5×10 7 49. The use according to any one of items 28 to 48, wherein one or more doses of said cells are used. (Item 50) 50. The use according to any one of items 28 to 49, wherein an antimicrobial agent, an antifungal agent, an antiviral agent or a combination thereof is administered simultaneously. (Item 51) 51. The use according to any one of items 28 to 50, wherein the cells are in a formulation comprising one or more other pharmaceutically active agents. (Item 52) 52. The use according to any one of items 28 to 51, wherein the cells are administered parenterally, intravenously or stereotaxically. (Item 53) 53. The use according to any one of items 28 to 52, wherein the cells are administered by intravenous method. (Item 54) 54. The use according to any one of items 28 to 53, wherein the medicament is for use without the concomitant use of an immunosuppressant.

Claims

1. A composition for treating intracerebral hemorrhage and reducing intracerebral hematoma volume in a subject, the composition comprising multipotent adult progenitor cells that express oct-4, rex-1, and rox-1, the multipotent adult progenitor cells being neither embryonic stem cells, nor embryonic germ cells, and capable of differentiating into at least one cell type of each of at least two of the endodermal, ectodermal, and mesodermal lineages, and being allogeneic or xenogeneic to the subject.

2. A composition for treating intracerebral hemorrhage and reducing intracerebral hematoma volume in a subject, the composition comprising multipotent adult progenitor cells that express oct-4, rex-1, and rox-1, the multipotent adult progenitor cells being neither embryonic stem cells, nor embryonic germ cells, nor germ cells, that express telomerase, and that are allogeneic or xenogeneic to the subject.

3. A composition for treating intracerebral hemorrhage and reducing intracerebral hematoma volume in a subject, the composition comprising multipotent adult progenitor cells that express oct-4, rex-1, and rox-1, the multipotent adult progenitor cells being neither embryonic stem cells, nor embryonic germ cells, nor germ cells, and being allogeneic or xenogeneic to the subject.

4. A composition for treating intracerebral hemorrhage and reducing intracerebral hematoma volume in a subject, said composition comprising multipotent adult progenitor cells that express oct-4, rex-1 and rox-1, which are not embryonic stem cells, embryonic germ cells or germ cells, which have undergone at least 40 cell doublings in culture prior to their use, and which are allogeneic or xenogeneic to the subject.

5. 5. The composition of any one of claims 2 to 4, wherein the cells are capable of differentiating into at least one cell type of each of at least two of the endodermal, ectodermal, and mesodermal lineages.

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

7. 4. The composition of claim 3, wherein the cells have undergone at least 40 cell doublings in culture prior to their use.

8. The composition of claim 1 , wherein the cells express telomerase.

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

10. 10. The composition of claim 1, wherein the cells have undergone at least 40 cell doublings in culture prior to their use.

11. The composition of any one of claims 1 to 10, wherein the cells have a normal karyotype.

12. The composition of any one of claims 1 to 11, wherein the cells are not tumorigenic.

13. The composition of any one of claims 1 to 12, wherein the cells are not immunogenic in the subject.

14. The composition of any one of claims 1 to 13, wherein the cells are capable of differentiating into at least one cell type of each of the endodermal, ectodermal, and mesodermal lineages.

15. The composition of any one of claims 1 to 14, wherein the cells are mammalian cells.

16. The composition of any one of claims 1 to 15, wherein the cells are human cells.

17. 17. The composition of any one of claims 1 to 16, wherein the cells are derived from cells isolated from any one of placental tissue, umbilical cord tissue, umbilical cord blood, bone marrow, blood, spleen tissue, thymus tissue, spinal cord tissue, adipose tissue, and liver tissue.

18. The composition of any one of claims 1 to 17, wherein the cells are derived from bone marrow.

19. The composition of any one of claims 1 to 18, wherein the subject is a human.

20. 10 per kg of the subject's mass ４ ~10 ８ The composition of any one of claims 1 to 19, wherein one or more doses of said cells are used.

21. 10 per kg of the subject's mass ６ ~5 x 10 ７ The composition of any one of claims 1 to 20, wherein one or more doses of said cells are used.

22. The composition of any one of claims 1 to 21, wherein an antimicrobial agent, an antifungal agent, an antiviral agent or a combination thereof is used simultaneously.

23. The composition of any one of claims 1 to 22, wherein the cells are in a formulation that also includes one or more other pharmaceutically active agents.

24. The composition according to any one of claims 1 to 23, characterized in that the composition is administered parenterally, intravenously or stereotaxically.

25. The composition according to any one of claims 1 to 24, characterized in that the composition is administered by intravenous means.

26. The composition of any one of claims 1 to 25, wherein an immunosuppressive treatment is not administered concomitantly with administration of the cells.

27. Use of cells in the manufacture of a medicament for treating intracerebral hemorrhage and reducing intracerebral hematoma volume in a subject, wherein the cells are multipotent adult progenitor cells that express oct-4, rex-1 and rox-1, that are not embryonic stem cells, embryonic germ cells or germ cells, that are capable of differentiating into at least one cell type of each of at least two of the endodermal, ectodermal and mesodermal lineages, and that are allogeneic or xenogeneic to the subject.

28. Use of cells in the manufacture of a medicament for treating intracerebral hemorrhage and reducing intracerebral hematoma volume in a subject, wherein the cells are multipotent adult progenitor cells that express oct-4, rex-1 and rox-1, and are not embryonic stem cells, embryonic germ cells or germ cells, that express telomerase, and are allogeneic or xenogeneic to the subject.

29. Use of cells in the manufacture of a medicament for treating intracerebral hemorrhage and reducing intracerebral hematoma volume in a subject, wherein the cells are multipotent adult progenitor cells that express oct-4, rex-1 and rox-1, and which are not embryonic stem cells, embryonic germ cells or germ cells, and which are allogeneic or xenogeneic to the subject.

30. 1. Use of cells in the manufacture of a medicament for treating intracerebral hemorrhage and reducing intracerebral hematoma volume in a subject, wherein the cells are multipotent adult progenitor cells that express oct-4, rex-1 and rox-1, which are not embryonic stem cells, embryonic germ cells or germ cells, which have undergone at least 40 cell doublings in culture prior to their use, and which are allogeneic or xenogeneic to the subject.

31. The use according to any one of claims 28 to 30, wherein the cells are capable of differentiating into at least one cell type of each of at least two of the endodermal, ectodermal and mesodermal lineages.

32. The use according to any one of claims 29 to 30, wherein the cells express telomerase.

33. 30. The use of claim 29, wherein the cells have undergone at least 40 cell doublings in culture prior to their use.

34. 28. The use of claim 27, wherein the cells express telomerase.

35. 28. The use of claim 27, wherein the cells express telomerase and have undergone at least 40 cell doublings in culture prior to their use.

36. 28. The use of claim 27, wherein the cells have undergone at least 40 cell doublings in culture prior to their use.

37. The use according to any one of claims 27 to 36, wherein the cells have a normal karyotype.

38. The use according to any one of claims 27 to 37, wherein the cells are not tumorigenic.

39. The use of any one of claims 27 to 38, wherein the cells are not immunogenic in the subject.

40. 40. The use according to any one of claims 27 to 39, wherein the cells are capable of differentiating into at least one cell type of each of the endodermal, ectodermal and mesodermal lineages.

41. The use according to any one of claims 27 to 40, wherein the cells are mammalian cells.

42. The use according to any one of claims 27 to 41, wherein the cells are human cells.

43. 43. The use of any of claims 27 to 42, wherein the cells are derived from cells isolated from any one of placental tissue, umbilical cord tissue, umbilical cord blood, bone marrow, blood, spleen tissue, thymus tissue, spinal cord tissue, adipose tissue, and liver tissue.

44. The use according to any one of claims 27 to 43, wherein the cells are derived from bone marrow.

45. The use according to any one of claims 27 to 44, wherein the subject is a human.

46. 10 per kg of the subject's mass ４ ~10 ８ 46. ​​The use according to any one of claims 27 to 45, wherein one or more doses of said cells are used.

47. 10 per kg of the subject's mass ６ ~5 x 10 ７ 47. The use according to any one of claims 27 to 46, wherein one or more doses of said cells are used.

48. 48. The use according to any one of claims 27 to 47, wherein an antimicrobial agent, an antifungal agent, an antiviral agent or a combination thereof is administered simultaneously.

49. The use according to any one of claims 27 to 48, wherein the cells are in a formulation comprising one or more other pharmaceutically active agents.

50. The use according to any one of claims 27 to 49, wherein the cells are administered parenterally, intravenously or stereotaxically.

51. The use according to any one of claims 27 to 50, wherein the cells are administered by intravenous means.

52. 52. The use according to any one of claims 27 to 51, wherein the medicament is for use without the concomitant use of immunosuppressants.

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