Functional recovery from cerebral infarction

Abstract

To solve the following problem that: there are no approved therapies for cerebral infarction except tissue plasminogen activator (TPA) if administered within three hours of presentation of symptom onset; given the lack of therapeutic options for the treatment of cerebral infarction, there is a strong need for additional therapies that promote reperfusion, or are neuroprotective.SOLUTION: The present disclosure provides methods of treating a subject who has suffered a cerebral infarction, the method comprising administering systemically to the subject a population of cells enriched for mesenchymal lineage precursor or stem cells (MLPSCs) such as STRO-1+ cells or progeny thereof to increase stimulus-induced cortical activation or reduce infarct volume.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to methods of treating cerebral infarction in a human subject. [Background technology]

[0002] Cerebral infarction remains a leading cause of morbidity and mortality in industrialized countries. It is the third leading cause of death. A stroke is a rapid loss of brain function caused by an obstruction of the brain's blood supply. There are two general types of stroke: (i) ischemic stroke, which is caused by a temporary or permanent blockage of blood flow to the brain and accounts for 85% of stroke cases, and (ii) hemorrhagic stroke, which is caused by a ruptured blood vessel and accounts for the majority of the remaining cases. Cerebral infarction often causes neuronal cell death and can lead to death. The most common cause of ischemic stroke is occlusion of the middle cerebral artery (an intracranial artery downstream from the internal carotid artery), which damages the cerebrum (e.g., the cerebral cortex), such as the motor cortex or sensory cortex of the brain. Such damage results in hemiplegia, hemisensory loss, and either language or visuospatial impairment, depending on the cerebral hemisphere damaged. The volume of the affected brain and its impaired function can be visualized by functional imaging techniques such as blood oxygenation level-dependent (BOLD) magnetic resonance imaging (MRI), which images the concomitant reduction in blood flow in affected brain regions.

[0003] A stroke can affect a subject physically, mentally, emotionally, or a combination of all three.

[0004] Some of the physical impairments that can result from a stroke include muscle weakness, numbness, pressure sores, pneumonia, incontinence, apraxia (the inability to perform familiar movements), difficulty performing daily activities, loss of appetite, loss of speech, loss of vision, and pain. If the stroke is severe enough or in certain locations, such as parts of the brain stem, it can result in coma or death.

[0005] Emotional problems resulting from a stroke may result directly from damage to the emotional centers of the brain or from frustration and difficulty adapting to new constraints. Emotional difficulties after a stroke include depression, anxiety, panic attacks, flat affect (inability to express emotions), mania, apathy, and psychosis.

[0006] Cognitive impairments resulting from stroke include perception disorders, speech disorders, dementia, and problems with attention and memory. Stroke patients may be unaware of their impairments, a condition called anosognosia. In a condition called hemispatial neglect, patients are unable to attend to anything in space opposite the damaged hemisphere. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2004 / 85630 Brochure [Patent Document 2] International Publication No. 01 / 04268 Brochure [Patent Document 3] International Publication No. 2004 / 085630 Brochure [Patent Document 4] International Publication No. 01 / 14268 Brochure [Patent Document 5] U.S. Patent No. 5,486,359 [Patent Document 6] International application PCT / US94 / 05700 [Patent Document 7] U.S. Patent No. 5,173,414 [Patent Document 8] U.S. Patent No. 5,139,941 [Patent Document 9] International Publication No. 92 / 01070 Brochure [Patent Document 10] International Publication No. 93 / 03769 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 2nd ed. (1989), Volumes I, II, and III, all volumes. [Non-patent document 2] DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover, ed., 1985), IRL Press, Oxford, full text [Non-patent document 3] Oligonucleotide Synthesis: A Practical Approach (ed. MJ Gait, 1984) IRL Press, Oxford, full text, especially Gait, pp. l~22; Atkinson et al., pp. 35~81; Sproat et al., pp. 83~115; and papers by Wu et al., pp. 135~151. [Non-patent document 4] 4. Nucleic Acid Hybridization: A Practical Approach (eds. B.D. Hames & S.J. Higgins, 1985) IRL Press, Oxford, full text [Non-Patent Document 5] Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, full text [Non-patent document 6] Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), all volumes [Non-Patent Document 7] JF Ramalho Ortigao, "The Chemistry of Peptide Synthesis" in "Knowledge database of Access to Virtual Laboratory website" (Interactiva, Germany) [Non-patent document 8] Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, RL (1976). Biochem. Biophys. Res. Commun. 73 336~342 [Non-Patent Document 9] Merrifield, R.B. (1963). J. Am. Chem. Soc. 85, 2149-2154 [Non-Patent Document 10] Barany, G. and Merrifield, R.B. (1979) In The Peptides (Gross, E. and Meienhofer, J., eds.), Vol. 2, pp. 1-284, Academic Press, New York. 12. [Non-Patent Document 11] Wunsch, E. (ed.) (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Muler, E. ed.), Vol. 15, 4th edition, Parts 1 and 2, Thieme, Stuttgart. [Non-Patent Document 12] Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg [Non-Patent Document 13] Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449~474

Non-patent document 14

Non-patent document 15

Non-patent document 16

Non-patent document 17

Non-patent document 18

Non-patent document 19

Non-patent document 20

Non-patent document 21

Non-patent document 22

Non-patent document 23

Non-patent document 24

Non-patented document 25

Non-patent document 26

Non-patent document 27

Non-patent document 28

Non-patent document 29

Non-patent document 30

Non-patent document 31

Non-patent document 32

Non-patent document 33

Non-patent document 34

Non-licensed Document 35

Non-licensed Document 36

Non-licensed Document 37

Non-licensed Document 38

Non-licensed Document 39

Non-licensed Document 40

Non-licensed Document 41

Non-licensed Document 42

Non-licensed Document 43

Non-licensed Document 44

Non-licensed Document 45

[0009] Other than tissue plasminogen activator (TPA) when administered within 3 hours of presenting symptoms, there is no approved treatment for cerebral infarction. Given the lack of therapeutic options for the treatment of cerebral infarction, additional therapies that promote reperfusion or provide neuroprotection are greatly needed. [Means for solving the problem]

[0010] The present disclosure provides human mesenchymal progenitor or stem cells (MLPSCs), e.g., STRO-1 + It is based on the inventor's surprising discovery that systemic administration of human mesenchymal progenitor cells (hMPCs) results in improved functional recovery within the infarcted cortical volume as assessed by functional imaging.

[0011] Accordingly, a first aspect described herein is a method for increasing cortical activation or reducing infarct volume after cerebral infarction, the method comprising systemic administration of a therapeutically effective amount of a human cell population enriched in mesenchymal progenitor or stem cells (MLPSCs) to a human subject in need thereof.

[0012] In some embodiments, cerebral infarction is ischemic cerebral infarction.In some embodiments, when cerebral infarction is ischemic cerebral infarction, the cerebral infarction of the subject to be treated is caused by hypoxic-ischemic encephalopathy (HIE).In other embodiments, cerebral infarction is hemorrhagic cerebral infarction.

[0013] In some embodiments, the cerebral infarction is in the motor cortex. In some embodiments, the affected volume is reduced after administration. In some embodiments, cortical activation is increased. In some embodiments, motor function is improved in a human subject. In some embodiments, the increased cortical activation after treatment is in response to contralateral tactile stimulation. In some embodiments, cortical activation is increased within the infarct volume.

[0014] In some embodiments, the systemic administration of the human cell population occurs within about 24 hours after the stroke, hi other embodiments, the systemic administration occurs within about 12 hours after the stroke.

[0015] In some embodiments, the MLPSCs are STRO-1 + In some embodiments, STRO-1 is an MPC. + MPC is STRO-1 bright In some embodiments, STRO-1 is an MPC. + MPC is a tissue nonspecific alkaline phosphatase (TNAP) + or CD146 + is.

[0016] In other embodiments, the MLPSCs are mesenchymal stem cells.

[0017] In some embodiments, the administered human cell population is an allogeneic human cell population, hi other embodiments, the human cell population is an autologous human cell population.

[0018] In some embodiments, the methods described herein provide a method for detecting approximately 2×10 6 cells / cm 3 Approximately 2 × 10 from the affected cortex 7 cells / cm 3 In other embodiments, the method comprises administering 0.1 x 10 6 5 x 10 cells / kg body weight 6 cells / kg body weight.

[0019] In some embodiments, the administered human cell population is in culture expanded prior to administration.

[0020] In some embodiments, the human cell population was derived from bone marrow, dental pulp, adipose, or pluripotent stem cells. In some embodiments, the human cell population was not derived from dental pulp or adipose. In some embodiments, the human cell population is a genetically modified human cell population.

[0021] In some embodiments, the systemic administration of the cell population is intra-arterial or intravenous administration.

[0022] In some embodiments, the methods described herein include administering a thrombolytic agent. In some embodiments, the methods described herein avoid administration of a thrombolytic agent. In other embodiments, the subject is not administered a thrombolytic agent before or after administration of the human cell population. In other embodiments, the method includes administering mannitol. In some embodiments, the method includes co-administering mannitol and temozolomide, either as a single formulation or separately. In other embodiments, the method includes administering an anti-inflammatory agent.

[0023] In some embodiments, the administered human cell population is administered multiple times, hi some embodiments, the human cell population is administered once every four weeks or more.

[0024] In other embodiments, the population of human cells is administered once.

[0025] In some embodiments, at least a portion of the cells of the human cell population are labeled for in vivo detection, hi some embodiments, when the labeled cells are administered to a subject, the method also includes tracking the location of the labeled cells in the subject after administration.

[0026] In some embodiments of any of the above-described methods, the method further includes determining a change in infarct volume and / or a change in activity within the infarct volume following administration.

[0027] The methods described herein shall apply mutatis mutandis to methods for reducing the risk of further cerebral infarction. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows a line graph depicting forelimb placing motor behavior scores in rat groups at various time points after internal carotid artery occlusion (MCAO), a model of stroke. Various groups were intravenously administered 1 x 106 human MPCs at the indicated time points after MCAO treatment. Note: Lower values indicate improved motor behavior. Administration of MPCs at 6 hours (p<0.01), 12 hours (p<0.01), 24 hours (p<0.001), 48 hours (p<0.01), and 7 days (p<0.01) after MCAO significantly improved forelimb recovery compared to vehicle administration. [Figure 2] Figure 1 shows a line graph depicting hindlimb placing motor behavior scores in rat groups at various time points after MCAO. Various groups were intravenously administered 1x106 human MPCs at the indicated time points after MCAO treatment. Administration of MPCs 6 hours (p<0.001), 12 hours (p<0.01), 24 hours (p<0.001), and 48 hours (p<0.001) after MCAO significantly improved hindlimb recovery compared to vehicle administration. [Figure 3] Figure 1 shows a line graph depicting the body swing motor behavior scores in rat groups at various time points after MCAO. Various groups were intravenously administered 1x10 human MPCs at the indicated time points after MCAO treatment. Administration of huMPCs at 6 hours (p<0.05), 12 hours (p<0.05), 48 hours (p<0.01), and 7 days (p<0.01) after MCAO significantly improved recovery of body swing compared to vehicle administration. [Figure 4] 1 is a line graph showing body weight after MCAO. There was no significant difference in body weight between the MPC-treated group and the vehicle group. [Figure 5] FIG. 1 shows a schematic of an MRI imaging study of cortical responsiveness to tactile stimulation in rats after MCAO. [Figure 6]FIG. 1 shows a schematic of the MRI imaging equipment for the MCAO rat study. [Figure 7] 1 is a bar graph showing measurements (mean ± SEM) of infarct volume (upper panel) and infarct volume as a percentage of the total brain (lower panel) at MRI on day 8. The MPC-treated group had statistically smaller infarct volumes compared to the vehicle-treated group (p<0.05). Furthermore, infarct volume as a percentage of the total brain was significantly smaller in the MPC-treated group (p<0.05). [Figure 8] 1 is a bar graph showing activation of the primary and secondary motor cortex (lower panel) and the primary and secondary somatosensory cortex (lower panel) by left (contralateral) forepaw stimulation in vehicle- and MPC-treated groups. Significantly higher levels of activation in the primary motor cortex were observed in the MPC-treated group than in the vehicle-treated group (p<0.05). [Figure 9] 1 is a bar graph showing the level of cortical activation within the infarct cortical volume in response to contralateral tactile stimulation in vehicle- and MPC-treated groups. The level of cortical activation within the infarct was significantly higher in the MPC-treated group than in the vehicle-treated group (p<0.01). DETAILED DESCRIPTION OF THE INVENTION

[0029] General Techniques and Selected Definitions Throughout this specification, unless otherwise specified or the context requires otherwise, references to a single step, composition, group of steps, or group of compositions shall be interpreted as encompassing one and more (i.e., one or more) of that step, composition, group of steps, or group of compositions.

[0030] Each example of this disclosure shall apply mutatis mutandis to each of all other examples unless otherwise stated.

[0031] Those skilled in the art will understand that the present disclosure is susceptible to variations and modifications other than those specifically described. The present disclosure is to be understood to include all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds referenced or indicated herein, individually or collectively, and any and all combinations of said steps or features, or any two or more thereof.

[0032] The present disclosure is intended to be illustrative only and is not limited in scope by the specific examples set forth herein. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0033] The present disclosure is carried out without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such techniques are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 2nd Edition (1989), Volumes I, II, and III, complete; DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover, ed., 1985), IRL Press, Oxford, complete; Oligonucleotide Synthesis: A Practical Approach (M.J. Gait, ed., 1984), IRL Press, Oxford, complete, in particular Gait, pp. 1-22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and the articles by Wu et al., pp. 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B.D. Hames & S.J. Higgins, eds., 1985), IRL Press, Oxford, complete; Immobilized Cells and Enzymes: A Practical Approach (1986), IRL Press, Oxford, complete. Oxford, full text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), full volume; JF Ramalho Ortigao, "The Chemistry of Peptide Synthesis" in the "knowledge database of Access to Virtual Laboratory website" (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, RL (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, RB (1963). J. Am. Chem. Soc. 85, 2149-2154; Barany, G. and Merrifield, RB (1979) in The Peptides (Gross, E. and Meienhofer, J., eds.), Vol. 2, pp. 1-284, Academic Press, New York. 12. Wunsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Muler, E, ed.), Vol. 15, 4th ed., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, 3rd ed. (John R.W. Masters, ed., 2000), ISBN 0199637970, full text.

[0034] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated step or element or integer or group of steps or elements or integers but not to the exclusion of other steps or elements or integers or groups of elements or integers.

[0035] As used herein, the term "cerebral infarction" shall be interpreted to mean a loss of brain function, usually of rapid onset, due to impaired blood flow to the brain or brainstem. The impairment may be ischemia (lack of blood) caused, for example, by thrombosis or embolism (referred to herein as "ischemic cerebral infarction"), or may be due to hemorrhage (referred to herein as "hemorrhagic cerebral infarction"). In one example, the loss of brain function is accompanied by neuronal cell death. In one example, cerebral infarction is caused by impaired or lost blood from the cerebrum or its regions. In one example, cerebral infarction is a neurological deficit of cerebrovascular origin that lasts for more than 24 hours or is terminated by death within 24 hours (as defined by the World Health Organization). Symptoms lasting for more than 24 hours distinguish cerebral infarction from transient ischemic attack (TIA), in which symptoms last less than 24 hours. Symptoms of a stroke include hemiplegia (paralysis on one side of the body), hemiparesis (weakness on one side of the body); facial weakness; paresthesia; decreased sensation; changes in smell, taste, hearing, or vision; loss of smell, taste, hearing, or vision; drooping eyelids (ptosis); detectable weakness of the eye muscles; decreased gag reflex; decreased ability to swallow; decreased pupillary response to light; decreased sensation in the face; decreased balance; nystagmus; changes in respiratory rate; changes in heart rate; sternocleidomastoid muscle weakness with decreased ability or inability to turn the head to one side; tongue weakness; aphasia (inability to speak or understand language); apraxia (changes in voluntary movement); visual field defects; memory deficits; hemineglect or hemispatial neglect Symptoms include: neglect (lack of attention to space on the side of the visual field opposite the lesion); disorganized thinking; confusion; the onset of hypersexual gestures; anosognosia (persistent denial of the existence of the deficit); difficulty walking; altered coordination of movements; dizziness; imbalance; loss of consciousness; headache; and / or vomiting.

[0036] Those skilled in the art will recognize that the term "cerebrum" includes the cerebral cortex (or cortex of the cerebral hemispheres), the basal ganglia (or basal ganglia), and the limbic system.

[0037] As used herein, the term "infarct" refers to the area or volume of the brain directly affected by the cerebral infarct process.

[0038] The term "brain function" includes · Reasoning, planning, parts of speech, behavior, emotions, and problem-solving (involves the frontal lobe); · Behavior, orientation, awareness, and perception of stimuli (involves the parietal lobe); visual processing (associated with the occipital lobe); and · This includes perception and recognition of auditory stimuli, memory, and speech (which involves the temporal lobe).

[0039] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of STRO-1 administered to alleviate one or more effects of cerebral infarction, e.g., motor function decline. + MPC and / or their progeny (as well as culture-expanded STRO-1 + "An effective amount" is intended to mean a sufficient amount of a population enriched for MPCs (referred to as MPCs). A single dose of an "effective amount" need not be sufficient to provide a therapeutic effect; for example, multiple administrations of an effective amount of the population may provide an enhanced therapeutic effect.

[0040] As used herein, the term "low dose" refers to a dose of STRO-1 + 1 x 10 cells and / or their progeny 6 A low dose is understood to mean less than 0.5×10 but still sufficient to be an "effective amount" as defined herein and / or a "therapeutically effective amount" as defined herein. For example, a low dose is 0.5×10 6 cells or less, or 0.4 x 10 6 cells or less, or 0.3 x 10 6 cells or less, or 0.1 x 10 6 Contains no more than 10 cells.

[0041] As used herein, the terms "treat" or "treatment" or "treating" shall be understood to mean administering (systemically) a quantity of cells to increase stimulus-evoked cortical activity (e.g., primary cortical activity) in response to sensory input above the corresponding activity in an untreated subject.

[0042] As used herein, the term "normal or healthy individual" shall be taken to mean a subject who has not suffered from a cerebral infarction.

[0043] As used herein, "STRO-1" + The term "STRO-1 cells" + Mesenchymal progenitor cells (MPC) or STRO-1 + They are equivalent to pluripotent cells.

[0044] As used herein, STRO-1 + cells, STRO-1 + MPC or STRO-1 + The term "progeny thereof" in reference to a pluripotent cell means any of the aforementioned cells after propagation in culture, and such culture-propagated (progeny) cells are not related to the initial "primary" STRO-1 + The pluripotency and therapeutic properties of the cells are maintained.

[0045] As used herein, the term "effects of cerebral infarction" is understood to include and provide literal support for one or more of increasing cortical activity (e.g., primary motor cortex activity), increasing cortical activity within the infarct volume, and / or reducing the infarct volume.

[0046] Mesenchymal progenitor or stem cells (MLPSCs) In some embodiments, the MLPSC-enriched human cell population administered in the methods described herein is obtained from bone marrow, dental pulp, adipose, or pluripotent stem cells. In some embodiments, the human cell population is not obtained from dental pulp or adipose. In some embodiments, the human cell population is not obtained from dental pulp. In some embodiments, the MLPSC-enriched human cell population is STRO-1. + Cells are enriched for STRO-1 + STRO-1 cells can be found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph nodes, thymus, bone, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germ lineages such as mesoderm and / or endoderm and / or ectoderm. + Exemplary sources of cells are obtained from bone marrow and / or dental pulp.

[0047] In one example, STRO-1 + STRO-1 cells are multipotent cells that can differentiate into numerous cell types, including, but not limited to, adipose, bone, cartilage, elastic, muscle, and fibrous connective tissue. The specific lineage commitment and differentiation pathways that these cells enter depend on various influences from mechanical influences, and / or endogenous bioactive factors such as growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue. Thus, STRO-1 + Multipotent cells are non-hematopoietic progenitor cells that divide to give rise to daughter multipotent stem cells.

[0048] In one example, STRO-1 + The cells are enriched from a sample obtained from a human subject, e.g., the subject to be treated or a related or unrelated subject. The terms "enriched," "enriched," or variations thereof, are used herein to refer to a cell population that has an increased proportion of one particular cell type or several particular cell types when compared to an untreated cell population (e.g., cells in their natural environment). In one example, STRO-1 +The enriched population of cells may comprise at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% STRO-1 + In this regard, "STRO-1 + The term "enriched cell population" refers to "STRO1 + This is interpreted as expressly supporting the term "cell population comprising X% of cells," where X% is a percentage recited herein. STRO-1+ cells may, in some instances, form clonogenic colonies, e.g., CFU-F (fibroblasts) or a subset thereof (e.g., 50%, 60%, 70%, 70%, 90%, or 95%) may possess this activity.

[0049] In one example, the cell population contains a selectable form of STRO-1 + In this context, the term "selectable morphology" refers to the cells that are enriched from a cell preparation containing STRO-1. + It will be understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for selection of the cells. This marker may be, but need not be, STRO-1. For example, as described and / or exemplified herein, cells (e.g., MPCs) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 also express STRO-1 (STRO-1 bright Therefore, cells expressing STRO-1 + The indication that a cell is selected does not imply that the cell is selected by STRO-1 expression. In one example, the cell is selected based on at least STRO-3 expression, e.g., the cell is selected by STRO-3 expression. + (TNAP + )

[0050] The selection of cells or populations thereof does not require selection from a particular tissue source. +The cells may be selected, isolated, or enriched from a wide variety of sources. Thus, in some instances, these terms may be used interchangeably with STRO-1. + Any tissue containing cells (e.g., MPCs), or vascular tissue, or pericytes (e.g., STRO-1 + The present invention provides support for selection from tissues containing pericytes, or any one or more of the tissues listed herein.

[0051] In one example, the cells + , VCAM-1 + , THY-1 + , CD146 + or any combination thereof.

[0052] In one example, the cells are STRO-1 + (or STRO-1 bright ) and CD146 + or cell populations expressing STRO-1 + (or STRO-1 bright ) and CD146 + The cells are enriched for mesenchymal progenitor cells expressing

[0053] "Individually" means that the present disclosure encompasses the listed markers or groups of markers separately, and that the appended claims may define such markers or groups of markers separately and separably from one another, regardless of the possibility that individual markers or groups of markers may not be separately listed herein.

[0054] By "collectively," it is meant that the present disclosure encompasses any number or combination of the listed markers or peptides, and that notwithstanding that such number or combination of markers or markers may not be specifically recited herein, the appended claims may define such combination or subcombination separately and severably from any other combination of markers or markers.

[0055] In one example, STRO-1 + STRO-1 cells bright (STRO-1 bri In one example, Stro-1 bri STRO-1 cells dim or STRO-1 intermediate Preferentially enriched over cells.

[0056] For example, STRO-1 bright The cells also + , VCAM-1 + , THY-1 + , and / or CD146 + For example, the cells may be selected for one or more of the aforementioned markers and / or shown to express one or more of the aforementioned markers. In this regard, it is not necessary to specifically test cells shown to express the markers; rather, previously enriched or isolated cells can be tested and subsequently used, and it may be reasonable to assume that the isolated or enriched cells also express the same markers.

[0057] In one example, the mesenchymal precursor cells are perivascular mesenchymal precursor cells as defined in WO 2004 / 85630.

[0058] Cells that are considered "positive" for a given marker, when the term relates to the intensity of fluorescence or other marker used in the cell sorting process, can represent either a low (lo or dim) or a high (bright, bri) level of the marker, depending on the extent to which the marker is present on the cell surface. The distinction between low (or dim or dull) and bri will be understood in the context of the marker used on the particular cell population being sorted. A cell that is considered "negative" for a given marker does not necessarily mean that the cell is completely absent. This term means that the marker is expressed at a relatively very low level by the cell, or that when detectably labeled, the marker gives off a very low signal or cannot be detected above background levels, e.g., levels detected using an isotype control antibody.

[0059] The term "bright," as used herein, refers to a marker on the cell surface that gives off a relatively high signal when detectably labeled. Without wishing to be bound by any theory, it is proposed that "bright" cells express more of the target marker protein (e.g., the antigen recognized by STRO-1) than other cells in the sample. For example, STRO-1 bri When cells were labeled with FITC-conjugated STRO-1 antibody, they were classified as non-bright cells (STRO-1) as determined by fluorescence-activated cell sorting (FACS) analysis. dull / dim ) yields a fluorescent signal greater than 0.1% of the most brightly labeled cells within the distribution of labeled cell intensities. In other examples, "bright" cells comprise at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the most brightly STRO-1 labeled cells in the starting sample. In some examples, STRO-1 brightThe cells have a 2-log higher surface expression of STRO-1 relative to "background," i.e., STRO-1- cells. dim and / or STRO-1 intermediate The cells have surface expression of STRO-1 above "background," but by less than 2 logs, typically about 1 log or less.

[0060] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, this term encompasses the liver isoform (LAP), bone isoform (BAP), and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule capable of binding to the STRO-3 antibody produced by the hybridoma cell line deposited with the American Type Culture Collection (ATCC) on December 19, 2005 under the provisions of the Budapest Treaty under accession number PTA-7282.

[0061] Additionally, in certain examples of the present disclosure, STRO-1 + The cells are capable of giving rise to clonogenic CFU-F.

[0062] In one example, a significant proportion of MLPSCs, e.g., STRO-1 +Multipotent cells can differentiate into at least two different germ lineages. Non-limiting examples of lineages to which multipotent cells can commit include bone progenitor cells; hepatocyte progenitor cells with the multipotential to differentiate into bile duct epithelial cells and hepatocytes; neural restricted cells that can give rise to glial cell precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; precursors of cardiac muscle and cardiac myocytes, and precursors of glucose-responsive insulin-secreting pancreatic beta cell lines. Other systems include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as the following progenitor cells: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal tubular epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes, and oligodendrocytes.

[0063] In another example, an MLPSC, e.g., STRO-1 + The cells are unable to give rise to hematopoietic cells in culture.

[0064] In one example, cells are obtained from a subject to be treated, cultured and expanded in vitro using standard techniques, and used to obtain expanded cells for administration to the subject as an autologous or allogeneic composition. In another example, cells of one or more established human cell lines are used. In some embodiments, MLPSCs, e.g., cells, are obtained by differentiation of pluripotent stem cells, e.g., human induced pluripotent stem cells (hiPSCs). See, e.g., Dayem et al. (2019), International Journal of Molecular Science, 20(8):E1922.

[0065] In some embodiments, the progeny (expanded) cells are obtained after about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passages from the parental population, although the progeny cells may be obtained after any number of passages from the parental population.

[0066] Progeny cells can be obtained by culturing in any suitable medium. The term "medium," when used in reference to cell culture, includes the components of the environment surrounding the cells. A medium may be a solid, liquid, gas, or a mixture of phases and substances. Medium includes liquid growth media and liquid media that do not support cell growth. The term "medium" also refers to a material intended for use in cell culture, even if it is not yet in contact with cells. In other words, a nutrient-rich liquid prepared for bacterial culture is a medium. A powder mixture that becomes suitable for cell culture when mixed with water or other liquid is sometimes called a "powdered medium."

[0067] In one example, progeny cells useful in the methods of the present disclosure can be isolated by TNAP analysis using magnetic beads labeled with STRO-3 antibody. + STRO-1 + They are obtained by isolating or enriching cells from bone marrow, followed by expanding the isolated cells in culture (for examples of suitable culture conditions, see Gronthos et al., Blood 85:929-940, 1995).

[0068] In some embodiments, the expanded MLPSCs are characterized by the following receptors: LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5, CD49a / CD49b / CD29, CD49c / CD29, CD49d / CD29, CD 90, CD29, CD18, CD61, integrin beta 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R, leptin-R (STRO-2 = leptin-R), RANKL, STRO-4 (HSP-90β), STRO-1 bright and CD146, collectively or individually, or any combination of these markers.

[0069] Some MLPSCs, e.g., STRO-1 +Methods for preparing enriched populations of pluripotent cells and their propagation in culture are described in WO 01 / 04268 and WO 2004 / 085630. + Multipotent cells rarely exist as completely pure preparations, and are generally present together with other cells, typically tissue-specific committed cells (TSCCs). WO 01 / 04268 refers to the recovery of such cells from bone marrow at purity levels of about 0.1% to 90%. The population containing MPCs from which the progeny are derived may be recovered directly from a tissue source or may be a population already expanded ex vivo.

[0070] For example, progeny may be recovered, non-expanded, substantially purified STRO-1 cells that comprise at least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, or 95% of the total cells of the population in which they reside. + The levels can be obtained from a population of pluripotent cells. These levels can be measured by, for example, TNAP, STRO-4 (HSP-90β), STRO-1 bright , 3G5 + This can be achieved by selecting cells that are positive for at least one marker selected individually or collectively from the group consisting of: VCAM-1, THY-1, CD146, and STRO-2.

[0071] STRO-1 + The starting population of cells may be derived from any one or more of the tissue types described in WO 01 / 04268 or WO 2004 / 085630, namely bone marrow, dental pulp cells, adipose tissue, and skin, or perhaps more broadly from adipose tissue, teeth, dental pulp, skin, liver, kidney, heart, retina, brain, hair follicles, intestine, lung, spleen, lymph nodes, thymus, pancreas, bone, ligament, bone marrow, tendon, and skeletal muscle. In some preferred embodiments, STRO-1 + The enriched population of cells is derived from bone marrow, dental pulp, adipose, or pluripotent stem cells.

[0072] When practicing the methods described in this disclosure, it will be understood that isolation of cells bearing any given cell surface marker can be achieved by several different methods, although exemplary methods rely on binding a binding agent (e.g., an antibody or antigen-binding fragment thereof) to the marker of interest, followed by isolation of those exhibiting either high levels of binding, or low levels or no binding. The most convenient binding agents are antibodies or antibody-based molecules, such as monoclonal antibodies or those based on monoclonal antibodies (e.g., proteins containing their antigen-binding sites), due to the specificity of these latter agents. While antibodies can be used in both steps, other agents can also be used, and thus ligands for these markers can also be used to enrich for cells bearing the marker or cells lacking the marker.

[0073] Antibodies or ligands can be attached to a solid support to allow for crude separation. In one example, the separation technique maximizes the retention of viability in the collected fraction. A variety of techniques with different efficiencies can be used to obtain relatively crude isolates. The particular technique used will depend on the separation efficiency, associated cytotoxicity, ease and speed of implementation, and the need for sophisticated equipment and / or technical skill. Separation techniques can include, but are not limited to, magnetic separation using antibody-coated magnetic beads, affinity chromatography, and "panning" with antibodies bound to a solid matrix. Techniques that provide accurate separation include, but are not limited to, FACS. Methods for performing FACS will be apparent to those skilled in the art.

[0074] Antibodies to each of the markers described herein are commercially available (e.g., monoclonal antibodies to STRO-1 are commercially available from R&D Systems, USA), are available from the ATCC or other depositories, and / or can be produced using art-recognized techniques.

[0075] In one example, STRO-1 + The method for isolating cells includes a first step, which is a solid-phase sorting step using, for example, magnetic activated cell sorting (MACS) to recognize high levels of STRO-1 expression. If desired, a second sorting step can be followed to generate higher levels of progenitor cell expression, as described in patent specification WO 01 / 14268. This second sorting step may include the use of two or more markers.

[0076] In some embodiments, the MLPSCs are mesenchymal stem cells (MSCs). The MSCs may be a homogenous composition or a mixed cell population enriched for MSCs. A homogenous MSC composition can be obtained by culturing adherent bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers recognized by unique monoclonal antibodies. Methods for obtaining MSC-enriched cell populations using plastic adherence techniques are described, for example, in U.S. Patent No. 5,486,359. MSCs prepared by conventional plastic adherence isolation rely on the nonspecific plastic-adherence properties of CFU-Fs. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium.

[0077] The mesenchymal precursor or stem cells can be cryopreserved prior to administration to a subject.

[0078] The method for obtaining MLPSCs, e.g., mesenchymal stem cells, may also include harvesting the cell source using known techniques prior to the first enrichment step. Thus, the tissue is surgically removed. The source tissue containing the cells is then separated into a so-called single cell suspension. This separation can be achieved by physical or enzymatic means.

[0079] Once a suitable population of MLPSCs is obtained, it can be cultured or expanded by any suitable means.

[0080] In some embodiments, cells are obtained from the subject to be treated, cultured in vitro using standard techniques, and used to obtain expanded cells for administration to the subject as an autologous composition, or to a different subject as an allogeneic composition.

[0081] Cells useful in the methods of the present disclosure can be preserved before use. Methods and procedures for preserving and storing eukaryotic cells, particularly mammalian cells, are known in the art (see, for example, Pollard, JW and Walker, JM (1997) Basic Cell Culture Protocols, 2nd ed., Humana Press, Totowa, NJ; Freshney, RI (2000) Culture of Animal Cells, 4th ed., Wiley-Liss, Hoboken, NJ). Any method for maintaining the biological activity of isolated stem cells, such as mesenchymal stem / progenitor cells or their progeny, can be used in connection with the present disclosure. In one example, the cells are maintained and stored using cryopreservation.

[0082] Modified cells In one example, MLPSCs and / or their progeny are genetically modified to express and / or secrete a protein of interest, e.g., the cells are engineered to express a protein useful for treating movement disorders or other effects of stroke, such as vascular endothelial growth factor (VEGF), erythropoietin, brain-derived growth factor (BDNF), or insulin-like growth factor (IGF-1), as reviewed in Larpthavesarp et al. (2015) Brain Science 5(2):165-177.

[0083] Methods for genetically modifying cells will be apparent to those skilled in the art. For example, the nucleic acid to be expressed in the cell is operably linked to a promoter for inducing expression in the cell. For example, the nucleic acid is linked to a promoter operable in various cells of interest, such as a viral promoter, for example, a CMV promoter (e.g., a CMV-IE promoter) or an SV-40 promoter. Further suitable promoters are known in the art and shall be construed as applicable to this example of the present disclosure.

[0084] In one example, the nucleic acid is provided in the form of an expression construct. As used herein, the term "expression construct" refers to a nucleic acid capable of expressing an operably linked nucleic acid (e.g., a reporter gene and / or a counterselectable reporter gene) in a cell. In the context of the present disclosure, it is understood that an expression construct may include or be a plasmid, bacteriophage, phagemid, cosmid, viral subgenomic or genomic fragment, or other nucleic acid capable of maintaining and / or replicating heterologous DNA in an expressible manner.

[0085] Methods for constructing suitable expression constructs for practicing the present disclosure will be clear to those skilled in the art and are described, for example, in Ausubel et al. (Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) or Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 3rd ed. 2001). For example, each component of the expression construct can be amplified from a suitable template nucleic acid, for example, using PCR, and then cloned into a suitable expression construct, such as a plasmid or phagemid.

[0086] Suitable vectors for such expression constructs are known in the art and / or described herein. For example, suitable expression vectors for use in the disclosed methods in mammalian cells include, for example, the pcDNA suite of vectors supplied by Invitrogen, the pCI suite of vectors (Promega), the pCMV suite of vectors (Clontech), the pM vectors (Clontech), the pSI vectors (Promega), the VP16 vectors (Clontech), or the pcDNA suite of vectors (Invitrogen).

[0087] Those of skill in the art will be aware of additional vectors and sources of such vectors, such as, for example, Life Technologies Corporation, Clontech, or Promega.

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

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

[0090] For example, retroviral vectors typically contain cis-acting long terminal repeats (LTRs) with packaging capacity for up to 6-10 kb of foreign sequence. Even the smallest cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate the expression construct into target cells for long-term expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SrV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 56:2731-2739 (1992); Johann et al., J. Virol. 65:1635-1640 (1992); Sommerfelt et al., Virol. 76:58-59 (1990); Wilson et al., J. Virol. 63:274-2318 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); International Application PCT / US94 / 05700; Miller and Rosman BioTechniques 7:980-990 (1989); Miller, AD Human Gene Therapy 7:5-14, 1990; Scarpa et al., Virology 75:849-852, 1991; Burns et al., Proc. Natl. Acad. Sci USA 90:8033-8037, 1993).

[0091] Various adeno-associated virus (AAV) vector systems have also been developed for nucleic acid delivery. AAV vectors can be readily constructed using techniques known in the art. See, for example, U.S. Pat. Nos. 5,173,414 and 5,139,941; WO 92 / 01070 and WO 93 / 03769; Lebkowski et al., Molec. Cell. Biol. 5:3988-3996, 1988; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter Current Opinion in Biotechnology 5:533-539, 1992; Muzyczka Current Topics in Microbiol, and Immunol. 158:97-129, 1992; Kotin, Human Gene Therapy 5:793-801, 1994; Shelling and Smith Gene Therapy 7:165-169, 1994; and Zhou et al., J Exp. Med. 179:1867-1875, 1994.

[0092] Additional viral vectors useful for delivering expression constructs of the present disclosure include those derived from viruses of the pox family, such as vaccinia virus and avian poxvirus, or alphaviruses, or composite viral vectors (e.g., as described in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 56:317-321, 1989).

[0093] In some embodiments, at least a portion of the administered cells are labeled to facilitate non-invasive detection, localization, and / or tracking of the administered labeled cells after administration. In some embodiments, the cells are genetically modified to express a reporter protein, e.g., a monomeric far-red fluorescent protein, that can be non-invasively detected in vivo. See, e.g., Wannier et al. (2018), PNAS, 115 (48) E11294-E11301. In other embodiments, the administered cells are labeled by non-genetic means, e.g., using a significant tracking label that is introduced into at least a portion of the administered cells and can then be non-invasively detected in vivo. One example of a suitable tracking label is Molady ION™ Rhodamine B (MIRB) (available from Biophysics Assay Laboratory, Inc.), an iron oxide-based superparamagnetic MRI contrast reagent with a colloidal size of 35 nm designed for cell labeling and MRI tracking that does not require transfection reagents for efficient cell labeling. The tracking can be visualized by MRI or fluorescence.

[0094] Stroke model Various techniques are known to induce ischemic stroke in non-human animal subjects, including aortic / vena cava occlusion, external neck tourniquet or pressure band, hemorrhage or hypotension, intracranial hypertension or common carotid artery occlusion, two-vessel occlusion and hypotension, four-vessel occlusion, unilateral common carotid artery occlusion (only in some species), endothelin-1-induced arterial and venous constriction, middle cerebral artery occlusion, spontaneous stroke (in spontaneously hypertensive rats), macrosphere embolization, clot embolization, or microsphere embolization. Hemorrhagic stroke can be modeled by injecting collagenase into the brain.

[0095] In one example, a model of cerebral infarction involves middle cerebral artery occlusion resulting in ischemic cerebral infarction.

[0096] To test the ability of the population and / or offspring to treat the effects of cerebral infarction, the population and / or offspring are administered after induction of cerebral infarction, e.g., within 1 hour to 1 day after cerebral infarction. Following administration, assessment of brain function and / or motor deficits is performed, e.g., at several times.

[0097] Methods for assessing brain function and / or motor impairments are readily apparent to those skilled in the art, including, for example, the rotarod, elevated plus maze, open field, Morris water maze, T-maze, radial arm maze, locomotor assessment (e.g., area over time), tail flick, or De Ryck behavioral tests (De Ryck et al., Cerebral infarction. 20:1383-1390, 1989). Additional tests will be readily apparent to those skilled in the art and / or are described herein. Similarly, models of HIE are known in the art. See, e.g., Millar et al. (2017), Frontiers in Cellular Neuroscience, 11(78):1-36.

[0098] In another example, the effect of cell administration on sensory stimulus-evoked cortical activity, infarct volume, or cortical activity within the infarct may be assessed by imaging techniques. In some preferred embodiments, magnetic resonance imaging (MRI), and in particular functional MRI (fMRI) techniques such as blood oxygen level-dependent (BOLD) imaging, are useful for making such assessments. PET and CT can also be used to make such assessments.

[0099] Functional assessment of motor behavior assays can be used alone or in combination with imaging techniques to assess the therapeutic efficacy of the cell compositions described herein. Such behavioral assays include, but are not limited to, limb placing, rotarod, grid walking, and elevated body swing. See, e.g., Schaar et al. (2010), Experimental & Translational Stroke Medicine, 2:13; and Borlongan et al. (1995), Physiology & Behavior, 58(5):909-917.

[0100] cell composition In one example of the present disclosure, MLPSCs, e.g., STRO-1 + The cells and / or their progeny are administered in the form of a composition, hi one example, such a composition comprises a pharmaceutically acceptable carrier and / or excipient.

[0101] The terms "carrier" and "excipient" refer to compositions conventionally used in the art to facilitate the storage, administration, and / or biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th ed., Mac Publishing Company (1980)). A carrier can also reduce any undesirable side effects of an active compound. A suitable carrier is, for example, stable and, for example, incapable of reacting with other ingredients in the carrier. In one example, a carrier causes no significant local or systemic adverse effects in a recipient at dosages and concentrations used therapeutically.

[0102] Suitable carriers for the present disclosure include those conventionally used, for example, water, saline, aqueous dextrose, lactose, Ringer's solution, buffers, hyaluronan, and glycols are exemplary liquid carriers, particularly for solutions (when isotonic). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

[0103] In another example, the carrier is, for example, a media composition in which the cells are grown or suspended. In one example, such a media composition does not induce any adverse effects in the subject to which it is administered. Further examples include cryopreservation media, e.g., physiological media containing one or more cryoprotective polyols such as dimethyl sulfoxide (DMSO), one or more cryoprotective agents such as trehalose, or combinations thereof.

[0104] Exemplary carriers and excipients do not adversely affect the viability of the cells and / or the ability of the cells to reduce, prevent or delay the effects of a stroke.

[0105] In one example, the carrier or excipient provides buffering to maintain the cells at the appropriate pH for biological activity, for example, phosphate buffered saline (PBS). PBS is an attractive carrier or excipient because it interacts minimally with cells and factors, allowing for rapid release of cells and factors, such as when the compositions of the present disclosure are formulated as liquids for direct application, e.g., by injection, into the bloodstream or tissues or areas surrounding or adjacent to tissues.

[0106] The cell compositions useful in the methods described herein can be administered alone or in mixtures with other cells. Cells that can be administered in conjunction with the compositions of the present disclosure include, but are not limited to, other multipotent or pluripotent cells or stem cells, or bone marrow cells. Different types of cells can be mixed with the compositions of the present disclosure immediately or shortly before administration, or they can be co-cultured together for a period of time before administration.

[0107] In one example, the composition comprises an effective amount or a therapeutically or prophylactically effective amount of cells. For example, the composition comprises about 1 x 10 5 MLPSCs / kg to approximately 1 × 10 7 MLPSC / kg or approximately 1 × 10 6 MLPSCs / kg to approximately 5 × 10 6 In another example, the composition comprises about 1 x 10 MLPSCs / kg. 5 STRO-1 + cells / kg to approximately 1 x 10 7 STRO-1 + cells / kg or approximately 1 x 10 6 STRO-1 + cells / kg to approximately 5 x 10 6 STRO-1 +The exact amount of cells administered will depend on various factors, such as the age, weight, and sex of the patient, as well as the extent and severity of the cerebral infarction and / or the location of the cerebral infarction.

[0108] In some embodiments, a low dose of cells is administered systemically to a subject. An exemplary dose is about 0.1 x 10 MLPSCs per kg. 6 From 2 x 10 6 Between pieces, for example, about 0.5 x 10 MLPSC per kg 5 From 2 x 10 6 For example, about 0.7 x 10 MLPSC per kg 5 to 1.5 x 10 6 Between 0.8 x 10 and 1.0 x 10, for example, 5 , 1.0×10 6 , 1.2 × 10 6 , or 1.4 × 10 6 Contains MLPSCs / kg.

[0109] In other embodiments, systemic administration is based on the estimated volume of the infarct, e.g., about 2×10 6 MLPSC / cm 3 Approximately 2 × 10 from the affected cortex 7 MLPSC / cm 3 Affected cortex, e.g., 3 × 10 6 , 4×10 6 , 5×10 6 , 8×10 6 , 1.2 × 10 7 , 1.5×10 7 , or approximately 2 × 10 6 MLPSC / cm 3 to approximately 2 × 10 7 MLPSC / cm 3 Up to a different number of cells / cm 3 is.

[0110] In some examples of the present disclosure, it may not be necessary or desirable to immunosuppress a patient prior to initiation of treatment with a cell composition. + Injection of the cells or their progeny may also be acceptable in some cases.

[0111] However, in other cases, it may be desirable or appropriate to pharmacologically immunosuppress the patient and / or reduce the subject's immune response to the cell composition prior to initiation of cell therapy. This can be accomplished by the use of systemic or local immunosuppressants. Alternatively, the cells can be genetically modified to reduce their immunogenicity.

[0112] Additional Components of the Composition The MLPSCs or their progeny can be administered together with other beneficial drugs or biomolecules (growth factors, trophic factors). When administered together with other agents, they can be administered together in a single pharmaceutical composition or in separate pharmaceutical compositions, either simultaneously with the other agents or sequentially (either before or after administration of the other agents). Bioactive factors that can be co-administered include anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory agents (e.g., p38MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors, pemirolast, tranilast, Remicade, sirolimus, and NSAIDs (non-steroidal anti-inflammatory drugs; e.g., tepoxalin, tolmen, suprofen); immunosuppressants / immunomodulators (e.g., calcineurin inhibitors such as cyclosporine, taclorimus; mTOR inhibitors (e.g., sirolimus, everolimus); anti-proliferative agents (e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g., prednisolone, hydrocortisone); monoclonal anti-IL-2R alpha receptor antibodies (e.g., basiliximab, daclizumab), polyclonal antibodies (e.g., cyclosporine, tolmen, suprofen), and cyclosporine-1 inhibitors (e.g., cyclosporine, tolmen, suprofen). monoclonal anti-T cell antibodies (e.g., antibodies such as antithymocyte globulin (ATG); antilymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3); antithrombogenic agents (e.g., heparin, heparin derivatives, urokinase, PPack (dextrophenylalanine proline arginine chloromethyl ketone), antithrombin compounds, platelet receptor antagonists, antithrombin antibodies, antiplatelet receptor antibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors); and antioxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine), and local anesthetics. In some embodiments, the administered cell composition comprises an anti-inflammatory agent. In other embodiments, the administered cell composition comprises a thrombolytic agent.

[0113] In some embodiments, the cell composition comprises an agent that temporarily disrupts the blood-brain barrier (BBB). In some embodiments, the administered cell composition comprises mannitol. Alternatively, the mannitol is administered shortly before or after administration of the cell composition, for example, within about an hour.

[0114] In some embodiments, the composition according to any of the examples described herein comprises factors for improving brain function and / or for regenerating brain neurons and / or for treating motor dysfunction, e.g., trophic factors.

[0115] Alternatively, or in addition, cells and / or compositions according to any of the examples described herein are combined with known treatments for the effects of stroke, such as physical and / or speech therapy.

[0116] In one example, a pharmaceutical composition according to any of the examples described herein includes a compound used to treat the effects of cerebral infarction. Alternatively, a method of treatment / prevention according to any of the examples of the present disclosure described herein further includes administering a compound used to treat the effects of cerebral infarction. Exemplary compounds are described herein and shall be construed to apply mutatis mutandis to these examples of the present disclosure.

[0117] In another example, the composition according to any of the examples described herein further comprises a factor that induces or promotes differentiation of progenitor cells into vascular cells. Exemplary factors include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF; e.g., PDGF-BB), and FGF.

[0118] medical devices The present disclosure also provides a medical device for use in or when used in a method according to any of the examples described herein. For example, the present disclosure provides a STRO-1 according to any of the examples described herein. +A syringe or catheter or other suitable delivery device is provided containing the cells and / or their progeny and / or the composition, optionally packaged with instructions for use in a method according to any of the examples described herein.

[0119] Administration In some embodiments, the subject to be treated is suffering from ischemic cerebral infarction. In certain embodiments, the subject to be treated is a neonatal subject suffering from hypoxic-ischemic encephalopathy (HIE). In other embodiments, the cerebral infarction is hemorrhagic cerebral infarction. In some preferred embodiments, the subject to be treated is a human subject.

[0120] In a preferred embodiment, an MLPSC, e.g., STRO-1 + The cells, MSCs, or progeny thereof are administered systemically.

[0121] In preferred embodiments, the MLPSCs are delivered to the subject's bloodstream, e.g., parenterally. Exemplary routes of parenteral administration include, but are not limited to, intra-arterial, intravenous, intraperitoneal, or intrathecal. In some preferred embodiments, the cell population enriched for MLPSCs or their progeny is delivered intra-arterially, intra-aortically, or into the atria or ventricles of the heart.

[0122] In the case of cell delivery to the atria or ventricles of the heart, the cells can be administered to the left atrium or left ventricle to avoid complications that may arise from rapid cell delivery to the lungs.

[0123] In one embodiment, the population is administered into the carotid artery.

[0124] The choice of administration regimen for a therapeutic formulation depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, and the immunogenicity of the entity.

[0125] In one example, the MLPSCs or their progeny are delivered as a single bolus dose. +The cells or their progeny are administered by continuous infusion or, for example, at daily or weekly intervals, or 1 to 7 times per week. Exemplary administration protocols include the maximum dosage or frequency of administration that avoids significant undesirable side effects. The total weekly dosage will depend on the type and activity of the agent / cells used. Determination of the appropriate dosage will be made by the clinician, for example, using parameters or factors known, suspected, or expected in the art to affect treatment. Generally, administration is initiated at a somewhat lower dose than optimal, and then increased by small increments until the desired or optimal effect is achieved, relative to any negative side effects.

[0126] In some embodiments, the cell compositions described herein (e.g., MLPSCs, e.g., STRO-1 + MPC, STRO-1 bright The MPC-enriched human cell population or MSCs are systemically administered within about 24 hours after the stroke, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 12 hours, 16 hours, 18 hours, or another time from about 1 hour to about 24 hours. In other embodiments, the cell composition is administered 24 hours, e.g., about 25 hours to 1 month, e.g., 26 hours, 28 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, or another time from about 24 hours to about 1 month after the stroke of the subject being treated. In some embodiments, the cell composition is systemically administered from 24 hours to about 48 hours. In other embodiments, the cell composition is systemically administered from about 48 hours to 2 weeks after the stroke. In some embodiments, when the cell compositions described herein are administered within about 24 hours after the stroke, the subject being treated does not receive a thrombolytic agent, either separately (before or after administration of the cells) or as part of the cell composition itself.

[0127] In some embodiments, after administration of cells suitably labeled for in vivo detection as described herein to a subject, the distribution of cells in the subject at one or more time points is determined from about 6 hours to 1 month, e.g., 12 hours, 24 hours, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 7 weeks, or another time point from about 6 hours to about 2 months after administration of the labeled cells.

[0128] In some embodiments, changes in infarct volume and / or infarct activity are determined in the treated subject over a period of at least 12 hours to 6 months after administration, e.g., 18 hours, 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or another time period from about 12 hours to about 6 months. Infarct volume and / or activity within the infarct can be determined using any of several methods known in the art, such as non-contrast head computed tomography (NCCT) and fMRI for volume determination and analysis of blood oxygen level dependent (BOLD) signal within the infarct region.

[0129] The present disclosure includes the following non-limiting examples. [Example]

[0130] Example 1 Treatment with human MPCs improves motor function in rodent models of infarction. Animals, husbandry, and diet Eighty-four male nude rats (RNU rats, Taconic, IBU051001C) weighing 250 to 275 g arrived 7 to 10 days before surgery. Food and water were available ad libitum throughout the study. Animals were assigned sequential identification numbers on their tails using a permanent marker pen. Animals were observed the day before surgery, and animals deemed unwell were excluded from the study.

[0131] Animals were housed in a room supplied with filtered air at a temperature of 21±2°C and a relative humidity of 50%±20%. The room was set on an automatic timer with a 12-hour light / 12-hour dark cycle with no dim light. Shepherd® (1 / 4 inch) premium corncob bedding was used, and one tube of Nylabone® (3.5 inches, Dura Bones Petite) was placed in each cage. Animals were fed Lab Diet® 5001 chow. Water was available ad libitum.

[0132] Animals were housed two per cage before and after surgery unless they showed severe aggression or injury, or if a caged animal died, leaving the animals housed alone.

[0133] Research Plan Animal preparation Seventy-two adult male nude rats were used in the study. All rats were housed for 7 days prior to surgery for acclimatization purposes and were habituated to behavioral assessment. At the end of the acclimatization period, rats were randomized and assigned to different groups.

[0134] Preparing for surgery Middle cerebral artery occlusion (MCAO), Tamura model Focal cerebral infarction was created by permanent occlusion of the proximal right middle cerebral artery (MCA) using a modified method from Tamura et al. Male nude rats (250–350 g at the time of surgery) were anesthetized with 2–3% isoflurane in a 2:1 NO:O mixture and maintained with 1–1.5% isoflurane in a 2:1 NO:O mixture. The temporalis muscle was bisected and reflected through an incision made midway between the eye and the tympanic canal. The proximal MCA was exposed by a subtemporal craniectomy without cutting the facial nerve or removing the zygomatic arch. The artery was then occluded and transected by microbipolar coagulation from just proximal to the olfactory tract to the inferior cerebral vein. Body temperature was maintained at 37.0 ± 1°C throughout the procedure. Buprenorphine SR (0.9-1.2 mg / kg, ZooPharm) and cefazolin (40-50 mg / kg, Hospira) were administered as analgesics at this time prior to the MCAO surgery.

[0135] dosage Cells (hMPC, TAN 2178, lot number 2011CC043) and vehicle (Cryomedia, lot number 2012CC034) were delivered by dry shipper from the supplier and stored in liquid nitrogen vapor. Cryopreserved hPMC were thawed immediately prior to injection as follows: hMPC (1 x 10 in 0.17 mL) 6 Cell suspension (0.17 mL) or vehicle (0.17 mL) was administered via tail vein injection 6, 12, 24, 48 hours, or 7 days after MCAO. The cell suspension was delivered over approximately 20 seconds. On days when behavioral testing and cell administration were to be performed on the same day, cells were always administered after behavioral testing.

[0136] Randomization and blinding At 24 hours, animals treated were randomly assigned to receive cells or vehicle using quickcalcs, available online at www.graphpad.com / quickcalcs / randomize2.cfm. Other animals were assigned to treatment groups in a manner that evenly distributed treatments on the day of surgery and maximized the number of animals that could receive cells from a single vial of thawed cells. The same investigator performed all animal surgeries and behavioral assessments and was blinded to the study design and treatment assignment of each animal.

[0137] Behavioral testing Functional activity was assessed with limb-placing and body-swing behavioral tests, which were performed 1 day before (day -1), 1 day (day 1), 3 days (day 3), 7 days (day 7), 14 days (day 14), 21 days (day 21), and 28 days (day 28) after MCAO (day 0 = day of MCAO).

[0138] Limb placement The limb placing test was divided into both forelimb and hindlimb tests. In the forelimb placing test, the examiner approached the rat to a tabletop and scored the rat's ability to place its forelimbs on the tabletop in response to whisker, visual, tactile, or proprioceptive stimuli. Similarly, in the hindlimb placing test, the examiner assessed the rat's ability to place its hindlimb on the tabletop in response to tactile and proprioceptive stimuli. Separate subscores (with half-point assignments) were obtained for each mode of sensory input and added to obtain a total score (for the forelimb placing test: 0 = normal, 12 = maximally impaired; for the hindlimb placing test: 0 = normal; 6 = maximally impaired).

[0139] 2.Body swing test The rat was held approximately 1 inch from the base of the tail. It was then elevated 1 inch above the surface of the table. The rat was held on the vertical axis, defined as 10° or less to either the left or right side. A swing was recorded whenever the rat moved its head to either side of the vertical axis. The rat had to return to a vertical position for the next swing to be counted. A total of 30 swings were counted. Normal rats usually have an equal number of swings on either side. After focal ischemia, rats tend to swing to the opposite side (left side).

[0140] culling Twenty-eight days after MCAO, rats were deeply anesthetized with a mixture of ketamine (50–100 mg / kg) and xylazine (5–10 mg / kg) intraperitoneally. They were then transcardially perfused with normal saline (2 units / ml heparin) followed by 10% formalin. Brains were removed and preserved in 10% formalin. Brains were sent to HistoTechnologies, Inc. and processed (H&E staining) to measure infarct volume.

[0141] Data analysis All data are expressed as mean ± SEM. Behavioral and body weight data were analyzed by repeated measures ANOVA (treatment x times). A positive p-value of the F-statistic from the overall ANOVA including all groups allowed pairwise ANOVA between groups. In Figures 1 to 3: * = different from vehicle-treated group at p<0.05 ** = p<0.01 different from vehicle-treated group *** = different from vehicle-treated group at p<0.001 For behavioral testing, day −1, the day before stroke, was intentionally excluded from the analysis to ensure normal distribution of the data.

[0142] result As shown in Figure 1, administration of hMPCs 6 hours (p<0.01), 12 hours (p<0.01), 24 hours (p<0.001), 48 hours (p<0.01), and 7 days (p<0.01) after MCAO significantly improved forelimb recovery compared to vehicle-treated animals. As shown in Figure 2, administration of hMPCs 6 hours (p<0.001), 12 hours (p<0.01), 24 hours (p<0.001), and 48 days (p<0.001) after MCAO significantly improved hindlimb recovery compared to vehicle-treated animals. Administration of hMPCs 6 hours (p<0.05), 12 hours (p<0.05), 48 hours (p<0.01), and 7 days (p<0.01) after MCAO significantly improved body swing recovery compared with vehicle administration (Figure 3). There was no significant difference in body weight between the MPC-treated and vehicle groups (Figure 4).

[0143] Example 2 Effect of intravenous hMPC or vehicle on functional imaging in a rat model of cerebral infarction. Infarction model Animals were anesthetized in an induction chamber containing 2–3% isoflurane in NO:O (2:1) and maintained with 1–1.5% isoflurane via a face mask. Once anesthetized, animals received cefazolin sodium (40 mg / kg, i.p.) and buprenorphine (0.1 mg / kg, sc). Lacrilube, an ophthalmic ointment, was applied to the eyes to prevent dryness. All animals were maintained at 37.0 ± 1°C throughout surgery. In all animals, a small focal stroke (infarction) was created on the right side of the brain (cerebral cortex) surface by middle cerebral artery occlusion (MCAO). Using aseptic technique, an incision was made midway between the eye and the tympanic canal. The temporalis muscle was isolated, bisected, and reflected.

[0144] To expose the MCA, a small bone window was removed using a drill and a longus (subtemporal craniectomy). Using a dissecting microscope, the dura was incised, and the MCA was permanently occluded by electrocoagulation using microbipolar electrocautery, from just proximal to the olfactory tract to the inferior cerebral vein (taking care not to rupture this vein). The MCA was then transected. The temporalis muscle was then repositioned, and the incision was closed subcutaneously using sutures. The skin incision was closed with surgical staples (two to three staples were required). After surgery, the animals were placed on a heating pad until they recovered from anesthesia. They were then returned to their clean home cages. Animals were observed frequently on the day of MCAO surgery (day 0) and at least once daily thereafter until they were sent to Ekam Imaging, Inc. for imaging on day 8. All image analysis was completed blinded, without any knowledge of treatment. Once image analysis was complete, the codes were unblinded.

[0145] Imaging Procedures - Functional Magnetic Resonance Imaging (fMRI) Rowlett nude (RNU) rats were obtained from Taconic. Animal health certificates were provided at the time of shipment on day 8. Animals were transported in temperature-controlled vehicles to the imaging facility on each imaging day (day 15). A total of two blinded groups (n = 9 / group), designated A and B, were studied. In addition, two non-operated animals were included and imaged on the first imaging day to serve as normal controls for comparison.

[0146] Imaging studies were performed using a Bruker Biospec 7.0T / 20cm USR horizontal magnet (Bruker, Billerica, MA USA) and a 20G / cm magnetic field gradient insert (ID = 12cm) (Bruker) with a 120μs rise time. Radiofrequency signals were transmitted and received by quad-coil electronics integrated into the animal restraint system. All animals were anesthetized, placed in the restraint system, and imaged to acquire the following anatomical and functional scans: 1) Pilot Scan (RARE Tripilot) 2) Whole-brain T2-weighted reference anatomical imaging (22 slices; 1.2 mm; FOV 3 cm) 2 ;256×256; RARE pulse sequence.) 3) fMRI (96 × 96 × 22, T2-weighted rapid acquisition with refocused echo (RARE) images; electrical foot shock, 0.6 mA, 3 min baseline, followed by 3 min stimulation in the left hind paw, another 3 min baseline, and another 3 min stimulation in the left forepaw. Stimulation was applied to the paw contralateral to the stroke (and the matched paw of the control). 4) fMRI (96x96x22, T2-weighted RARE images; 10% CO2 stress).

[0147] A schematic diagram of the imaging experimental procedure and equipment is shown in Figures 5 and 6. Respiration was monitored during imaging using a multi-animal monitoring and gating system (SAII, Stony Brook, NY).

[0148] Image analysis Because this study consisted of six different MR imaging modalities, data were analyzed using a variety of software and platforms. Data were compiled into a document format consisting of figures / images and tables with reported figures. Functional MR images (fMRI) were analyzed using in-house software MIVA, where each subject was registered to a segmented rat brain atlas (Ekam Imaging). The alignment method was facilitated by an interactive graphic user interface. Composite statistics were generated using the inverse transformation matrix. [Ti] -1 Each composite pixel location (i.e., row, column, and slice), pre-multiplied by, was mapped into the voxels of subject (i). The statistical contribution of subject (i) to the composite (row, column, and slice) location was determined by three linear interpolations of the subject's voxel values (rate of change). [Ti] -1The use of [subject] ensured that the entire set of composites was loaded with subject involvement. The average value across all subjects in a group was used to determine the composite value. The average number of activated pixels with the highest composite percent change value in a particular ROI was displayed on the composite map. The activated composite pixels were calculated as follows:

[0149]

number

[0150] The composite rate of change for the time history graphs of each region was based on a weighted average for each subject as follows:

[0151]

number

[0152] where N is the number of subjects.

[0153] Tissue sampling Eight days after MCAO, rats were deeply anesthetized with CO2 after imaging. The heart was exposed, and an 18G needle was inserted and connected to the left ventricle, toward the top where the ascending aorta connects, via an infusion pump while the heart was still beating. An incision was made in the atrium to allow blood / perfusate to escape. Perfusion began with saline containing approximately 2 units / mL heparin at a rate of 40 mL / min for 5 minutes, followed by 10% formalin at the same rate for 5 minutes. After decapitation, the brain was carefully removed and placed in a labeled tube containing at least 10 mL of 10% formalin.

[0154] result Infarct volume Figure 7 shows measurements (mean ± SEM) of infarct volume (upper graph) and percent of total brain infarct volume (lower graph) at MRI on day 8. The MPC-treated group had statistically smaller infarct volumes compared to the vehicle-treated group (p<0.05). Furthermore, infarct volume as a percentage of total brain was significantly smaller in the MPC-treated group (p<0.05).

[0155] Cortical activation by foot stimulation Figure 8 shows activation in the primary and secondary motor cortices and primary and secondary somatosensory cortices ipsilateral to the infarct following forepaw stimulation on the contralateral side of the infarct in vehicle and hMPC-treated groups, as demonstrated by BOLD imaging. As shown in Figure 8 (top panel), the volume of activation in the primary cortex (but not the secondary motor cortex) was significantly higher in the hMPC-treated group. No significant differences were observed between groups for activation of the somatosensory cortex (bottom panel). Similarly, no differences were observed in activation of the motor or somatosensory cortices on the contralateral side of the infarct (data not shown).

[0156] fMRI analysis of neural activity in the ischemic core and penumbra Post-hoc imaging analysis identified the ischemic area using anatomical scans. fMRI activation was measured after forepaw stimulation on the side opposite the infarct. The volume of activation was significantly greater in animals treated with MPC (Figure 9).

Claims

[Claim 1] A method for increasing cortical activation or reducing infarct volume after cerebral infarction, comprising systemically administering a therapeutically effective amount of a human cell population enriched in mesenchymal progenitor or stem cells (MLPSCs) to a human subject in need thereof.

Images