Methods for Treating Brain Injury

Abstract

The present invention relates generally to compositions and methods useful for treating brain injuries, such as stroke, optic neuropathy, traumatic brain injury, and cerebral palsy. The methods involve administering to a subject HMCs obtained by in vitro differentiation of pluripotent stem cells and / or extracellular vesicles (EVs) derived from such HMCs (HMC-EVs).

Description

[Technical Field]

[0001] Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 390,044, filed July 18, 2022, the entire contents of which are incorporated herein by reference.

[0002]

[0002] The present invention relates to a method for treating brain injury using mesenchymal stem cells and / or extracellular vesicles secreted from mesenchymal stem cells. [Background technology]

[0003] Brain injuries are complex and can have multiple severe clinical outcomes. An acquired brain injury is an injury to the brain that is not genetic, congenital, degenerative, or induced by birth trauma. The injury results in changes in the brain's neural activity, and such changes affect the physical integrity, metabolic activity, or functional capacity of neurons within the brain. There are two major types of acquired brain injury: traumatic and non-traumatic.

[0004] Traumatic brain injury (TBI) is a leading cause of death and disability in the United States. More than 1.7 million Americans suffer from TBI each year. TBI is caused by an external force, such as a blow, blow, or shake to the head, disrupting the brain's normal function. The severity of TBI ranges from "mild" (i.e., a temporary change in mental status or consciousness) to "severe" (i.e., prolonged loss of consciousness or memory after the injury). TBI accounts for approximately 30% of all injury deaths (Taylor et al., MMWR Surveill. Summ. 2017; Vol. 66 (No. SS-9): pp. 1-16). Approximately 153 people die each day in the United States from injuries, including TBI (ibid.). Those who survive TBI may face lasting effects that last for days or the rest of their lives. The effects of TBI can include impairments in thinking or memory, movement, sensation (e.g., vision or hearing), or emotional functioning (e.g., personality changes, depression).

[0005] Approximately 20–40% of people with TBI experience associated visual impairment (Houston KE et al., Am J Phys. Med. Rehabil. 2017, 96:e70–4). This can include blurred vision, visual field defects, and reduced visual acuity. These symptoms can occur acutely or chronically depending on the type, location, and severity of injury. TBI can affect various parts of the visual system, including the optic nerve and optic tracts, the lateral geniculate nucleus, and the optic radiation, resulting in a variety of visual problems (Barnett BP et al., Curr Treat Options Neurol., 2015, 17:329). Known sites of afferent pathway damage include the optic nerve and optic tracts. Structurally, the optic nerve is vulnerable to compression, traction, crush, laceration, and avulsion injuries. Sudden acceleration or deceleration of the head can indirectly cause traction on the optic nerve or shearing of the axons, which can lead to optic neuropathy.

[0006]

[0006] To date, several treatment options for TBI include hyperbaric oxygen therapy, non-invasive brain stimulation, task-oriented functional electrical stimulation, and behavioral therapy (Dang et al., Neural Plasticity 2017, Vol. 2017, Article ID 1582182, Item 6). However, there is still a need for improved treatment methods for TBI.

[0007]

[0007] Non-traumatic brain injury is usually caused by damage to the brain due to internal factors, such as oxygen deprivation, exposure to toxins, or compression by a tumor. Stroke is one example of a non-traumatic brain injury. Stroke is the fifth leading cause of death in the United States, with approximately 800,000 people experiencing a stroke each year. Stroke occurs when blood supply to the brain is cut off or reduced due to a blocked blood vessel or bleeding. When this occurs, the brain does not receive enough oxygen and nutrients, and brain cells begin to die. People who experience a stroke require emergency treatment, such as medications to break down blood clots and prevent further clot formation. While some strokes are treatable, some can result in disability or death.

[0008]

[0008] Cerebral palsy occurs as a result of sustained brain injury during fetal development or at birth. Cerebral palsy is caused by damage to the motor cortex of the brain, which affects muscle control and coordination, including an individual's ability to move, grasp objects, and speak. Cerebral palsy is a leading cause of disability in young children, affecting approximately 500,000 children and adults. Currently, there is no known cure for cerebral palsy. Summary of the Invention [Problem to be solved by the invention]

[0009]

[0009] Nerve cells and brain cells damaged in brain injury are generally irreparable because brain tissue cannot regenerate. Stem cell therapy has shown some promise in neural regeneration treatment. However, there remains a need for improved treatments for brain injury. [Means for solving the problem]

[0010]

[0010] The present invention provides mesenchymal stem cells (MSCs, also referred to herein as "HMCs") obtained by in vitro differentiation of pluripotent stem cells, and extracellular vesicles ("EVs") secreted from the HMCs of the present invention (HMC-EVs), and their use in methods for treating brain injury. Specifically, the inventors have discovered that the HMCs and HMC-EVs of the present invention are distinct from MSCs and EVs derived from other sources (e.g., adipose tissue-derived MSCs, bone marrow-derived MSCs, and / or umbilical cord blood-derived MSCs). Specifically, the HMCs of the present invention have a distinct expression profile when compared to other MSCs (e.g., adipose tissue-derived MSCs, bone marrow-derived MSCs, and / or umbilical cord blood-derived MSCs). Proteins / genes involved in neuroprotection and cell viability / survival pathways are upregulated in the HMCs of the present invention, suggesting that the HMCs of the present invention may confer neuroprotective effects and provide neurotrophic factors (i.e., factors involved in supporting neuronal survival, outgrowth, health, and / or recovery). Similarly, the HMC-EVs of the present invention share a similar profile to the HMCs from which they are secreted. Compared with MSCs and EVs derived from other tissues, similar signaling pathways enriched in HMCs are also enriched in HMC-EVs. This distinct profile makes HMCs and HMC-EVs particularly useful and effective in treating diseases, such as brain injury. Examples of brain injuries treatable using the HMCs and / or HMC-EVs of the present invention include stroke, traumatic brain injury, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, cerebral aneurysm, coma, optic neuropathy, and cerebral palsy.

[0011]

[0011] Therefore, in one aspect, the present invention provides a method for treating brain injury in a subject suffering from or suspected of suffering from brain injury, the method comprising the step of administering to the subject an effective amount of EVs (HMC-EVs) secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, thereby treating the subject's brain injury.

[0012] In some embodiments, the brain injury is selected from the group consisting of stroke, traumatic brain injury, optic neuropathy, cerebral palsy, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, cerebral aneurysm, and coma. In some embodiments, the brain injury is stroke. In some embodiments, the brain injury is optic neuropathy.

[0013] In some embodiments, the method includes increasing oligodendrocytes and progenitor cells in the brain after administering HMC-EVs to a subject. In some embodiments, the method includes preserving myelin in the brain after administering HMC-EVs to a subject. In some embodiments, the method includes preventing oxidative damage in neurons after administering HMC-EVs to a subject. In some embodiments, the method includes preventing neuronal death due to glutamate excitotoxicity injury after administering HMC-EVs to a subject. In some embodiments, the method includes reducing tissue loss in the brain after administering EVs to a subject. In some embodiments, the method includes reducing cell death in the brain after administering HMC-EVs to a subject. In some embodiments, the method includes stimulating pathways involved in the development of neuronal lineages after administering HMC-EVs to a subject.

[0014] In some embodiments, the HMC-EVs are administered systemically. In some embodiments, the HMC-EVs are administered intracerebrally. In some embodiments, the HMC-EVs are administered intrathecally. In some embodiments, the HMC-EVs are administered intracisternally. In some embodiments, the HMC-EVs are administered intraperitoneally.

[0015]

[0015] In some embodiments, the subject is a human.

[0016] In some embodiments, the HMCs are obtained by in vitro differentiation of human pluripotent stem cells. In some embodiments, the pluripotent stem cells are further differentiated into hemangioblasts. In some embodiments, the pluripotent stem cells are embryonic stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the induced pluripotent stem cells are generated by contacting cells with one or more reprogramming factors.

[0016]

[0017] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 9 at higher levels compared to EVs secreted from umbilical cord blood-derived MSCs (UCB-MSC-EVs).

[0017]

[0018] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 10 at a lower level compared to UCB-MSC-EVs.

[0019] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 11 at higher levels compared to EVs secreted from bone marrow-derived MSCs (BM-MSC-EVs).

[0018]

[0020] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 12 at a lower level compared to BM-MSC-EVs.

[0021] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 13 at higher levels compared to EVs secreted from adipose tissue-derived MSCs (AD-MSC-EVs).

[0019]

[0022] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 14 at a lower level compared to AD-MSC-EVs.

[0023] In some embodiments, HMC-EVs express at least one of the proteins in Table 15 at higher levels compared to UCB-MSC-EVs.

[0020]

[0024] In some embodiments, HMC-EVs express at least one of the proteins in Table 16 at a lower level compared to UCB-MSC-EVs.

[0025] In some embodiments, HMC-EVs express at least one of the proteins in Table 17 at higher levels compared to BM-MSC-EVs.

[0021]

[0026] In some embodiments, HMC-EVs express at least one of the proteins in Table 18 at a lower level compared to BM-MSC-EVs.

[0027] In some embodiments, HMC-EVs express at least one of the proteins in Table 19 at higher levels compared to AD-MSC-EVs.

[0022]

[0028] In some embodiments, HMC-EVs express at least one of the proteins in Table 20 at a lower level compared to AD-MSC-EVs.

[0029] In some embodiments, HMC-EVs express at least one miRNA selected from the group consisting of hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-199b-5p, hsa-miR-21-5p, hsa-miR-23a-3p, hsa-miR-125a-5p, hsa-miR-106a-5p+hsa-miR-17-5p, and hsa-miR-221-3p at higher levels compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0023]

[0030] In some embodiments, HMC-EVs have higher levels of ALDOC, ANXA5, APBB2, BASP1, CAV1, CD81, CD99, CKM, EPB41L3, FDPS, GNAQ, GNG12, GP9, H2AC20, H2AC21, H3-3A, H3-7, H4-16, HLA-A, ITGA2, KPNA2, KRAS, KRT4, LRRC5 compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs. 9, MAMDC2, MARCKSL1, MDGA1, MERTK, MFGE8, MMP14, MVP, PCDH1, PDGFRB, PDIA3, RPL13, RPS18, RPS3A, RPS4X, SDCBP, SLC2A1, SLC3A2, TAGLN2, TNC, TSPAN14, TSPAN33, TSPAN9, TTYH3, UCHL1, VAT1, YWHAB, and YWHAQ.

[0024]

[0031] In some embodiments, HMC-EVs have higher levels of ADGRG6, AGRN, ANXA6, APOC4, ARHGAP1, ARGHDIA, ARL8A, ARPC5, B2M, BBS1, BLVRA, BST1, CA2, CCN2, CCNB3, CD34, CD36, CD47, CORO1A, DTD1, EEF1D, EEF1G, ENG, ESD, GNAI2, GNB1, H1-3, H2BC15, HIP1, KIF11, LAMP1, LAP3, LGALS1, LTBP3, MAPK3, MARC, and / or IL-1. The cell expresses at least one protein selected from the group consisting of KS, MBTD1, MDH1, MOB1B, MYL12B, MYO1F, MYO3A, NIBAN2, PEBP1, PF4, PGAP1, PLOD1, PPP2R1A, PRSS23, PXDN, RALA, RAP2A, RPS13, RPS3, RPSA, S100A11, SLC44A1, SLC44A2, SLTM, SMG1, SPARC, SRSF8, STRADB, STX11, STXBP2, TGM2, TPP1, TPTE2, TRIM5, TRPM2, TUBA8, TUBB3, VCAN, YWHAE, and ZFN607.

[0025]

[0032] In some embodiments, HMC-EVs express at least one protein selected from the group consisting of ADIPOQ, CAT, CEP290, IGLV6-57, TAS2R33, and TMEM198 at lower levels compared to EVs secreted from BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0026]

[0033] In some embodiments, HMC-EVs express at least one protein selected from the group consisting of AKAP9, ALB, ALOX5, APLP2, CD109, CDSN, CHST9, ERC1, F11, ARMCX5, LAMB4, LRRTM2, LTF, MSH6, OAF, OLFML3, PAK6, RGS14, SEMA7A, SURF1, and TRIM4 at lower levels compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0027]

[0034] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 21 at higher levels compared to HMCs.

[0035] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 22 at a lower level compared to HMCs.

[0028]

[0036] In some embodiments, about 1×10 6 ~Approx. 1×10 13 HMC-EVs are administered to a subject. In some embodiments, about 10 x 10 10 pieces or approximately 30 x 10 10 HMC-EVs are administered to a subject.

[0029]

[0037] In some embodiments, the HMC-EVs are administered in a pharmaceutical composition.

[0038] In some embodiments, the pharmaceutical composition comprises (a) a buffer that maintains the solution at physiological pH, (b) at least 2 mM or at least 0.05% (w / v) glucose, and (c) an osmotically active agent that maintains the solution at physiological osmolality.

[0030]

[0039] In some embodiments, the glucose is D-glucose (dextrose). In some embodiments, the osmotically active agent is a salt. In some embodiments, the osmotically active agent is a magnesium salt, a phosphate salt, a sulfate salt, a chloride salt, a poorly absorbed disaccharide such as lactulose, a sugar alcohol such as mannitol or sorbitol, and polyethylene glycol, or a combination thereof. In some embodiments, the osmotically active agent is CaCl2, KCl, NaCl, KH2PO4, Na3HPO4, MgCl2, MgSO4, HEPES, NaHCO3, or a combination thereof. In some embodiments, the salt is sodium chloride.

[0031]

[0040] In some embodiments, the method further comprises administering to the subject an effective amount of HMCs obtained by in vitro differentiation of pluripotent stem cells.

[0041] In one aspect, the present invention provides a method for treating brain injury in a subject suffering from or suspected of suffering from brain injury, the method comprising administering to the subject an effective amount of HMCs obtained by in vitro differentiation of pluripotent stem cells, thereby treating the brain injury in the subject.

[0032]

[0042] In some embodiments, the brain injury is selected from the group consisting of stroke, traumatic brain injury, cerebral palsy, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, cerebral aneurysm, optic neuropathy, and coma.

[0033]

[0043] In some embodiments, the brain injury is a stroke.

[0044] In some embodiments, the brain injury is optic neuropathy.

[0045] In some embodiments, the method includes preserving myelin in the brain after administering HMC to a subject. In some embodiments, the method includes suppressing a neuroinflammatory response after administering HMC to a subject. In some embodiments, the method includes reducing microglial activation and astrocytic activation in the brain after administering HMC to a subject. In some embodiments, the method includes stimulating a pathway involved in cell survival after administering HMC to a subject. In some embodiments, the method includes stimulating expression of a neuroprotective gene in the brain after administering HMC to a subject. In some embodiments, the neuroprotective gene is selected from the group consisting of heat shock protein family B member 1 (HSPB1), insulin-like growth factor 1 (IGF2), and secreted phosphoprotein 1 (SPP1). In some embodiments, the method includes stimulating a pathway involved in synaptic transmission in the brain after administering HMC to a subject. In some embodiments, the method includes stimulating a pathway involved in neuronal lineage development after administering HMC to a subject. In some embodiments, the method includes reducing apoptosis after administering HMC to a subject.

[0034]

[0046] In some embodiments, the brain injury is a traumatic brain injury.

[0047] In some embodiments, the method includes reducing tissue loss in the brain after administering HMC to a subject. In some embodiments, the method includes reducing cell death in the brain after administering HMC to a subject. In some embodiments, the method includes increasing neurogenesis after administering HMC to a subject. In some embodiments, the method includes reducing the abundance of microglia and macrophages in the cortex and striatum after administering HMC to a subject. In some embodiments, the method includes reducing inflammation in the spleen after administering HMC to a subject. In some embodiments, the method includes migration of HMC across the blood-brain barrier to the cortex, striatum, and / or hippocampus.

[0035]

[0048] In some embodiments, the brain injury is cerebral palsy.

[0049] In some embodiments, the method includes reducing apoptosis in the brain after administering HMC to a subject. In some embodiments, the method includes reducing lesion size in the brain after administering HMC to a subject. In some embodiments, the method includes reducing microglial activation and astrocytic activation in the brain after administering HMC to a subject. In some embodiments, the method includes preserving myelin in the corpus callosum after administering HMC to a subject. In some embodiments, the method includes at least partial rescue of Olig2 in the brain after administering HMC to a subject.

[0036]

[0050] In some embodiments, the HMC is administered systemically. In some embodiments, the HMC is administered intracerebrally. In some embodiments, the HMC is administered intrathecally. In some embodiments, the HMC is administered intracisternally. In some embodiments, the HMC is administered intraperitoneally. In some embodiments, the mesenchymal stem cells are human cells.

[0037]

[0051] In some embodiments, the subject is a human.

[0052] In some embodiments, the pluripotent stem cells are further differentiated into hemangioblasts. In some embodiments, the pluripotent stem cells are embryonic stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are human pluripotent stem cells.

[0038]

[0053] In some embodiments, the HMCs have been passaged in vitro no more than five times prior to administration to a subject.

[0054] In some embodiments, HMCs express at least one of the genes in Table 3 at a higher level compared to bone marrow-derived MSCs (BM-MSCs).

[0039]

[0055] In some embodiments, HMCs express at least one of the genes in Table 4 at a lower level compared to BM-MSCs.

[0056] In some embodiments, HMCs express at least one of the genes in Table 5 at a higher level compared to umbilical cord blood-derived MSCs (UCB-MSCs).

[0040]

[0057] In some embodiments, HMCs express at least one of the genes in Table 6 at a lower level compared to UCB-MSCs.

[0058] In some embodiments, HMCs express at least one of the genes in Table 7 at higher levels compared to adipose tissue-derived MSCs (AD-MSCs).

[0041]

[0059] In some embodiments, HMCs express at least one of the genes in Table 8 at a lower level compared to AD-MSCs.

[0060] In some embodiments, HMCs basally express mRNA encoding interleukin-6 (IL-6) at a level that is less than 10 percent of the IL-6 mRNA level expressed by BM-MSCs basally, and HMCs basally express mRNA encoding CD24 at a level that is greater than the CD24 mRNA level expressed by BM-MSCs basally.

[0042]

[0061] In some embodiments, HMCs express at least one gene selected from the group consisting of CALR, UBB, PKM, CXCL8, C15orf48, PSME2, TPM3, ANKRD1, PFN1, SRGN, ACTB, MDK, TAGLN2, CFL1, HSP90AA1, HSPA8, CXCL12, UCHL1, HMGA2, HMGA1, HN1, PTMA, SP90AB1, PRDX1, GSTP1, KRT18, IGFBP4, CALD1, COL4A1, COL4A2, and GAPDH at higher levels compared to adipose tissue-derived MSCs (AD-MSCs).

[0043]

[0062] In some embodiments, HMCs express at least one gene selected from the group consisting of TMSB4X, ACTG1, GSTP1, KRT18, IGFBP5, NPY, KRT8, PRDX6, MDK, DKK3, UCHL1, TUBB3, HN1, PTMA, HSP90AB1, HMGA1, HSPA8, TAGLN2, ANKRD1, PFN1, CYBA, and UBB at higher levels compared to AD-MSCs.

[0044]

[0063] In some embodiments, HMCs express lower levels of SERPINE1, ACTA2, TPM2, CTGF, SERPINE2, CRYAB, ELN, MFGE8, ANXA2, POSTN, VIM, MFAP5, ISLR, THBS1, TIMP3, DKK1, COL6A3, COL6A1, TPT1, BCYRN1, COL1A1, SPARC, TPM1, BGN, COL1A2, COL3A1, TGFBI, CRLF1, C The cells express at least one gene selected from the group consisting of OMP, NEAT1, MT-CO3, MT-CO2, MT-ATP8, MT-CYB, MT-CO1, MT-ATP6, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-ND3, MT-ND1, MT-ND2, GREM1, TMSB4X, ITGB1, LMNA, H2AFZ, FTL, EEF1G, NPM1, EEF1A1, RACK1, ACTG1, and TPM4.

[0045]

[0064] In some embodiments, HMCs express at least one gene selected from the group consisting of SERPINE1, S100A6, CD59, POSTN, VIM, MFAP5, ISLR, THBS1, COL6A3, TIMP3, ELN, ANXA2, COL1A1, BCYRN1, CCDC80, COL6A1, COL6A2, BGN, COL1A2, COL3A1, TGFB1, CRLF1, COMP, and GREM1 at lower levels compared to AD-MSCs.

[0046]

[0065] In some embodiments, HMCs express at least one gene selected from the group consisting of MT1X, MT1G, TMSB10, CCL8, INHBA, CTSB, SERPINB2, ADM, APOL1, FTH1, CCL2, CCL5, CSF1, IL1B, IGFBP3, P4HB, DCN, FSTL1, ANXA5, LOX, CD63, CTSZ, FN1, LGALS1, LDHA, RCN3, MMP2, and TIMP1 at lower levels compared to AD-MSCs.

[0047]

[0066] In some embodiments, HMCs express at least one gene selected from the group consisting of PPIA, NPM1, HNRNPA1, IGFBP5, KRT19, KRT18, GSTP1, TUBB, TUBA1B, KRT8, HN1, PTMA, TUBA1C, HSPA8, HMGA1, CFL1, MYL6, ACTB, UCHL1, TAGLN2, MDK, GREM1, MMP1, and CTSC at higher levels compared to bone marrow-derived MSCs (BM-MSCs).

[0048]

[0067] In some embodiments, HMCs express at least one gene selected from the group consisting of ANXA2, TPT1, VIM, COL6A1, BGN, COL6A2, CTGF, TIMP3, ACTA2, COL3A1, SPARC, ITGB1, SERPINH1, TPM2, TGFBI, COL1A1, TPM1, COL6A3, TPM4, SERPINE2, CALD1, COL1A2, TAGLN, MYL9, MT-RNR2, POSTN at lower levels compared to BM-MSCs.

[0049]

[0068] In some embodiments, HMCs express at least one of the miRNAs in Table 21 at a lower level compared to HMC-EVs secreted from HMCs.

[0069] In some embodiments, HMCs express at least one of the miRNAs in Table 22 at higher levels compared to HMC-EVs secreted from HMCs.

[0050]

[0070] In some embodiments, about 1×10 6 ~Approx. 1×10 13 HMC are administered to the subject.

[0071] In some embodiments, the HMC is administered in a pharmaceutical composition.

[0051]

[0072] In some embodiments, the pharmaceutical composition comprises (a) a buffer that maintains the solution at physiological pH, (b) at least 2 mM or at least 0.05% (w / v) glucose, and (c) an osmotically active agent that maintains the solution at physiological osmolality.

[0052]

[0073] In some embodiments, the glucose is D-glucose (dextrose). In some embodiments, the osmotically active agent is a salt. In some embodiments, the salt is sodium chloride.

[0053]

[0074] In another aspect, the present invention provides a method of treating brain injury in a subject suffering from or suspected of suffering from brain injury, comprising administering to the subject an effective amount of EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, and an effective amount of HMCs obtained by in vitro differentiation of pluripotent stem cells, thereby treating the brain injury in the subject.

[0054]

[0075] In one aspect, the invention provides a composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of CALR, UBB, PKM, CXCL8, C15orf48, PSME2, TPM3, ANKRD1, PFN1, SRGN, ACTB, MDK, TAGLN2, CFL1, HSP90AA1, HSPA8, CXCL12, UCHL1, HMGA2, HMGA1, HN1, PTMA, SP90AB1, PRDX1, GSTP1, KRT18, IGFBP4, CALD1, COL4A1, COL4A2, and GAPDH at a higher level compared to AD-MSCs.

[0055]

[0076] In one aspect, the present invention provides a composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of TMSB4X, ACTG1, GSTP1, KRT18, IGFBP5, NPY, KRT8, PRDX6, MDK, DKK3, UCHL1, TUBB3, HN1, PTMA, HSP90AB1, HMGA1, HSPA8, TAGLN2, ANKRD1, PFN1, CYBA, and UBB at a higher level compared to AD-MSCs.

[0056]

[0077] In one aspect, the invention provides a composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of PPIA, NPM1, HNRNPA1, IGFBP5, KRT19, KRT18, GSTP1, TUBB, TUBA1B, KRT8, HN1, PTMA, TUBA1C, HSPA8, HMGA1, CFL1, MYL6, ACTB, UCHL1, TAGLN2, MDK, GREM1, MMP1, and CTSC at a higher level compared to BM-MSCs.

[0057]

[0078] In one embodiment, the present invention provides a composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at lower levels, as compared to AD-MSCs, SERPINE1, ACTA2, TPM2, CTGF, SERPINE2, CRYAB, ELN, MFGE8, ANXA2, POSTN, VIM, MFAP5, ISLR, THBS1, TIMP3, DKK1, COL6A3, COL6A1, TPT1, BCYRN1, COL1A1, SPARC, TPM1, BGN, COL1A2, COL3A1, TGFBI, C Compositions are provided that express at least one gene selected from the group consisting of RLF1, COMP, NEAT1, MT-CO3, MT-CO2, MT-ATP8, MT-CYB, MT-CO1, MT-ATP6, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-ND3, MT-ND1, MT-ND2, GREM1, TMSB4X, ITGB1, LMNA, H2AFZ, FTL, EEF1G, NPM1, EEF1A1, RACK1, ACTG1, and TPM4.

[0058]

[0079] In one aspect, the invention provides a composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of SERPINE1, S100A6, CD59, POSTN, VIM, MFAP5, ISLR, THBS1, COL6A3, TIMP3, ELN, ANXA2, COL1A1, BCYRN1, CCDC80, COL6A1, COL6A2, BGN, COL1A2, COL3A1, TGFB1, CRLF1, COMP, and GREM1 at a lower level compared to AD-MSCs.

[0059]

[0080] In one aspect, the invention provides a composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of MT1X, MT1G, TMSB10, CCL8, INHBA, CTSB, SERPINB2, ADM, APOL1, FTH1, CCL2, CCL5, CSF1, IL1B, IGFBP3, P4HB, DCN, FSTL1, ANXA5, LOX, CD63, CTSZ, FN1, LGALS1, LDHA, RCN3, MMP2, and TIMP1 at a lower level compared to AD-MSCs.

[0060]

[0081] In one aspect, the invention provides a composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of ANXA2, TPT1, VIM, COL6A1, BGN, COL6A2, CTGF, TIMP3, ACTA2, COL3A1, SPARC, ITGB1, SERPINH1, TPM2, TGFBI, COL1A1, TPM1, COL6A3, TPM4, SERPINE2, CALD1, COL1A2, TAGLN, MYL9, MT-RNR2, POSTN at a lower level compared to BM-MSCs.

[0061]

[0082] In some embodiments, HMCs further express at least one of the genes in Table 3 at a higher level compared to BM-MSCs.

[0083] In some embodiments, HMCs further express at least one of the genes in Table 4 at a lower level compared to BM-MSCs.

[0062]

[0084] In some embodiments, HMCs further express at least one of the genes in Table 5 at a higher level compared to UCB-MSCs.

[0085] In some embodiments, HMCs further express at least one of the genes in Table 6 at a lower level compared to UCB-MSCs.

[0063]

[0086] In some embodiments, HMCs further express at least one of the genes in Table 7 at a higher level compared to AD-MSCs.

[0087] In some embodiments, HMCs further express at least one of the genes in Table 8 at a lower level compared to AD-MSCs.

[0064]

[0088] In one aspect, the present invention provides a pharmaceutical composition comprising an HMC of the present invention and a pharmaceutically acceptable carrier.

[0089] In one aspect, the present invention provides a population of HMC-EVs of the present invention.

[0065]

[0090] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 9 at higher levels compared to UCB-MSC-EVs.

[0091] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 10 at a lower level compared to UCB-MSC-EVs.

[0066]

[0092] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 11 at higher levels compared to BM-MSC-EVs.

[0093] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 12 at a lower level compared to BM-MSC-EVs.

[0067]

[0094] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 13 at higher levels compared to AD-MSC-EVs.

[0095] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 14 at a lower level compared to AD-MSC-EVs.

[0068]

[0096] In some embodiments, HMC-EVs express at least one of the proteins in Table 15 at higher levels compared to UCB-MSC-EVs.

[0097] In some embodiments, HMC-EVs express at least one of the proteins in Table 16 at a lower level compared to UCB-MSC-EVs.

[0069]

[0098] In some embodiments, HMC-EVs express at least one of the proteins in Table 17 at higher levels compared to BM-MSC-EVs.

[0099] In some embodiments, HMC-EVs express at least one of the proteins in Table 18 at a lower level compared to BM-MSC-EVs.

[0070]

[0100] In some embodiments, HMC-EVs express at least one of the proteins in Table 19 at higher levels compared to AD-MSC-EVs.

[0101] In some embodiments, HMC-EVs express at least one of the proteins in Table 20 at a lower level compared to AD-MSC-EVs.

[0071]

[0102] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 21 at higher levels compared to HMCs.

[0103] In some embodiments, HMC-EVs express at least one of the miRNAs in Table 22 at a lower level compared to HMCs.

[0072]

[0104] In some embodiments, HMC-EVs express at least one miRNA selected from the group consisting of hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-199b-5p, hsa-miR-21-5p, hsa-miR-23a-3p, hsa-miR-125a-5p, hsa-miR-106a-5p+hsa-miR-17-5p, and hsa-miR-221-3p at higher levels compared to EVs secreted from BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0073]

[0105] In some embodiments, HMC-EVs have higher levels of ALDOC, ANXA5, APBB2, BASP1, CAV1, CD81, CD99, CKM, EPB41L3, FDPS, GNAQ, GNG12, GP9, H2AC20, H2AC21, H3-3A, H3-7, H4-16, HLA-A, ITGA2, KPNA2, KRAS, KRT4, LRRC5 compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs. 9, MAMDC2, MARCKSL1, MDGA1, MERTK, MFGE8, MMP14, MVP, PCDH1, PDGFRB, PDIA3, RPL13, RPS18, RPS3A, RPS4X, SDCBP, SLC2A1, SLC3A2, TAGLN2, TNC, TSPAN14, TSPAN33, TSPAN9, TTYH3, UCHL1, VAT1, YWHAB, and YWHAQ.

[0074]

[0106] In some embodiments, HMC-EVs have higher levels of ADGRG6, AGRN, ANXA6, APOC4, ARHGAP1, ARGHDIA, ARL8A, ARPC5, B2M, BBS1, BLVRA, BST1, CA2, CCN2, CCNB3, CD34, CD36, CD47, CORO1A, DTD1, EEF1D, EEF1G, ENG, ESD, GNAI2, GNB1, H1-3, H2BC15, HIP1, KIF11, LAMP1, LAP3, LGALS1, LTBP3, MAPK3, MARC, and / or IL-1. The cell expresses at least one protein selected from the group consisting of KS, MBTD1, MDH1, MOB1B, MYL12B, MYO1F, MYO3A, NIBAN2, PEBP1, PF4, PGAP1, PLOD1, PPP2R1A, PRSS23, PXDN, RALA, RAP2A, RPS13, RPS3, RPSA, S100A11, SLC44A1, SLC44A2, SLTM, SMG1, SPARC, SRSF8, STRADB, STX11, STXBP2, TGM2, TPP1, TPTE2, TRIM5, TRPM2, TUBA8, TUBB3, VCAN, YWHAE, and ZFN607.

[0075]

[0107] In some embodiments, HMC-EVs express at least one protein selected from the group consisting of ADIPOQ, CAT, CEP290, IGLV6-57, TAS2R33, and TMEM198 at a lower level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0076]

[0108] In some embodiments, HMC-EVs express at least one protein selected from the group consisting of AKAP9, ALB, ALOX5, APLP2, CD109, CDSN, CHST9, ERC1, F11, ARMCX5, LAMB4, LRRTM2, LTF, MSH6, OAF, OLFML3, PAK6, RGS14, SEMA7A, SURF1, and TRIM4 at lower levels compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0077]

[0109] In one aspect, the present invention provides a pharmaceutical composition comprising the HMC-EV of the present invention and a pharmaceutically acceptable carrier.

[0110] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one miRNA in Table 9 at a higher level compared to UCB-MSC-EVs.

[0078]

[0111] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one miRNA in Table 10 at a lower level compared to UCB-MSC-EVs.

[0079]

[0112] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 11 at higher levels compared to BM-MSC-EVs.

[0080]

[0113] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 12 at a lower level compared to BM-MSC-EVs.

[0081]

[0114] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 13 at higher levels compared to AD-MSC-EVs.

[0082]

[0115] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 14 at a lower level compared to EVs secreted from AD-MSC-EVs.

[0083]

[0116] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 15 at a higher level compared to UCB-MSC-EVs.

[0084]

[0117] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 16 at a lower level compared to UCB-MSC-EVs.

[0085]

[0118] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 17 at higher levels compared to BM-MSC-EVs.

[0086]

[0119] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 18 at a lower level compared to BM-MSC-EVs.

[0087]

[0120] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 19 at a higher level compared to AD-MSC-EVs.

[0088]

[0121] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 20 at a lower level compared to AD-MSC-EVs.

[0089]

[0122] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 21 at a higher level compared to HMCs.

[0090]

[0123] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 22 at a lower level compared to HMCs.

[0091]

[0124] In one embodiment, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one miRNA selected from the group consisting of hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-199b-5p, hsa-miR-21-5p, hsa-miR-23a-3p, hsa-miR-125a-5p, hsa-miR-106a-5p+hsa-miR-17-5p, and hsa-miR-221-3p at higher levels compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0092]

[0125] In one embodiment, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express higher levels of ALDOC, ANXA5, APBB2, BASP1, CAV1, CD81, CD99, CKM, EPB41L3, FDPS, GNAQ, GNG12, GP9, H2AC20, H2AC21, H3-3A, H3-7, H4-16, HLA-A, ITGA2, KPNA2, KRAS, KR, and / or BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs. A population of HMC-EVs is provided, which express at least one protein selected from the group consisting of T4, LRRC59, MAMDC2, MARCKSL1, MDGA1, MERTK, MFGE8, MMP14, MVP, PCDH1, PDGFRB, PDIA3, RPL13, RPS18, RPS3A, RPS4X, SDCBP, SLC2A1, SLC3A2, TAGLN2, TNC, TSPAN14, TSPAN33, TSPAN9, TTYH3, UCHL1, VAT1, YWHAB, and YWHAQ.

[0093]

[0126] In one embodiment, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at higher levels, as compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs, ADGRG6, AGRN, ANXA6, APOC4, ARHGAP1, ARGHDIA, ARL8A, ARPC5, B2M, BBS1, BLVRA, BST1, CA2, CCN2, CCNB3, CD34, CD36, CD47, CORO1A, DTD1, EEF1D, EEF1G, ENG, ESD, GNAI2, GNB1, H1-3, H2BC15, HIP1, KIF11, LAMP1, LAP3, LGALS1, LTBP3, MA A population of HMC-EVs is provided, which express at least one protein selected from the group consisting of PK3, MARCKS, MBTD1, MDH1, MOB1B, MYL12B, MYO1F, MYO3A, NIBAN2, PEBP1, PF4, PGAP1, PLOD1, PPP2R1A, PRSS23, PXDN, RALA, RAP2A, RPS13, RPS3, RPSA, S100A11, SLC44A1, SLC44A2, SLTM, SMG1, SPARC, SRSF8, STRADB, STX11, STXBP2, TGM2, TPP1, TPTE2, TRIM5, TRPM2, TUBA8, TUBB3, VCAN, YWHAE, and ZFN607.

[0094]

[0127] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one protein selected from the group consisting of ADIPOQ, CAT, CEP290, IGLV6-57, TAS2R33, and TMEM198 at a lower level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0095]

[0128] In one aspect, the present invention provides a population of HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one protein selected from the group consisting of AKAP9, ALB, ALOX5, APLP2, CD109, CDSN, CHST9, ERC1, F11, ARMCX5, LAMB4, LRRTM2, LTF, MSH6, OAF, OLFML3, PAK6, RGS14, SEMA7A, SURF1, and TRIM4 at lower levels compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0096]

[0129] In one aspect, the present invention provides a pharmaceutical composition comprising the HMC-EV of the present invention and a pharmaceutically acceptable carrier.

[0130] The present invention also provides a method for determining neurite outgrowth of HMC and / or HMC-EV populations. The method includes the steps of (a) preparing a mixed neuronal culture from isolated cerebral cortex, (b) seeding the HMC and / or HMC-EV populations onto a permeable membrane, (c) applying strain to the mixed neuronal culture, (d) overlaying the strained mixed neuronal culture with the permeable membrane of step (b), and (e) measuring neurite outgrowth of the mixed neuronal culture. In one embodiment, step (d) is performed in a medium substantially lacking serum. In another embodiment, the method further includes determining gene expression of the mixed neuronal culture in the presence and absence of HMC and / or HMC-EV populations. In another embodiment, the strain is a physical scratch applied to the mixed neuronal culture. In another embodiment, the strain is a vacuum pressure and positive air pressure applied to the mixed neuronal culture. In another embodiment, the strain is applied as a stretching vibration between 15% and 0%.

[0097]

[0131] The present invention also provides a method for determining neurite outgrowth of HMC and / or HMC-EV populations. The method includes the steps of: (a) preparing a mixed neuronal culture from isolated cerebral cortex; (b) seeding the HMC and / or HMC-EV populations onto a permeable membrane; (c) applying strain to the mixed neuronal culture; (d) overlaying the strained mixed neuronal culture with the permeable membrane of step (b); and (e) measuring neurite outgrowth of the mixed neuronal culture. In one embodiment, the method further includes determining gene expression in the mixed neuronal culture in the presence and absence of HMC and / or HMC-EV populations. In another embodiment, the strain is a physical scratch applied to the mixed neuronal culture. In another embodiment, the strain is a vacuum pressure and positive air pressure applied to the mixed neuronal culture. In another embodiment, the strain is applied as a stretching oscillation between 15% and 0%. [Brief explanation of the drawings]

[0098] [Figure 1]

[0132] Figure 1 shows the results of the elevating body swing test (EBST) in rats in which TBI was induced by controlled cortical impact (CCI) and HMC or vehicle was administered intracerebrally (IC) or intravenously (IV). [Figure 2]

[0133] FIG. 2 shows forelimb immobility in rats in which TBI was induced by controlled cortical impact (CCI) and HMC or vehicle was administered intracerebrally (IC) or intravenously (IV). [Figure 3]

[0134] FIG. 3 shows paw grasping in rats in which TBI was induced by controlled cortical impact (CCI) and HMC or vehicle was administered intracerebrally (IC) or intravenously (IV). [Figure 4]

[0135] Figure 4A shows H&E staining of the brains of rats that underwent controlled cortical impact (CCI) TBI and were administered HMC or vehicle intracerebrally (IC) or intravenously (IV). Figure 4B shows a bar graph of the TBI impact area in rats measured by H&E staining. [Figure 5A]

[0136] Figure 5A shows Nissl staining of the peri-impact cortex of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5B shows a bar graph of the percentage of viable cells in the peri-impact cortex of rats, as determined by Nissl staining. Figure 5C shows Nissl staining of the striatum of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5D shows a bar graph of the percentage of viable cells in the striatum of rats, as determined by Nissl staining. Figure 5E shows Nissl staining of the hippocampus of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5F shows a bar graph of the percentage of viable cells in the hippocampus of rats, as determined by Nissl staining. [Figure 5B] Figure 5A shows Nissl staining of the peri-impact cortex of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5B shows a bar graph of the percentage of viable cells in the peri-impact cortex of rats, as determined by Nissl staining. Figure 5C shows Nissl staining of the striatum of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5D shows a bar graph of the percentage of viable cells in the striatum of rats, as determined by Nissl staining. Figure 5E shows Nissl staining of the hippocampus of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5F shows a bar graph of the percentage of viable cells in the hippocampus of rats, as determined by Nissl staining. [Figure 5C] Figure 5A shows Nissl staining of the peri-impact cortex of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5B shows a bar graph of the percentage of viable cells in the peri-impact cortex of rats, as determined by Nissl staining. Figure 5C shows Nissl staining of the striatum of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5D shows a bar graph of the percentage of viable cells in the striatum of rats, as determined by Nissl staining. Figure 5E shows Nissl staining of the hippocampus of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5F shows a bar graph of the percentage of viable cells in the hippocampus of rats, as determined by Nissl staining. [Figure 5D] Figure 5A shows Nissl staining of the peri-impact cortex of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5B shows a bar graph of the percentage of viable cells in the peri-impact cortex of rats, as determined by Nissl staining. Figure 5C shows Nissl staining of the striatum of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5D shows a bar graph of the percentage of viable cells in the striatum of rats, as determined by Nissl staining. Figure 5E shows Nissl staining of the hippocampus of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5F shows a bar graph of the percentage of viable cells in the hippocampus of rats, as determined by Nissl staining. [Figure 5E]Figure 5A shows Nissl staining of the peri-impact cortex of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5B shows a bar graph of the percentage of viable cells in the peri-impact cortex of rats, as determined by Nissl staining. Figure 5C shows Nissl staining of the striatum of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5D shows a bar graph of the percentage of viable cells in the striatum of rats, as determined by Nissl staining. Figure 5E shows Nissl staining of the hippocampus of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5F shows a bar graph of the percentage of viable cells in the hippocampus of rats, as determined by Nissl staining. [Figure 5F] Figure 5A shows Nissl staining of the peri-impact cortex of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5B shows a bar graph of the percentage of viable cells in the peri-impact cortex of rats, as determined by Nissl staining. Figure 5C shows Nissl staining of the striatum of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5D shows a bar graph of the percentage of viable cells in the striatum of rats, as determined by Nissl staining. Figure 5E shows Nissl staining of the hippocampus of rats that underwent TBI via controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5F shows a bar graph of the percentage of viable cells in the hippocampus of rats, as determined by Nissl staining. [Figure 6A]

[0137] Figure 6A shows doublecortin (DCX) staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6B shows a bar graph of the number of DCX cells in the cortical region of rats. Figure 6C shows DCX staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6D shows a bar graph of the number of DCX cells in the striatum of rats. Figure 6E shows DCX staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6F shows a bar graph of the number of DCX cells in the hippocampus of rats. [Figure 6B] Figure 6A shows doublecortin (DCX) staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6B shows a bar graph of the number of DCX cells in the cortical region of rats. Figure 6C shows DCX staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6D shows a bar graph of the number of DCX cells in the striatum of rats. Figure 6E shows DCX staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6F shows a bar graph of the number of DCX cells in the hippocampus of rats. [Figure 6C]Figure 6A shows doublecortin (DCX) staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6B shows a bar graph of the number of DCX cells in the cortical region of rats. Figure 6C shows DCX staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6D shows a bar graph of the number of DCX cells in the striatum of rats. Figure 6E shows DCX staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6F shows a bar graph of the number of DCX cells in the hippocampus of rats. [Figure 6D] Figure 6A shows doublecortin (DCX) staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6B shows a bar graph of the number of DCX cells in the cortical region of rats. Figure 6C shows DCX staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6D shows a bar graph of the number of DCX cells in the striatum of rats. Figure 6E shows DCX staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6F shows a bar graph of the number of DCX cells in the hippocampus of rats. [Figure 6E]Figure 6A shows doublecortin (DCX) staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6B shows a bar graph of the number of DCX cells in the cortical region of rats. Figure 6C shows DCX staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6D shows a bar graph of the number of DCX cells in the striatum of rats. Figure 6E shows DCX staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6F shows a bar graph of the number of DCX cells in the hippocampus of rats. [Figure 6F] Figure 6A shows doublecortin (DCX) staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6B shows a bar graph of the number of DCX cells in the cortical region of rats. Figure 6C shows DCX staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6D shows a bar graph of the number of DCX cells in the striatum of rats. Figure 6E shows DCX staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 6F shows a bar graph of the number of DCX cells in the hippocampus of rats. [Figure 7A]

[0138] Figure 7A shows Iba1 staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 7B shows a bar graph of Iba1 cell counts in the cortex of rats. Figure 7C shows Iba1 staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 7D shows a bar graph of Iba1 cell counts in the striatum of rats. [Figure 7B] Figure 7A shows Iba1 staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 7B shows a bar graph of Iba1 cell counts in the cortex of rats. Figure 7C shows Iba1 staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 7D shows a bar graph of Iba1 cell counts in the striatum of rats. [Figure 7C] Figure 7A shows Iba1 staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 7B shows a bar graph of Iba1 cell counts in the cortex of rats. Figure 7C shows Iba1 staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 7D shows a bar graph of Iba1 cell counts in the striatum of rats. [Figure 7D] Figure 7A shows Iba1 staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 7B shows a bar graph of Iba1 cell counts in the cortex of rats. Figure 7C shows Iba1 staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 7D shows a bar graph of Iba1 cell counts in the striatum of rats. [Figure 8A]

[0139] Figure 8A shows OX6 staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and were administered intracerebrally (IC) or intravenously (IV) with HMC or vehicle. Figure 8B shows a bar graph of the number of OX6 cells in the cortex of rats. Figure 8C shows OX6 staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and were administered intracerebrally (IC) or intravenously (IV) with HMC or vehicle. Figure 8D shows a bar graph of the number of OX6 cells in the striatum of rats. [Figure 8B] Figure 8A shows OX6 staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and were administered intracerebrally (IC) or intravenously (IV) with HMC or vehicle. Figure 8B shows a bar graph of the number of OX6 cells in the cortex of rats. Figure 8C shows OX6 staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and were administered intracerebrally (IC) or intravenously (IV) with HMC or vehicle. Figure 8D shows a bar graph of the number of OX6 cells in the striatum of rats. [Figure 8C] Figure 8A shows OX6 staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and were administered intracerebrally (IC) or intravenously (IV) with HMC or vehicle. Figure 8B shows a bar graph of the number of OX6 cells in the cortex of rats. Figure 8C shows OX6 staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and were administered intracerebrally (IC) or intravenously (IV) with HMC or vehicle. Figure 8D shows a bar graph of the number of OX6 cells in the striatum of rats. [Figure 8D] Figure 8A shows OX6 staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and were administered intracerebrally (IC) or intravenously (IV) with HMC or vehicle. Figure 8B shows a bar graph of the number of OX6 cells in the cortex of rats. Figure 8C shows OX6 staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and were administered intracerebrally (IC) or intravenously (IV) with HMC or vehicle. Figure 8D shows a bar graph of the number of OX6 cells in the striatum of rats. [Figure 9]

[0140] Figure 9A shows IL6 staining in the spleens of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 9B shows a bar graph of IL6 staining intensity in the spleens of rats. [Figure 10]

[0141] Figure 10A shows TNF-alpha staining in the spleens of rats that underwent controlled cortical impact (CCI) TBI and were administered HMC or vehicle intracerebrally (IC) or intravenously (IV). Figure 10B shows a bar graph of TNF-alpha staining intensity in the spleens of rats. [Figure 11A]

[0142] Figure 11A shows HuNu staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11B shows a bar graph of HuNu cell counts in the cortex of rats. Figure 11C shows HuNu staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11D shows a bar graph of HuNu cell counts in the striatum of rats. Figure 11E shows HuNu staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11F shows a bar graph of HuNu cell counts in the hippocampus of rats. [Figure 11B]Figure 11A shows HuNu staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11B shows a bar graph of HuNu cell counts in the cortex of rats. Figure 11C shows HuNu staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11D shows a bar graph of HuNu cell counts in the striatum of rats. Figure 11E shows HuNu staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11F shows a bar graph of HuNu cell counts in the hippocampus of rats. [Figure 11C] Figure 11A shows HuNu staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11B shows a bar graph of HuNu cell counts in the cortex of rats. Figure 11C shows HuNu staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11D shows a bar graph of HuNu cell counts in the striatum of rats. Figure 11E shows HuNu staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11F shows a bar graph of HuNu cell counts in the hippocampus of rats. [Figure 11D]Figure 11A shows HuNu staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11B shows a bar graph of HuNu cell counts in the cortex of rats. Figure 11C shows HuNu staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11D shows a bar graph of HuNu cell counts in the striatum of rats. Figure 11E shows HuNu staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11F shows a bar graph of HuNu cell counts in the hippocampus of rats. [Figure 11E] Figure 11A shows HuNu staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11B shows a bar graph of HuNu cell counts in the cortex of rats. Figure 11C shows HuNu staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11D shows a bar graph of HuNu cell counts in the striatum of rats. Figure 11E shows HuNu staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11F shows a bar graph of HuNu cell counts in the hippocampus of rats. [Figure 11F]Figure 11A shows HuNu staining in the cortex of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11B shows a bar graph of HuNu cell counts in the cortex of rats. Figure 11C shows HuNu staining in the striatum of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11D shows a bar graph of HuNu cell counts in the striatum of rats. Figure 11E shows HuNu staining in the hippocampus of rats that underwent controlled cortical impact (CCI) TBI and received intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 11F shows a bar graph of HuNu cell counts in the hippocampus of rats. [Figure 12A]

[0143] Figure 12A shows the migration of unstimulated hESC-MSCs ("HMCs"), BM-MSCs, and UCB-MSCs into approximately 500 μm wide gaps at 0 and 6 hours. Figure 12B shows a bar graph of the number of unstimulated and stimulated cells that migrated into the gap. [Figure 12B] Figure 12A shows the migration of unstimulated hESC-MSCs ("HMCs"), BM-MSCs, and UCB-MSCs into approximately 500 μm wide gaps at 0 and 6 hours. Figure 12B shows a bar graph of the number of unstimulated and stimulated cells that migrated into the gap. [Figure 13]

[0144] FIG. 13 shows images of neurite outgrowth staining at days 1 and 7 after scratch and co-culture of hESC-MSCs (“HMCs”) with mixed neuronal cultures. [Figure 14]

[0145] Figure 14A shows the TUNEL rankings for each rat tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 14B shows a bar graph of the average TUNEL rankings for each group of rats tested. The TUNEL rankings were as follows: 1 = no structural damage and no TUNEL, 2 = structural damage and low TUNEL, 3 = structural damage and moderate TUNEL, 4 = structural damage and high TUNEL, and 5 = severe damage / tissue loss. Comparison of rats in the Sham group vs. the HI group showed a t-test score of 0.006284 and a Mann-Whitney score of 0.0256. Comparison of the Sham group vs. the Lot B group showed a t-test score of 0.148904 and a Mann-Whitney score of 0.2. Comparison of the HI group vs. the Lot B group showed a t-test score of 0.101453 and a Mann-Whitney score of 0.1841. [Figure 15]

[0146] FIG. 15 shows H&E staining of rat brains tested in an in vivo neonatal hypoxic-ischemic model of cerebral palsy. [Figure 16]

[0147] Figure 16A shows images of Iba-1 staining in peri-infarct tissues of rats tested in an in vivo neonatal hypoxic-ischemic model of cerebral palsy. Figure 16B shows the average signal intensity of Iba-1 staining in each rat tested in an in vivo neonatal hypoxic-ischemic model of cerebral palsy. Figure 16C shows the average average signal intensity of Iba-1 staining in each group of rats tested. Comparison of rats in the Sham group vs. the HI group showed a t-test of 0.039335 and a Mann-Whitney of 0.065, comparison of rats in the Sham group vs. the Lot B group showed a t-test of 0.129562 and a Mann-Whitney of 0.1949, and comparison of rats in the HI group vs. the Lot B group showed a t-test of 0.353204 and a Mann-Whitney of 0.4418. [Figure 17]

[0148] Figure 17A shows images of GFAP staining in peri-infarct tissues of rats tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 17B shows the average signal intensity of GFAP staining in each rat tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 17C shows the average average signal intensity of GFAP staining in each group of tested rats. Comparison of rats in the Sham group vs. the HI group showed a t-test of 0.011749 and a Mann-Whitney of 0.0047; comparison of rats in the Sham group vs. the Lot B group showed a t-test of 0.070012 and a Mann-Whitney of 0.0207; and comparison of rats in the HI group vs. the Lot B group showed a t-test of 0.57941 and a Mann-Whitney of 0.7984. [Figure 18]

[0149] Figure 18A shows images of MBP staining in the corpus callosum of rats tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 18B shows the average signal intensity of MBP staining in each rat tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 18C shows the average average signal intensity of MBP staining in each group of rats tested. Comparison of rats in the Sham group vs. the HI group showed a t-test of 0.012963 and a Mann-Whitney of 0.007, comparison of rats in the Sham group vs. the Lot B group showed a t-test of 0.189251 and a Mann-Whitney of 0.3282, and comparison of rats in the HI group vs. the Lot B group showed a t-test of 0.172857 and a Mann-Whitney of 0.2345. [Figure 19A]

[0150] Figure 19A shows images of Olig2 staining in the hippocampus of the ipsilateral hemisphere of the lesion of rats tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 19B shows the mean signal intensity of Olig2 staining in the SVZ, cortex, hippocampus, and region average of each rat tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 19C shows the mean mean signal intensity of Olig2 staining in the SVZ, cortex, hippocampus, and region average of each group of rats tested. Comparison of rats in lot B vs. HI for Olig2 staining showed 0.3962 for t-test in the SVZ, 0.4399 for t-test in the cortex, 0.5435 for t-test in the hippocampus, and 0.3597 for region average. [Figure 19B] Figure 19A shows images of Olig2 staining in the hippocampus of the ipsilateral hemisphere of the lesion of rats tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 19B shows the mean signal intensity of Olig2 staining in the SVZ, cortex, hippocampus, and region average of each rat tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 19C shows the mean mean signal intensity of Olig2 staining in the SVZ, cortex, hippocampus, and region average of each group of rats tested. Comparison of rats in lot B vs. HI for Olig2 staining showed 0.3962 for t-test in the SVZ, 0.4399 for t-test in the cortex, 0.5435 for t-test in the hippocampus, and 0.3597 for region average. [Figure 19C]Figure 19A shows images of Olig2 staining in the hippocampus of the ipsilateral hemisphere of the lesion of rats tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 19B shows the mean signal intensity of Olig2 staining in the SVZ, cortex, hippocampus, and region average of each rat tested in an in vivo neonatal hypoxia-ischemia model of cerebral palsy. Figure 19C shows the mean mean signal intensity of Olig2 staining in the SVZ, cortex, hippocampus, and region average of each group of rats tested. Comparison of rats in lot B vs. HI for Olig2 staining showed 0.3962 for t-test in the SVZ, 0.4399 for t-test in the cortex, 0.5435 for t-test in the hippocampus, and 0.3597 for region average. [Figure 20]

[0151] Figure 20 shows the results of the body swing test in rats with middle cerebral artery occlusion (MCAO) stroke and receiving HMC via three routes of administration: intravenous (IV), intracerebral (IC), and intrathecal (IT). Two-way ANOVA with Tukey's MCT was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 21]

[0152] Figure 21 shows the results of the forelimb placing, hindlimb placing, and body swing tests in rats with middle cerebral artery occlusion (MCAO) stroke and receiving HMC and HMC-EV via intravenous, intracerebral, and intracisternal administration. Two-way ANOVA with Tukey's MCT was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 22]

[0153] Figure 22 shows the results of the forelimb placing, hindlimb placing, and body swing tests in rats with middle cerebral artery occlusion (MCAO) stroke and receiving HMC-EV via intracisternal administration. Two-way ANOVA with Tukey's MCT was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 23]

[0154] Figure 23 shows the results of the forelimb placing, hindlimb placing, and body swing tests in rats with middle cerebral artery occlusion (MCAO) stroke and receiving HMC-EV via intrathecal administration. Two-way ANOVA using Turkey's MCT was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 24]

[0155] Figure 24A shows images of MBP staining in the cortex and striatum of rats undergoing MCAO stroke and receiving HMCs (obtained from C-GS1 cells and N-lineage cells) by intravenous administration. Figure 24B shows the average signal intensity of MBP staining in the cortex of rats tested in an in vivo MCAO stroke model. Figure 24C shows the average signal intensity of MBP staining in the striatum of rats tested in an in vivo MCAO stroke model. For comparisons between the sham group and the vehicle group, a Welch test was used for statistical analysis. ***p<0.001. For comparisons between the vehicle group and the treatment group, a one-way ANOVA with Dunnett's multiple comparison test was used for statistical analysis. *p<0.05, **p<0.01, and ***p<0.001. [Figure 25]

[0156] Figure 25A shows images of Iba1 staining in the cortex and striatum of rats undergoing MCAO stroke and receiving HMCs (obtained from C-GS1 cells and N-lineage cells) by intravenous administration. Figure 25B shows the average signal intensity of Iba1 staining in the cortex of rats tested in an in vivo MCAO stroke model. Figure 25C shows the average signal intensity of Iba1 staining in the striatum of rats tested in an in vivo MCAO stroke model. For comparisons between the sham group and the vehicle group, a Welch test was used for statistical analysis. ***p<0.001. For comparisons between the vehicle group and the treatment group, a one-way ANOVA with Dunnett's multiple comparison test was used for statistical analysis. *p<0.05, **p<0.01, and ***p<0.001. [Figure 26]

[0157] Figure 26A shows images of GFAP staining in the cortex and striatum of rats undergoing MCAO stroke and receiving HMCs (obtained from C-GS1 cells and N-lineage cells) by intravenous administration. Figure 26B shows the average signal intensity of GFAP staining in the cortex of rats tested in an in vivo MCAO stroke model. Figure 26C shows the average signal intensity of GFAP staining in the striatum of rats tested in an in vivo MCAO stroke model. For comparisons between the sham group and the vehicle group, a Welch test was used for statistical analysis. ***p<0.001. For comparisons between the vehicle group and the treatment group, a one-way ANOVA with Dunnett's multiple comparison test was used for statistical analysis. *p<0.05, **p<0.01, and ***p<0.001. [Figure 27]

[0158] Figure 27A shows images of MBP staining in rats with MCAO stroke receiving HMC-EVs (obtained from N-lineage cells and treated with 50 ng / mL of IFN-gamma for 96 hours) via intracisternal administration. Figure 27B shows the average signal intensity of MBP staining in rats tested in an in vivo MCAO stroke model. cc: corpus callosum, ec: external capsule, cg: cingulate gyrus. For comparisons between vehicle and treatment groups, Bonferroni comparisons were used for statistical analysis. **p<0.01. [Figure 28]

[0159] Figure 28A shows images of Iba1 staining in rats with MCAO stroke receiving HMC-EVs (obtained from N-lineage cells and treated with 50 ng / mL gamma interferon for 96 hours) via intracisternal administration. Figure 28B shows the average signal intensity of Iba1 staining in rats tested in an in vivo MCAO stroke model. cc: corpus callosum, ec: external capsule, cg: cingulate gyrus. For comparisons between vehicle and treatment groups, Bonferroni comparisons were used for statistical analysis. **p<0.01. [Figure 29]

[0160] Figure 29A shows images of GFAP staining in rats with MCAO stroke receiving HMC-EVs (obtained from N-lineage cells and treated with 50 ng / mL gamma interferon for 96 hours) via intracisternal administration. Figure 29B shows the average signal intensity of GFAP staining in rats tested in an in vivo MCAO stroke model. cc: corpus callosum, ec: external capsule, cg: cingulate gyrus. For comparisons between vehicle and treatment groups, Bonferroni comparisons were used for statistical analysis. **p<0.01. [Figure 30]

[0161] Figure 30A shows images of Olig2 staining in rats with MCAO stroke receiving HMC-EVs (obtained from N-lineage cells and treated with 50 ng / mL gamma interferon for 96 hours) via intracisternal administration. Figure 30B shows the mean signal intensity of Olig2 staining in rats tested in an in vivo MCAO stroke model. cc: corpus callosum, ec: external capsule, cg: cingulate gyrus. For comparisons between vehicle and treatment groups, Bonferroni comparisons were used for statistical analysis. **p<0.01. [Figure 31]

[0162] Figure 31A shows images of NG2 staining in rats with MCAO stroke receiving HMC-EVs (obtained from N-lineage cells and treated with 50 ng / mL gamma interferon for 96 hours) via intracisternal administration. Figure 30B shows the average signal intensity of NG2 staining in rats tested in an in vivo MCAO stroke model. cc: corpus callosum, ec: external capsule, cg: cingulate gyrus. For comparisons between vehicle and treatment groups, Bonferroni comparisons were used for statistical analysis. **p<0.01. [Figure 32]

[0163] FIG. 32 is a schematic diagram of the study design for an in vitro oxygen glucose deprivation (OGD) assay to model stroke. [Figure 33]

[0164] Figure 33A shows TUNEL staining and imaging of primary rat neurons treated with or without HMC after 0, 1, 2, and 3 hours of oxygen glucose deprivation (OGD) injury. Figure 33B shows average TUNEL quantification of primary rat neurons treated with or without MSC after 0, 1, 2, and 3 hours of OGD injury. [Figure 34A]

[0165] Figures 34A-F show pathway enrichment analysis of differential expression between neurons grown on HMC-enriched medium and control medium after 3 hours of oxygen-glucose deprivation injury. Figures 34A-B show pathways enriched by differential expression. Figures 34C-F show differential expression between OGD neurons grown on HMC-enriched medium and control medium for the gene oncology section. Figure 34C shows upregulation of pathways involved in cell survival, neuroprotection, and synaptic transmission in OGD neurons grown on HMC-enriched cultures. Figure 34D shows upregulation of genes involved in neuroprotection in OGD neurons grown on HMC-enriched cultures. Figure 34E shows downregulation of pathways involved in apoptosis in OGD neurons grown on HMC-enriched cultures. Figure 34F shows downregulation of genes involved in the general response to apoptosis or cell death in OGD neurons grown on HMC-enriched cultures. [Figure 34B]Figures 34A-F show pathway enrichment analysis of differential expression between neurons grown on HMC-enriched medium and control medium after 3 hours of oxygen-glucose deprivation injury. Figures 34A-B show pathways enriched by differential expression. Figures 34C-F show differential expression between OGD neurons grown on HMC-enriched medium and control medium for the gene oncology section. Figure 34C shows upregulation of pathways involved in cell survival, neuroprotection, and synaptic transmission in OGD neurons grown on HMC-enriched cultures. Figure 34D shows upregulation of genes involved in neuroprotection in OGD neurons grown on HMC-enriched cultures. Figure 34E shows downregulation of pathways involved in apoptosis in OGD neurons grown on HMC-enriched cultures. Figure 34F shows downregulation of genes involved in the general response to apoptosis or cell death in OGD neurons grown on HMC-enriched cultures. [Figure 34C] Figures 34A-F show pathway enrichment analysis of differential expression between neurons grown on HMC-enriched medium and control medium after 3 hours of oxygen-glucose deprivation injury. Figures 34A-B show pathways enriched by differential expression. Figures 34C-F show differential expression between OGD neurons grown on HMC-enriched medium and control medium for the gene oncology section. Figure 34C shows upregulation of pathways involved in cell survival, neuroprotection, and synaptic transmission in OGD neurons grown on HMC-enriched cultures. Figure 34D shows upregulation of genes involved in neuroprotection in OGD neurons grown on HMC-enriched cultures. Figure 34E shows downregulation of pathways involved in apoptosis in OGD neurons grown on HMC-enriched cultures. Figure 34F shows downregulation of genes involved in the general response to apoptosis or cell death in OGD neurons grown on HMC-enriched cultures. [Figure 34D]Figures 34A-F show pathway enrichment analysis of differential expression between neurons grown on HMC-enriched medium and control medium after 3 hours of oxygen-glucose deprivation injury. Figures 34A-B show pathways enriched by differential expression. Figures 34C-F show differential expression between OGD neurons grown on HMC-enriched medium and control medium for the gene oncology section. Figure 34C shows upregulation of pathways involved in cell survival, neuroprotection, and synaptic transmission in OGD neurons grown on HMC-enriched cultures. Figure 34D shows upregulation of genes involved in neuroprotection in OGD neurons grown on HMC-enriched cultures. Figure 34E shows downregulation of pathways involved in apoptosis in OGD neurons grown on HMC-enriched cultures. Figure 34F shows downregulation of genes involved in the general response to apoptosis or cell death in OGD neurons grown on HMC-enriched cultures. [Figure 34E] Figures 34A-F show pathway enrichment analysis of differential expression between neurons grown on HMC-enriched medium and control medium after 3 hours of oxygen-glucose deprivation injury. Figures 34A-B show pathways enriched by differential expression. Figures 34C-F show differential expression between OGD neurons grown on HMC-enriched medium and control medium for the gene oncology section. Figure 34C shows upregulation of pathways involved in cell survival, neuroprotection, and synaptic transmission in OGD neurons grown on HMC-enriched cultures. Figure 34D shows upregulation of genes involved in neuroprotection in OGD neurons grown on HMC-enriched cultures. Figure 34E shows downregulation of pathways involved in apoptosis in OGD neurons grown on HMC-enriched cultures. Figure 34F shows downregulation of genes involved in the general response to apoptosis or cell death in OGD neurons grown on HMC-enriched cultures. [Figure 34F]Figures 34A-F show pathway enrichment analysis of differential expression between neurons grown on HMC-enriched medium and control medium after 3 hours of oxygen-glucose deprivation injury. Figures 34A-B show pathways enriched by differential expression. Figures 34C-F show differential expression between OGD neurons grown on HMC-enriched medium and control medium for the gene oncology section. Figure 34C shows upregulation of pathways involved in cell survival, neuroprotection, and synaptic transmission in OGD neurons grown on HMC-enriched cultures. Figure 34D shows upregulation of genes involved in neuroprotection in OGD neurons grown on HMC-enriched cultures. Figure 34E shows downregulation of pathways involved in apoptosis in OGD neurons grown on HMC-enriched cultures. Figure 34F shows downregulation of genes involved in the general response to apoptosis or cell death in OGD neurons grown on HMC-enriched cultures.

[0166] [Figure 35]

[0167] Figure 35A shows in vitro OGD assay RNAseq analysis of primary rat neurons treated with or without HMC after 0, 1, 2, and 3 hours of oxygen-glucose deprivation (OGD) injury. Figure 35B shows qPCR analysis of primary rat neurons treated with or without HMC after 0, 1, 2, and 3 hours of oxygen-glucose deprivation (OGD) injury. Two-way ANOVA with Sidak's multiple comparison test was used for statistical analysis: *p<0.05, **p<0.01, and ****p<0.0001. [Figure 36]

[0168] Figure 36A shows the attenuation of cell death by HMC-EV. The percentage of cell death was determined as the number of PI+ cells among all Hoechst+ cells. Two-way ANOVA was used for statistical significance analysis. ****p<0.0001. Figure 36B shows the dose-dependent attenuation of cell death by HMC-EV treatment. The percentage of cell death was determined as the number of PI+ cells among all Hoechst+ cells. One-way [Figure 37]

[0169] Figure 37 shows maintenance of mitochondrial membrane potential in HMC-EV-treated cells undergoing nuclear swelling. HMC-EV treatment sustained cells in the nuclear swelling stage after glutamate-induced injury. [Figure 38]

[0170] Figure 38 shows principal component analysis of the transcriptomes of HMCs (obtained from N-lineage cells) and adipose tissue-derived MSCs, demonstrating that HMCs differ from adipose tissue-derived MSCs in both basal and interferon-gamma stimulated states. AMSC-B-1, 2, 3: adipose tissue-derived MSCs obtained from three different adult donors, two technical replicates for each biological replicate. AMSC-S-1, 2, 3: adipose tissue-derived MSCs, but stimulated with gamma interferon. NHMC-B: three technical replicates of MSCs derived from N-lineage cells, basal state. NHMC-S: MSCs derived from N-lineage cells, but stimulated with gamma interferon. [Figure 39]

[0171] FIG. 39 shows the weights of the different genes that contribute to the second principal component that determines the variance between HMCs (obtained from N-lineage cells) and adipose tissue-derived MSCs. [Figure 40]

[0172] Figure 40 shows a hierarchical clustering map demonstrating that HMCs (obtained from N-lineage cells) are distinct from adipose tissue-derived MSCs in both basal and gamma interferon-stimulated states. AB1, AB2, AB3 - adipose tissue-derived MSCs obtained from three different adult donors, two technical replicates per donor, basal cell state. AS1, AS2, AS3 - adipose tissue-derived MSCs stimulated with gamma interferon. NB - MSCs derived from N-lineage cells, basal state, three technical replicates. NS - MSCs derived from N-lineage cells and stimulated with gamma interferon. [Figure 41]

[0173] Figure 41 shows the basal HMC-specific cluster of genes. [Figure 42]

[0174] FIG. 42 shows the basal adipose tissue-derived MSC-specific cluster of genes. [Figure 43]

[0175] Figure 43 shows pathway enrichment of differential expression patterns between HMCs (obtained from N-lineage cells) and adipose tissue-derived MSCs, demonstrating a significant HMC-specific upregulation of several pathways (indicated by arrows) involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, and synaptogenesis signaling. [Figure 44]

[0176] FIG. 44 shows the top 15 most strongly differentially expressed genes that contribute to the activation of neuronal CREB signaling in HMCs (obtained from N-lineage cells). [Figure 45]

[0177] FIG. 45 shows the top 15 most strongly upregulated genes that contribute to the enrichment of axon guidance pathways in HMCs (obtained from N-lineage cells). [Figure 46]

[0178] FIG. 46 shows the top 15 most highly expressed genes that contribute to the activation of synaptogenesis signaling pathways in HMCs (obtained from N-lineage cells). [Figure 47]

[0179] FIG. 47 shows the top 15 most upregulated genes that contribute to the activation of neuroinflammatory signaling pathways in HMCs (obtained from N-lineage cells). [Figure 48]

[0180] Figure 48 shows principal component analysis of the transcriptomes of HMCs derived from N-lineage cells, HMCs derived from GMP1 cells, and adipose tissue-derived MSCs. AMSC-B - 1, 2, 3 - adipose tissue-derived MSCs obtained from three different adult donors, basal state, two technical replicates for each biological replicate. AMSC-S - 1, 2, 3 - adipose tissue-derived MSCs obtained from three different adult donors but stimulated with gamma interferon. NHMC-B - HMCs derived from N-lineage cells, basal state. HMCs derived from NHMC-SN-lineage cells but stimulated with gamma interferon. GMP-B - HMCs derived from the GMP1 cell line, basal state. GMP-S - HMCs derived from the GMP1 cell line but stimulated with gamma interferon. [Figure 49]

[0181] Figure 49 shows hierarchical clustering maps demonstrating that HMCs (obtained from N-lineage cells) and HMCs (obtained from GMP1 cells) are distinct from adipose tissue-derived MSCs in both basal and gamma interferon-stimulated cell states. AB1, AB2, AB3 - Adipose tissue-derived MSCs obtained from three different adult donors, two technical replicates per donor, basal cell state. AS1, AS2, AS3 - Adipose tissue-derived MSCs obtained from three different adult donors and stimulated with gamma interferon. HMCs derived from NB-N-lineage cells, basal state, three technical replicates. HMCs derived from NS-N-lineage cells and stimulated with gamma interferon. HMCs derived from GB-GMP1 cell line, basal state, three technical replicates. HMCs derived from GS-GMP1 cell line and stimulated with gamma interferon. [Figure 50]

[0182] Figure 50 shows the HMC-specific cluster of genes. [Figure 51]

[0183] Figure 51 shows the basal adipose tissue-derived MSC-specific cluster of genes. [Figure 52]

[0184] FIG. 52 shows stimulated adipose tissue-derived MSC-specific clusters of genes. [Figure 53A]

[0185] Figure 53A shows pathway enrichment of differential expression patterns between HMCs (obtained from GMP1 cells) and adipose tissue-derived MSCs, demonstrating significant HMC-specific upregulation of several pathways involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, and synaptogenesis signaling. Figure 53B shows the top canonical pathways differentially regulated in HMCs. Figure 53C shows exemplary regulators that are activated and inhibited in HMCs. [Figure 53B] Figure 53A shows pathway enrichment of differential expression patterns between HMCs (obtained from GMP1 cells) and adipose tissue-derived MSCs, demonstrating significant HMC-specific upregulation of several pathways involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, and synaptogenesis signaling. Figure 53B shows the top canonical pathways differentially regulated in HMCs. Figure 53C shows exemplary regulators that are activated and inhibited in HMCs. [Figure 53C] Figure 53A shows pathway enrichment of differential expression patterns between HMCs (obtained from GMP1 cells) and adipose tissue-derived MSCs, demonstrating significant HMC-specific upregulation of several pathways involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, and synaptogenesis signaling. Figure 53B shows the top canonical pathways differentially regulated in HMCs. Figure 53C shows exemplary regulators that are activated and inhibited in HMCs. [Figure 54A]

[0186] Figure 54A shows pathway enrichment of differential expression patterns between HMCs (obtained from N-lineage cells) and adipose tissue-derived MSCs, demonstrating significant HMC-specific upregulation of several pathways involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, and synaptogenesis signaling. Figure 54B shows the top canonical pathways differentially regulated in HMCs. Figure 54C shows exemplary regulators that are activated and inhibited in HMCs. [Figure 54B]Figure 54A shows pathway enrichment of differential expression patterns between HMCs (obtained from N-lineage cells) and adipose tissue-derived MSCs, demonstrating significant HMC-specific upregulation of several pathways involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, and synaptogenesis signaling. Figure 54B shows the top canonical pathways differentially regulated in HMCs. Figure 54C shows exemplary regulators that are activated and inhibited in HMCs. [Figure 54C] Figure 54A shows pathway enrichment of differential expression patterns between HMCs (obtained from N-lineage cells) and adipose tissue-derived MSCs, demonstrating significant HMC-specific upregulation of several pathways involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, and synaptogenesis signaling. Figure 54B shows the top canonical pathways differentially regulated in HMCs. Figure 54C shows exemplary regulators that are activated and inhibited in HMCs. [Figure 55]

[0187] Figure 55 shows principal component analysis of the transcriptomes of HMCs (obtained from N-lineage cells) and bone marrow-derived MSCs, demonstrating that HMCs differ from bone marrow-derived MSCs in both basal and interferon-gamma stimulated states. BM-B - bone marrow-derived MSCs from three different adult donors, basal state, two technical replicates for each biological replicate. BM-S - bone marrow-derived MSCs but stimulated with gamma interferon. Three technical replicates of HMCs derived from NBN-lineage cells, basal state. HMCs derived from NSN-lineage cells but stimulated with gamma interferon. [Figure 56]

[0188] Figure 56 shows the weights of the various genes that contribute to the second principal component that determines the variance between HMCs and bone marrow-derived MSCs. [Figure 57]

[0189] Figure 57 shows a hierarchical clustering map demonstrating that HMCs (obtained from N-lineage cells) are distinct from bone marrow-derived MSCs in both basal and gamma interferon-stimulated cell states. BMB1, BMB2, BMB3 - bone marrow-derived MSCs obtained from 3 different adult donors, 2 technical replicates per donor, basal cell state. BMS1, BMS2, BMS3 - bone marrow-derived MSCs stimulated with gamma interferon. NB - HMCs derived from N-lineage cells, basal state, 3 technical replicates. NS - HMCs derived from N-lineage cells and stimulated with gamma interferon. [Figure 58]

[0190] Figure 58 shows the basal HMC-specific cluster of genes. [Figure 59]

[0191] Figure 59 shows the basal bone marrow-derived MSC-specific cluster of genes. [Figure 60]

[0192] Figure 60 shows pathway enrichment of differential expression patterns between HMCs (obtained from N-lineage cells) and bone marrow-derived MSCs, demonstrating a significant HMC-specific upregulation of several pathways (indicated by arrows) involved in neuronal lineage development, e.g., CREB signaling in neurons. [Figure 61]

[0193] FIG. 61 shows the top 15 most strongly differentially expressed genes that contribute to the activation of neuronal CREB signaling in HMCs (derived from N-lineage cells). [Figure 62]

[0194] FIG. 62 shows the top 15 most strongly upregulated genes that contribute to the activation of synaptogenic signaling in HMCs (derived from N-lineage cells). [Figure 63A]

[0195] Figure 63A shows pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from bone marrow-derived MSCs (BM-MSC-EVs). Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange bars. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue bars. White / gray bars represent pathways enriched in HMC-EVs, i.e., proteins contributing to these pathways are enriched. Figure 63B shows the disease or function annotation of proteins with higher expression levels in HMC-EVs compared to BM-MSC-EVs. Figure 63C shows the disease or function annotation of proteins with lower expression levels in HMC-EVs compared to BM-MSC-EVs. An activation z-score greater than 2 or less than -2 is considered the threshold. [Figure 63B] Figure 63A shows pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from bone marrow-derived MSCs (BM-MSC-EVs). Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange bars. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue bars. White / gray bars represent pathways enriched in HMC-EVs, i.e., proteins contributing to these pathways are enriched. Figure 63B shows the disease or function annotation of proteins with higher expression levels in HMC-EVs compared to BM-MSC-EVs. Figure 63C shows the disease or function annotation of proteins with lower expression levels in HMC-EVs compared to BM-MSC-EVs. An activation z-score greater than 2 or less than -2 is considered the threshold. [Figure 63C]Figure 63A shows pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from bone marrow-derived MSCs (BM-MSC-EVs). Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange bars. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue bars. White / gray bars represent pathways enriched in HMC-EVs, i.e., proteins contributing to these pathways are enriched. Figure 63B shows the disease or function annotation of proteins with higher expression levels in HMC-EVs compared to BM-MSC-EVs. Figure 63C shows the disease or function annotation of proteins with lower expression levels in HMC-EVs compared to BM-MSC-EVs. An activation z-score greater than 2 or less than -2 is considered the threshold. [Figure 64A]

[0196] Figure 64A shows pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from adipose tissue-derived MSCs (AD-MSC-EVs). Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange bars. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue bars. White / gray bars represent pathways enriched in HMC-EVs, i.e., proteins contributing to these pathways are enriched. Figure 64B shows the disease or function annotation of proteins with higher expression levels in HMC-EVs compared to AD-MSC-EVs. Figure 64C shows the disease or function annotation of proteins with lower expression levels in HMC-EVs compared to AD-MSC-EVs. An activation z-score greater than 2 or less than -2 is considered the threshold. [Figure 64B]Figure 64A shows pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from adipose tissue-derived MSCs (AD-MSC-EVs). Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange bars. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue bars. White / gray bars represent pathways enriched in HMC-EVs, i.e., proteins contributing to these pathways are enriched. Figure 64B shows the disease or function annotation of proteins with higher expression levels in HMC-EVs compared to AD-MSC-EVs. Figure 64C shows the disease or function annotation of proteins with lower expression levels in HMC-EVs compared to AD-MSC-EVs. An activation z-score greater than 2 or less than -2 is considered the threshold. [Figure 64C] Figure 64A shows pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from adipose tissue-derived MSCs (AD-MSC-EVs). Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange bars. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue bars. White / gray bars represent pathways enriched in HMC-EVs, i.e., proteins contributing to these pathways are enriched. Figure 64B shows the disease or function annotation of proteins with higher expression levels in HMC-EVs compared to AD-MSC-EVs. Figure 64C shows the disease or function annotation of proteins with lower expression levels in HMC-EVs compared to AD-MSC-EVs. An activation z-score greater than 2 or less than -2 is considered the threshold. [Figure 65A]

[0197] Figure 65A shows pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from umbilical cord blood-derived MSCs (UCB-MSC-EVs). Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange bars. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue bars. White / gray bars represent pathways enriched in HMC-EVs, i.e., proteins contributing to these pathways are enriched. Figure 65B shows the disease or function annotation of proteins with higher expression levels in HMC-EVs compared to UCB-MSC-EVs. Figure 65C shows the disease or function annotation of proteins with lower expression levels in HMC-EVs compared to UCB-MSC-EVs. An activation z-score greater than 2 or less than -2 is considered a threshold. [Figure 65B] Figure 65A shows pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from umbilical cord blood-derived MSCs (UCB-MSC-EVs). Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange bars. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue bars. White / gray bars represent pathways enriched in HMC-EVs, i.e., proteins contributing to these pathways are enriched. Figure 65B shows the disease or function annotation of proteins with higher expression levels in HMC-EVs compared to UCB-MSC-EVs. Figure 65C shows the disease or function annotation of proteins with lower expression levels in HMC-EVs compared to UCB-MSC-EVs. An activation z-score greater than 2 or less than -2 is considered a threshold. [Figure 65C]Figure 65A shows pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from umbilical cord blood-derived MSCs (UCB-MSC-EVs). Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange bars. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue bars. White / gray bars represent pathways enriched in HMC-EVs, i.e., proteins contributing to these pathways are enriched. Figure 65B shows the disease or function annotation of proteins with higher expression levels in HMC-EVs compared to UCB-MSC-EVs. Figure 65C shows the disease or function annotation of proteins with lower expression levels in HMC-EVs compared to UCB-MSC-EVs. An activation z-score greater than 2 or less than -2 is considered a threshold. DETAILED DESCRIPTION OF THE INVENTION

[0099] definition

[0198] As used herein, the terms "pluripotent cell," "pluripotent stem cell," and "PSC" refer broadly to cells that have the ability to prolong or virtually indefinitely reproduce in vitro while maintaining an undifferentiated state, exhibit a stable (preferably normal) karyotype, and have the ability to differentiate into all three germ layers (i.e., ectoderm, mesoderm, and endoderm) under appropriate conditions. Typical pluripotent cells (a) have the ability to induce teratomas when transplanted into immunodeficient (SCID) mice, (b) have the ability to differentiate into cell types of all three germ layers (e.g., ectodermal, mesodermal, and endodermal cell types), and (c) express at least one hES cell marker (e.g., Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, NANOG, TRA1 60, TRA1 81, SOX2, REX1). Exemplary pluripotent cells may express Oct-4, alkaline phosphatase, SSEA-3 surface antigen, SSEA4 surface antigen, TRA1 60, and / or TRA1 81. Additional exemplary pluripotent cells include, but are not limited to, embryonic stem cells, induced pluripotent (iPS) cells, embryo-derived cells, pluripotent cells produced from embryonic germ (EG) cells (e.g., by culturing in the presence of FGF-2, LIF, and SCF), parthenogenetic ES cells, ES cells generated from cultured inner cell mass cells (ICM), ES cells generated from blastomeres, and ES cells generated by nuclear transfer (e.g., transferring a somatic cell nucleus into a recipient oocyte). Exemplary pluripotent cells can be generated without destroying an embryo. For example, induced pluripotent cells can be generated from cells obtained without destroying an embryo. As a further example, pluripotent cells can be generated from biopsied blastomeres (which can be accomplished without harming the remaining embryo), which can optionally be cryopreserved, cultured, and / or transplanted into a suitable host. Pluripotent cells (regardless of their origin) can be genetically or otherwise modified to enhance lifespan, potency, homing, or to deliver desired factors into cells differentiated from such pluripotent cells (e.g., MSCs or hemangioblasts).By way of non-limiting example, pluripotent cells may express Sirt1 (thereby extending lifespan); optionally express one or more telomerase subunit genes under the control of an inducible or repressible promoter; incorporate a fluorescent label; incorporate iron oxide particles or other such reagents (which may be used for cell tracking by in vivo imaging, MRI, etc. See Thu et al., Nat Med. 2012 Feb. 26; 18(3):463-7); express bFGF, which may improve lifespan (See Go et al., J. Biochem. 142:741-748 (2007)); express CXCR4 for homing (Shi et al., Haematologica. 2007 July; 92(7):897-904); or express recombinant TRAIL to induce caspase-mediated apoptosis in cancer cells such as gliomas (Sasportas et al., Proc Natl Acad Sci USA. 2009 Mar. 24; 106(12):4822-7).

[0100]

[0199] "Embryo" or "embryonic," as used herein, refers broadly to a developing mass of cells that has not implanted into the uterine membrane of a maternal host. An "embryonic cell" is a cell isolated from or contained within an embryo. This includes blastomeres (which may be obtained as early as the two-cell stage) and aggregated blastomeres.

[0101]

[0200] "Embryonic stem cells" (ES cells or ESCs) encompass pluripotent cells generated from embryonic cells (e.g., from cultured inner cell mass cells or cultured blastomeres, etc.). Often, such cells are subcultured or are being cultured as cell lines. Embryonic stem cells can be used as pluripotent stem cells in the process of generating hemangioblasts, as described herein. For example, ES cells can be generated by any method (including sexual or asexual means) known in the art, including derivation from embryos generated by, for example, fertilization of egg cells with sperm or sperm DNA, nuclear transfer (including somatic cell nuclear transfer), or parthenogenesis. As a further example, embryonic stem cells also include cells generated by somatic cell nuclear transfer (even when non-embryonic cells are used in the process). For example, ES cells can be derived from the ICM of a blastocyst-stage embryo, or from embryonic stem cells derived from one or more blastomeres. Such embryonic stem cells can be generated from embryonic material produced by fertilization or asexual means (including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgeny). As discussed further above (see "Pluripotent Cells"), ES cells can be genetically or otherwise modified to enhance lifespan, potency, homing, or to deliver desired factors into cells differentiated from such pluripotent cells (e.g., MSCs or hemangioblasts).

[0102]

[0201] ES cells can be generated homozygous or hemizygous in one or more HLA genes, for example, through genetic manipulation, screening for spontaneous loss of heterozygosity, etc. ES cells can be genetically or otherwise modified to enhance lifespan, potency, homing, or to deliver desired factors into cells differentiated from such pluripotent cells (e.g., MSCs or hemangioblasts). Regardless of their origin or the method used to generate them, embryonic stem cells generally possess one or more of the following characteristics: (i) the ability to differentiate into cells of all three germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and (iii) the ability to generate teratomas when transplanted into immunodeficient animals. Embryonic stem cells that can be used in embodiments of the present invention include, but are not limited to, human ES cells ("hESCs" or "hES cells"), such as CT2, MA01, MA09, ACT-4, No. 3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Additional exemplary cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. See also the NIH Human Embryonic Stem Cell Registry. An exemplary human embryonic stem cell line that may be used is MA09 cells. The isolation and preparation of MA09 cells was previously described in Klimanskaya et al. (2006) "Human Embryonic Stem Cell lines Derived from Single Blastomeres." Nature 444:481-485. Human ES cells used in accordance with exemplary embodiments of the present invention may be derived and maintained in accordance with GMP standards.

[0103]

[0202] Exemplary hES cell markers include, but are not limited to, alkaline phosphatase, Oct-4, Nanog, early embryonic specific antigen-3 (SSEA-3), early embryonic specific antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth and differentiation factor 3 (GDF3), down-regulated protein 1 (REX1), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency-associated factor 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-cadherin, Cluster designation 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand). Additionally, the embryonic stem cells may express Oct-4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, TRA 1 60, and / or TRA 1 81.

[0104]

[0203] ESCs can be initially co-cultured with or without feeder cells, such as mouse embryonic feeder (MEF) cells or human feeder cells (e.g., human dermal fibroblasts (HDFs)), in any medium known in the art that maintains ESC pluripotency. The MEF cells or human feeder cells can be mitotically inactivated, for example, by exposure to mitomycin C, gamma irradiation, or other known methods, before seeding the ESCs into the co-culture, so that the MEF cells do not proliferate in the culture. Additionally, ESC cell cultures can be observed microscopically, and colonies containing non-ESC cell morphology can be removed and discarded, for example, by laser ablation using a stem cell cutting tool or other methods. Generally, no additional MEF cells or human feeder cells are used after harvesting ESCs for seeding to form embryoid bodies.

[0105]

[0204] Alternatively, hES cells can be cultured under feeder-free conditions on a solid surface (e.g., extracellular matrix, etc.) by any method known in the art, for example, by Klimanskaya et al., Lancet, 365:1636-1641 (2005). Thus, hES cells used in the methods described herein can be cultured in feeder-free cultures.

[0106]

[0205] "Embryo-derived cells" (EDCs), as used herein, broadly refer to cells derived from the pluripotent morula, cells derived from the blastocyst (including the inner cell mass, embryonic shield, or epiblast), or other pluripotent stem cells of the early embryo (including primitive endoderm, ectoderm, mesoderm, and their derivatives). "EDCs" also include blastomeres and cell masses obtained from single blastomeres or aggregates of embryos from various stages of development, but exclude human embryonic stem cells that have been passaged as cell lines.

[0107]

[0206] As used herein, "potency" broadly refers to the concentration, e.g., number of cells (e.g., hemangioblast-derived MSCs, etc.), that produces a defined effect. Potency does not involve measuring the maximum effect, but rather can be defined in terms of an effective concentration (EC50) based on effects at various points on the concentration axis of a dose-response curve. Potency can also be determined from a metric dose-response curve (EC50) or a quantitative dose-response curve (ED50, TD50, and LD50), although potency is preferably measured by EC50. The term "EC50" refers to the concentration of a drug, antibody, or toxicant that elicits a response midway between the baseline and the maximum effect after a given exposure time. Thus, the EC50 in a metric dose-response curve represents the concentration of a compound at which 50% of the compound's maximum effect is observed. The EC50 in a quantitative dose-response curve represents the concentration of a compound at which 50% of the population shows a response after a given exposure period. EC50 can be determined using animal tests (defined animal models show measurable physiological changes in response to drug application), cell-based assays (using a given cell system that shows a measurable biological response when a drug is added), and / or enzymatic reactions (the biological activity of a drug is measured by the accumulation of products produced after a drug-promoted chemical reaction). Preferably, immunomodulatory assays are used to determine EC50. Non-limiting examples of such immunomodulatory assays include intracellular cytokine, cytotoxicity, regulatory potential, cell signaling potential, proliferation potential, apoptosis assessment, and other assays.

[0108]

[0207] "Mesenchymal stem cells" (MSCs), as used herein, refer to pluripotent stem cells that have the capacity for self-renewal and differentiation into osteoblasts, chondrocytes, and adipocytes, among other mesenchymal cell lineages. Unless otherwise specifically indicated, the MSCs of the present invention are MSCs generated by in vitro differentiation of pluripotent stem cells, and may also be referred to herein as HMCs. In one embodiment, HMCs may be generated by in vitro differentiation of pluripotent stem cells followed by differentiation into hemangioblasts, which then differentiate into HMCs. HMCs may be identified by the expression of one or more markers further described herein. HMCs may also have any of the characteristics described in WO2013 / 082543, U.S. Patent No. 8,962,321, and U.S. Patent No. 8,961,956, the entire contents of which are hereby incorporated by reference.

[0109]

[0208] HMCs can be genetically or otherwise modified to enhance lifespan, potency, homing, or to deliver desired factors into the HMCs or cells differentiated from such HMCs. By way of non-limiting example, HMCs may express Sirt1 (thereby extending lifespan); optionally express one or more telomerase subunit genes under the control of an inducible or repressible promoter; incorporate fluorescent labels; incorporate iron oxide particles or other such reagents (which may be used for cell tracking by in vivo imaging, MRI, etc. See Thu et al., Nat Med. 2012 Feb. 26; 18(3):463-7); express bFGF, which may improve lifespan (See Go et al., J. Biochem. 142:741-748 (2007)); express CXCR4 for homing (Shi et al., Haematologica. 2007 July; 92(7):897-904); or express recombinant TRAIL to induce caspase-mediated apoptosis in cancer cells such as gliomas (Sasportas et al., Proc Natl Acad Sci USA. 2009 Mar. 24; 106(12):4822-7).

[0110]

[0209] As used herein, the term "extracellular vesicles" or "EVs" refers to lipid-bound vesicles secreted by cells into the extracellular space. There are three major subtypes of EVs: microvesicles (MVs), exosomes, and apoptotic bodies, which are distinguished based on their biogenesis, release pathway, size, contents, and function (Zaborowski MP et al., Bioscience. 2015; 65:783-797). Typically, extracellular vesicles range in diameter from 20 nm to 5000 nm and can contain a variety of macromolecular payloads, either contained within the internal space (i.e., lumen), displayed on the external surface of the EV, and / or present across the membrane. The payloads can include nucleic acids (e.g., microRNAs (miRNAs), long non-coding RNAs (lncRNAs), mRNAs, DNA fragments), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example, but not limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived from / secreted by living or dead organisms, isolated tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells.

[0111]

[0210] "Optic neuropathy," as used herein, includes any disease, disorder, or condition involving damage to the optic nerve. Optic neuropathies include hereditary optic neuropathies (e.g., autosomal dominant optic atrophy (Kjer's disease) and maternally inherited Leber's hereditary optic atrophy) and non-hereditary optic neuropathies (e.g., ischemic optic neuropathy). In one embodiment, the optic neuropathy is glaucoma / glaucomatous optic neuropathy.

[0112]

[0211] "Therapeutic," "therapeutic," "treating," "treating," or "treatment," as used herein, broadly refers to treating a disease, arresting or suppressing the onset of a disease or its clinical symptoms, and / or alleviating the disease or causing regression of a disease or its clinical symptoms. "Therapeutic," "therapeutic," "treating," "treating," or "treatment" includes prevention, prevention, treatment, cure, amelioration, reduction, relief, and / or providing relief from a disease, signs, and / or symptoms of a disease. "Therapeutic," "therapeutic," "treating," "treating," or "treatment" includes the reduction of signs and / or symptoms of ongoing disease in a patient with ongoing signs and / or symptoms of a disease. "Therapeutic," "therapeutic," "treating," "treating," or "treatment" also encompasses "prophylaxis" and "prevention." Prevention includes preventing a disease from occurring following treatment of a disease in a patient, or reducing the incidence or severity of a disease in a patient. The term "reduced," when used in reference to "therapy," "therapeutic," "treating," "treating," or "treatment," broadly refers to a clinically significant decrease in signs and / or symptoms. "Treatment," "therapeutic," "treating," "treating," or "treatment" includes treatment of recurrent or relapsing signs and / or symptoms. "Treatment," "therapeutic," "treating," "treating," or "treatment" includes, but is not limited to, eliminating the onset of signs and / or symptoms, regardless of time, as well as reducing existing signs and / or symptoms and eliminating existing signs and / or symptoms. "Treatment," "therapeutic," "treating," "treating," or "treatment" includes treating chronic diseases ("maintenance") and treating acute diseases. For example, treatment includes treating or preventing recurrence or relapse of signs and / or symptoms.

[0113]

[0212] As used herein, the term "effective amount" is intended to include an amount of HMC and / or HMC-EV sufficient to effectively treat the disease when administered to a subject with brain injury (e.g., by reducing, alleviating, or maintaining an existing disease or one or more symptoms of the disease). Alleviating the disease includes slowing the course of the disease or reducing the severity of subsequent disease. The "effective amount" may vary depending on the nature of the HMC and / or HMC-EV, the method of administration of the HMC and / or HMC-EV, the disease and its severity, and the medical history, age, weight, family history, genetic makeup, type of prior or current treatment (if any), and other individual characteristics of the subject being treated.

[0114]

[0213] An "effective amount" also includes the amount of HMC and / or HMC-EV that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The HMC and / or HMC-EV employed in the methods of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0115]

[0214] "Normalization of pathology," as used herein, refers to restoring abnormal structure and / or function resulting from disease to a more normal state. Normalization suggests that the progression of pathology can be controlled or ameliorated by correcting abnormalities in structure and / or function resulting from disease in tissues, organs, cell types, etc. For example, following treatment with the HMCs of the present invention, brain abnormalities resulting from brain injury, e.g., traumatic brain injury, can be improved, corrected, or reversed.

[0116]

[0215] "Induced pluripotent stem cells" or "iPSCs" or "iPS cells," as used herein, refer to pluripotent stem cells generated by reprogramming somatic cells. iPS cells can be generated by expressing or inducing the expression of a combination of factors ("reprogramming factors"). iPS cells can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. iPS cells can also be obtained from cell banks. Alternatively, iPS cells can be generated de novo (by processes known in the art) before differentiation into MSCs or another cell type begins. iPS cell generation can be the first step in the generation of differentiated cells. iPS cells can be specifically generated using material obtained from a particular patient or a matched donor, with the ultimate goal of generating histocompatible MSC cells. iPS cells can be generated from cells that are substantially non-immunogenic to the intended recipient, such as from autologous cells, or from cells that are histocompatible with the intended recipient. As further discussed above (see "Pluripotent Cells"), pluripotent cells (including iPS cells) can be genetically or otherwise modified to enhance lifespan, potency, homing, or to deliver desired factors into cells differentiated from such pluripotent cells (e.g., MSCs or hemangioblasts).

[0117]

[0216] As a further example, induced pluripotent stem cells (iPS cells) can be generated by contacting somatic or other cells with one or more reprogramming factors. For example, the reprogramming factor(s) can be expressed by a cell, for example, from an exogenous nucleic acid added to the cell, or from an endogenous gene in response to a factor (e.g., a small molecule, microRNA, etc.) that promotes or induces expression of that gene (Suh and Blelloch, Development, Vol. 138, pp. 1653-1661 (2011); Miyoshi et al., Cell Stem Cell (2011), doi:10.1016 / j.stem.2011.05.001; Sancho-Martinez et al., Journal of Molecular Cell Biology (2011), pp. 1-3; Anokye-Danso et al., Cell Stem (See, e.g., Cell, 8, 376-388, April 8, 2011; Orkin and Hochedlinger, Cell, 145, 835-850, June 10, 2011). Reprogramming factors can be provided from exogenous sources, for example, by addition to culture medium, or can be introduced into cells by methods known in the art, for example, via coupling with cell-penetrating peptides, protein or nucleic acid transfection agents, lipofection, electroporation, biolistic particle delivery systems (gene guns), microinjection, etc. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4 (sometimes called Oct3 / 4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4, Sox2, Nanog, and Lin28. In other embodiments, somatic cells can be reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors.In another embodiment, somatic cells are reprogrammed by expressing Oct4, Sox2, MYC, Klf4, Nanog, and Lin28. In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells. iPS cells can generally be identified by the expression of the same markers as embryonic stem cells, although specific iPS cell lines may differ in their expression profiles.

[0118]

[0217] Induced pluripotent stem cells can be generated by expressing or inducing the expression of one or more reprogramming factors in somatic cells. In one embodiment, the somatic cells are fibroblasts (e.g., skin fibroblasts, synovial fibroblasts, or lung fibroblasts) or non-fibroblastic somatic cells. In one embodiment, as described above, the somatic cells are reprogrammed by expressing at least one, two, three, four, or five reprogramming factors. In another embodiment, the expression of the reprogramming factors can be induced by contacting the somatic cells with at least one agent, e.g., a small organic molecule agent, that induces the expression of the reprogramming factors.

[0119]

[0218] Somatic cells can also be reprogrammed using a combinatorial approach in which reprogramming factors are expressed (e.g., using viral vectors, plasmids, etc.) and expression of the reprogramming factors is induced (e.g., using small organic molecules). For example, reprogramming factors can be expressed in somatic cells by infection with viral vectors, such as retroviral or lentiviral vectors. Reprogramming factors can also be expressed in somatic cells using non-integrated vectors, such as episomal plasmids or mRNA. See, for example, Yu et al., Science. 2009, May 8; 324(5928):797-801, which is hereby incorporated by reference in its entirety. When non-integrated vectors are used to express reprogramming factors, the factors can be expressed in cells using electroporation, transfection, or somatic cell transduction with the vectors.

[0120]

[0219] Once the reprogramming factors are expressed in the cells, the cells can be cultured by any method known in the art. Over time, cells with ES characteristics will appear in the culture dish. Cells can be selected and subcultured, for example, based on ES morphology or on the expression of selectable or detectable markers. Cells can be cultured to generate a culture of cells that resemble ES cells (these are putative iPS cells). iPS cells can generally be identified by the expression of the same markers as other embryonic stem cells, although specific iPS cell lines may differ in their expression profiles. Exemplary iPS cells can express Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, TRA160, and / or TRA181.

[0121]

[0220] To confirm the pluripotency of iPS cells, cells can be tested in one or more pluripotency assays.For example, cells can be tested for the expression of ES cell markers; cells can be evaluated for the ability to generate teratomas when transplanted into SCID mice; cells can be evaluated for the differentiation ability to generate all three germ layers of cell types.Once pluripotent iPS cells are obtained, they can be used to generate hemangioblasts and MSCs.

[0122]

[0221] "Hemangioblasts" or "HBs," as used herein, refer to pluripotent cells that serve as common progenitors for both hematopoietic and endothelial cell lineages. In embryonic development, they are thought to arise as a transitional cell type that appears during early mesoderm development and populates primitive blood islands (Choi et al., Development, 125(4):725-732 (1998)). Hemangioblasts then have the ability to give rise to both primitive and definitive hematopoietic cells, HSCs, and endothelial cells (Mikkola et al., J. Hematother. Stem Cell Res, 11(1):9-17 (2002)).

[0123]

[0222] Hemangioblasts can be derived in vitro from both mouse PSCs (Kennedy et al., Nature (386):488-493 (1997); Perlingeiro et al., Stem Cells (21):272-280 (2003)) and human PSCs (References 14, 15; Yu et al., Blood 2010, Vol. 116:4786-4794). Other studies have claimed to have isolated hemangioblasts from umbilical cord blood (Bordoni et al., Hepatology, 45(5):1218-1228), from peripheral blood-derived circulating CD34-lin-CD45-CD133- cells (Ciraci et al., Blood, 118:2105-2115), and from mouse uterus (Sun et al., Blood, 116(16):2932-2941 (2010)). Both mouse and human PSC-derived hemangioblasts have been obtained through the culture and differentiation of clusters of cells grown in liquid culture, followed by expansion of the cells in semi-solid media containing various cytokines and growth factors (Kennedy and Perlingeiro, 14, 15). See also U.S. Pat. No. 8,017,393, incorporated herein by reference in its entirety. In one embodiment, hemangioblasts can be generated in vitro from pluripotent stem cells according to the methods described in, for example, U.S. Patent No. 9,938,500, U.S. Patent No. 9,410,123, and WO2013 / 082543 (all of which are incorporated herein by reference in their entireties). The term hemangioblast also includes hemangio-colony-forming cells, described in U.S. Patent No. 8,017,393 (incorporated herein by reference in their entireties), which have the ability to differentiate into hematopoietic and endothelial cell lineages as well as the ability to become smooth muscle cells that are negative for CD34, CD31, KDR, and CD133. In another embodiment, hemangioblasts are positive for the blood markers CD43 and CD45 and express low levels or are negative for the pericyte markers CD146, PDGRb, and / or NG2.

[0124]

[0223] Hemangioblasts useful in the methods described herein can be derived or obtained by any of these known methods or those described herein. For example, embryoid bodies can be formed by culturing pluripotent cells under non-adherent conditions, such as on low-adhesion substrates, in the "hanging drop" method, or through an Able Biott spin bioreactor. In these cultures, PSCs can form aggregates or clusters of cells called embryoid bodies. See Itskovitz-Eldor et al., Mol Med. 2000 Feb. 6(2):88-95, the entire contents of which are incorporated by reference. Generally, embryoid bodies initially form as solid aggregates or clusters of pluripotent cells; however, over time, some embryoid bodies contain fluid-filled cavities; the former are referred to in the literature as "simple" embryoid bodies, and the latter as "cyst" embryoid bodies (ibid.). Cells within EBs (both solid and cyst forms) can differentiate, generating increasing numbers of cells over time. Optionally, the EBs are then cultured as adherent cultures, allowing for the formation of outgrowths. Similarly, pluripotent cells capable of overgrowth and the formation of multilayered cell populations can differentiate over long periods of time.

[0125]

[0224] In one embodiment, hemangioblasts are generated by a process comprising: (a) culturing a PSC line for 2, 3, 4, 5, 6, or 7 days to form clusters of cells (embryoid bodies; EBs); and (b) inducing the clusters of cells or EBs to differentiate into hemangioblasts. In a further embodiment, the clusters of cells or EBs obtained in step (b) are cultured in a cytokine-enriched, serum-free, methylcellulose-based medium. In one embodiment, hemangioblasts are generated by inducing differentiation of any pluripotent cell as described herein.

[0126]

[0225] In one embodiment, the clusters of cells or EBs are cultured in serum-free methylcellulose medium containing one or more components selected from the group including penicillin / streptomycin (pen / strp), EX-CYTE® growth cofactors (aqueous concentrate containing 9.0-11.0 g / L cholesterol, 13.0-18.0 g / L lipoproteins and fatty acids, pH 7-8.4), Flt3 ligand (FL), vascular endothelial growth factor (VEGF), thrombopoietin (TPO), basic fibroblast growth factor (bFGF), stem cell-derived factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL3), and interleukin-6 (IL6) for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days to generate hemangioblasts. In a preferred embodiment of the invention, hemangioblasts are harvested between days 6 and 14 of culture in, for example, serum-free methylcellulose plus one or more of the components of the preceding embodiments. In a preferred embodiment, one or more components may be present in the medium at the following concentrations: 50 ng / ml Flt3 ligand (FL), 50 ng / ml vascular endothelial growth factor (VEGF), 50 ng / ml thrombopoietin (TPO), 20-30 ng / ml basic fibroblast growth factor (bFGF), 50 ng / ml stem cell-derived factor (SCF), 20 ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng / ml interleukin-3 (IL-3), and 20 ng / ml interleukin-6 (IL-6). In vitro generation of mesenchymal stem cells

[0226] One embodiment of the present invention includes a method for generating mesenchymal stem cells (hereinafter "HMCs") by in vitro differentiation of hemangioblasts. The hemangioblasts are obtained by any of the methods described herein. In one form, the hemangioblasts are obtained by in vitro differentiation of pluripotent stem cells. The pluripotent stem cells can be cultured on feeders (e.g., human dermal fibroblasts or mouse embryonic fibroblasts) or in feeder-free conditions. In some embodiments, the hemangioblasts are cultured in feeder-free conditions and then seeded on an extracellular matrix. In another embodiment, the extracellular matrix is selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, heparan sulfate, a soluble preparation obtained from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, Matrigel, and human basement membrane extract. In still further embodiments, the extracellular matrix may be from any mammalian source, including human.

[0127]

[0227] In another embodiment, the hemangioblasts are replated and cultured for at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 days to form a preparation of HMCs. In one embodiment, initial seeding of hemangioblasts onto a substrate-coated tissue culture dish is at about 50,000 to about 100,000 cells / cm. 2 During the hemangioblast culture period, some hemangioblasts adhere to the culture plate and begin to differentiate into HMCs. The adhered cells are passaged every 3 to 6 days, or more than 6 days, for example, about 6 to 10 days, or about 10 to 15 days, depending on their growth rate, seeding density, and perceived degree of confluence. When passaged, the harvesting density is about 5,000 to about 20,000 cells / cm. 2 or approximately 20,000 to 40,000 cells / cm 2After the cells are harvested, the cells are counted and found to be approximately 2,500-6,000 cells / cm. 2 In one embodiment, HMCs are generated by the steps of (a) culturing ESCs for 8-12 days to generate hemangioblasts, (b) harvesting the hemangioblasts, (c) reseeding the hemangioblasts of step (b), and (d) culturing the hemangioblasts of step (c) for 14-30 days.

[0128]

[0228] In one embodiment, hemangioblasts are harvested, replated, and cultured in liquid medium under feeder-free conditions, i.e., without a feeder layer of cells (e.g., mouse embryonic fibroblasts, OP9 cells, or other cell types known to those skilled in the art). In a preferred embodiment, hemangioblasts are cultured on an extracellular matrix. In a more preferred embodiment, hemangioblasts are cultured on an extracellular matrix, wherein the matrix comprises a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells that gels at room temperature to form a reconstituted basement membrane (Matrigel). In an even more preferred embodiment, hemangioblasts are formed by the steps of: (a) culturing the hemangioblasts on the extracellular matrix for at least 7 days; and (b) transferring the hemangioblasts of step (a) to uncoated tissue culture plates and further culturing the hemangioblasts of step (b) for approximately 7 to 14 days. Hemangioblasts may be cultured in the presence of one or more factors selected from the group consisting of transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and / or platelet-derived growth factor (PDGF). In one embodiment, the extracellular matrix is selected from the group consisting of human basement membrane extract (BME) (e.g., Cultrex BME, Trevigen) or EHS matrix, laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen (e.g., collagen I, collagen IV), and heparan sulfate. The extracellular matrix or matrix component may be of mammalian, more specifically human, origin. In one embodiment, hemangioblasts are cultured on extracellular matrix protein-coated plates in a liquid medium containing serum, in which case the culture medium may contain components selected from αMEM (Sigma-Aldrich) supplemented with 10-20% fetal calf serum (FCS) (αMEM + 20% FCS), αMEM supplemented with 10-20% heat-inactivated human AB serum, and IMDM supplemented with 10-20% heat-inactivated AB human serum.

[0129]

[0229] In another embodiment, the hemangioblasts are cultured in medium containing serum or a serum replacement, such as αMEM supplemented with 20% fetal bovine serum, hi another embodiment, the hemangioblasts are cultured in serum-free medium.

[0130]

[0230] In further embodiments, the hemangioblasts are cultured on the extracellular matrix for about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In still further embodiments of the invention, HMCs are generated by a process comprising: (a) culturing hemangioblasts on the extracellular matrix for about 7 days; and (b) transferring the hemangioblasts from step (a) to uncoated tissue culture dishes and culturing them for an additional period of about 9 to 100 days, i.e., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 days. In yet another embodiment, HMCs are generated by a process comprising: (a) culturing hemangioblasts on an extracellular matrix for about 7 days; and (b) transferring the hemangioblasts from step (a) to a coated tissue culture dish and culturing them for an additional period of about 9-100 days, i.e., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 days.

[0131]

[0231] In one embodiment of the present invention, hemangioblasts are produced by the following steps: (a) culturing PSCs in the presence of vascular endothelial growth factor (VEGF) and / or bone morphogenetic protein 4 (BMP-4) (by way of non-limiting example) to form clusters of cells or EBs; (b) culturing the clusters of cells or EBs in the presence of at least one growth factor (e.g., basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), Flt3L (FL), thrombopoietin (TPO), and / or tPTD-HOXB4) in an amount sufficient to induce differentiation of the clusters of cells or EBs into hemangioblasts; and (c) culturing the hemangioblasts. and culturing the PSCs in a medium containing at least one additional growth factor (e.g., insulin, transferrin, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), and / or tPTD-HOXB4), wherein the at least one additional growth factor is provided in an amount sufficient to allow the proliferation of clusters of the cells in the culture, and optionally adding copper to any of steps (a)-(c).

[0132]

[0232] In one embodiment of the present invention, HMCs are generated by culturing hemangioblasts, wherein the hemangioblasts are produced by the steps of: (a) culturing PSCs in the presence of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 4 (BMP-4) to form cell clusters or EBs within 0 to 48 hours of initiating the culture; and (b) treating the cell clusters or EBs with at least one growth factor selected from the group consisting of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), Flt3L (FL), thrombopoietin (TPO), and tPTD-HOXB4 to induce differentiation of the cell clusters or EBs into hemangioblasts. and (c) culturing the hemangioblasts in a medium comprising at least one additional growth factor selected from the group consisting of insulin, transferrin, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), or tPTD-HOXB4, wherein the at least one additional growth factor is provided in the culture in an amount sufficient to propagate the hemangioblasts.

[0133]

[0233] In another embodiment, HMCs are produced by a step comprising: (a) harvesting hemangioblasts at least 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after inducing PSCs to differentiate into said hemangioblasts; and (b) harvesting HMCs produced within about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days after inducing said hemangioblasts from step (a) to differentiate into said mesenchymal cells.

[0134]

[0234] In yet another embodiment, a preparation of at least 80, 85, 90, 95, 100, 125, or 125 million HMCs is generated from about 200,000 hemangioblasts within about 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days of culturing the hemangioblasts, wherein said preparation of HMCs comprises about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 10%, 125, or 130 million HMCs. %, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% human embryonic stem cells. In yet another embodiment, at least 80, 85, 90, 100, 125, or 150 million HMCs are generated from about 200,000 hemangioblasts within about 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days of culturing the hemangioblasts. Extracellular vesicles secreted by mesenchymal stem cells

[0235] The present invention also provides extracellular vesicles isolated from, derived from, secreted from, or released from cells, such as HMCs of the present invention.

[0135]

[0236] As used herein, the term "extracellular vesicles" or "EVs" refers to lipid-bound vesicles secreted by cells into the extracellular space. There are three major subtypes of EVs: microvesicles (MVs), exosomes, and apoptotic bodies, which are distinguished based on their biogenesis, release pathway, size, contents, and function (Zaborowski MP et al., Bioscience. 2015; 65:783-797). Typically, extracellular vesicles range in diameter from 20 nm to 5000 nm and can contain a variety of macromolecular payloads, either contained within the internal space (i.e., lumen), displayed on the external surface of the EV, and / or present across the membrane. The payloads can include nucleic acids (e.g., microRNAs (miRNAs), long non-coding RNAs (lncRNAs), mRNAs, DNA fragments), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example, but not limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from / secreted by cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by viable cells (e.g., by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived from / secreted by living or dead organisms, isolated tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells.

[0136]

[0237] As used herein, the term "exosome" refers to small cell-derived vesicles containing a membrane surrounding an internal space (i.e., a lumen) that are formed from cells by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane (Yanez-Mo M. et al., J. Extracell. Vesicles. 2015;4:27066). Specifically, exosomes are involved in protein sorting, recycling, storage, transport, and release. Exosomes typically range in diameter from 20 to 300 nm. Exosomes are secreted by all cell types and have been found in plasma, urine, semen, saliva, bronchial fluid, cerebrospinal fluid (CSF), breast milk, serum, amniotic fluid, synovial fluid, tears, lymph, bile, and gastric acid.

[0137]

[0238] Exosomes have been found to be involved in cell-cell communication, cell maintenance, and tumor progression. In addition, exosomes have been found to stimulate immune responses by acting as antigen-presenting vesicles (Bobrie A. et al., Traffic. 2011;12:1659-1668). In the nervous system, exosomes have been found to help promote myelination, neurite outgrowth, and neuronal survival, thus playing a role in tissue repair and regeneration (Faure J. et al., Mol. Cell. Neurosci. 2006;31:642-648). At the same time, exosomes in the central nervous system (CNS) have been found to contain pathogenic proteins (e.g., beta-amyloid peptide, superoxide dismutase, and alpha-synuclein) that may contribute to disease progression (Fevrier B. et al., Proc. Natl. Acad. Sci. USA. 2004;101:9683-9688). Exosomes have also been identified as carriers of disease markers. The use of exosomes as carriers of biomarkers is ideal because these vesicles are found in bodily fluids (e.g., blood and urine), allowing for non-invasive or minimally invasive "liquid biopsy"-type approaches to diagnose patients and even monitor their response to treatment.

[0138]

[0239] In addition to their natural role in cell-cell interactions, exosomes can be loaded with different cargoes, such as drugs or exogenous nucleic acids or proteins, and deliver this cargo to different cells. The cargo can be bound to, embedded within, encapsulated within, or otherwise carried by the extracellular vesicles, or any combination thereof. Thus, as used herein, references to cargo being "present within" the extracellular vesicles or their lumen are understood to encompass any of the above means of delivering the cargo.

[0139]

[0240] The cargo may be endogenous cargo, exogenous cargo, or a combination thereof. Non-limiting examples of cargo that can be bound to, embedded in, encapsulated in, or otherwise transported by the extracellular vesicles described herein include nucleic acid molecules (e.g., DNA, cDNA, antisense oligonucleotides, mRNA, inhibitory RNA (antisense RNA, miRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), agomiR), antagomir, primary miRNA (pri-miRNA), long non-coding RNA (lncRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and microbial RNA), polypeptides (e.g., enzymes, antibodies), lipids, hormones, vitamins, minerals, small molecules, and pharmaceuticals, or any combination thereof. Importantly, exosomes are natural carriers of miRNA and other non-coding RNA, and can directly fuse with target cells via membrane fusion, allowing their contents to be directly delivered into the cytosol. This makes exosomes an excellent delivery system for small molecules (Lai RC et al., Biotechnol. Adv. 2013;31:543-551).

[0140]

[0241] Microvesicles are EVs formed by direct outward budding or shedding of the cell's plasma membrane. Microvesicles typically range in size from 100 to 1000 nm in diameter. The pathway of microvesicle formation is not fully understood, but it is thought to require cytoskeletal components, such as actin and microtubules, along with molecular motors (kinesins and myosins) and fusion machinery (SNAREs and tethering factors) (Cai H. et al., Dev. Cell. 2007;12:671-682). The number of microvesicles produced depends on the physiological state and microenvironment of the donor cells (Zaborowski MP et al., Bioscience. 2015;65:783-797). Similarly, it has been demonstrated that the number of microvesicles consumed depends on the physiological state and microenvironment of the recipient cells. Similar to exosomes, microvesicles are also involved in intercellular communication between local and distant cells. The ability of such EVs to alter recipient cells has been well documented (Harding CV et al., J. Cell Biol. 2013;200:367-371; White IJ et al., EMBO J. 2006;25:1-12). The uniqueness of EVs lies in their ability to package active cargo (proteins, nucleic acids, lipids) and deliver them to other cells, either nearby or distant, thereby altering the function of the recipient cells.

[0141]

[0242] Apoptotic bodies are secreted into the extracellular space by dying cells. Their diameters range from 50 nm to 5000 nm, with most apoptotic bodies tending toward the larger end (Borges F. et al., Braz. J. Med. Biol. Res. 2013;46:824-830). These bodies form when the cell's plasma membrane separates from the cytoskeleton as a result of increased hydrostatic pressure following cell shrinkage (Wickman G. et al., Cell Death Differ. 2012;19:735-742). The composition of apoptotic bodies is directly distinct from exosomes and microvesicles. Unlike exosomes and microvesicles, apoptotic bodies contain intact organelles, chromatin, and small amounts of glycosylated proteins (Borges F. et al., Braz. J. Med. Biol. Res. 2013;46:824-830; Thery C. et al., J. Immunol. 2001;166:7309-7318). Methods for isolating extracellular vesicles

[0243] The EVs of the present invention can be isolated, secreted, derived, or separated from culture media or other source material, e.g., HMCs of the present invention, using routine methods known in the art (see, e.g., Taylor et al., Serum / Plasma Proteomics, Chapter 15, "Extracellular vesicle Isolation for Proteomic Analyses and RNA Profiling," Springer Science, 2011; and Tauro et al., Methods 56 (2012) pp. 293-304, and references cited therein), and methods described in the Examples section below. The most commonly used methods involve multiple centrifugation and ultracentrifugation steps.

[0142]

[0244] Physical properties of EVs (e.g., HMC-EVs) can be used to isolate, purify, or enrich EVs, including separation based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieves, etc.), density (e.g., conventional or gradient centrifugation), or Svedberg constant (e.g., sedimentation with or without external force, etc.). Alternatively or additionally, isolation can be based on one or more biological properties, including methods that utilize surface markers (e.g., precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, nonspecific ligand binding, immunomagnetic capture of EVs using magnetic beads coated with antibodies targeting proteins exposed on the EV membrane, etc.).

[0143]

[0245] Methods based on the use of volume-excluding polymers, such as PEG, have been described recently by several different groups (US Patent Application Nos. 20130273544 and 20130337440). Two such products include ExoQuick (System Biosciences, Mountain View, USA) and Total Exosome Isolation Reagent (Life Technologies, Carlsbad, USA). These polymers function by binding water molecules and displacing poorly soluble components, such as extracellular endoplasmic reticulum and proteins, from solution, allowing their collection by brief, low-speed centrifugation.

[0144]

[0246] In some embodiments, isolation, purification, and enrichment can be performed in a general and non-selective manner (typically involving sequential centrifugation). Alternatively, isolation, purification, and enrichment can be performed in a more specific and selective manner (e.g., using producer cell-specific surface markers). For example, specific surface markers can be used in immunoprecipitation, FACS sorting, affinity purification, or bead-bound ligands for magnetic separation.

[0145]

[0247] In some embodiments, tangential flow filtration may be used to isolate or purify EVs (e.g., HMC-EVs).

[0248] In some embodiments, size-exclusion chromatography can be used to isolate or purify EVs (e.g., HMC-EVs). Size-exclusion chromatography techniques are known in the art. In some embodiments, density gradient centrifugation can be used to separate EVs. In some embodiments, isolation of EVs (e.g., HMC-EVs) can involve ion chromatography, such as anion exchange chromatography, cation exchange chromatography, or mixed-mode chromatography. In some embodiments, isolation of EVs (e.g., HMC-EVs) can involve desalting, dialysis, tangential flow filtration, ultrafiltration, or diafiltration, or any combination thereof. In some embodiments, isolation of EVs (e.g., HMC-EVs) can involve a combination of methods, including, but not limited to, differential centrifugation, size-based membrane filtration, concentration, and / or rate-zonal centrifugation. In some embodiments, isolation of EVs (e.g., HMC-EVs) can involve one or more centrifugation steps. Centrifugation can be performed at approximately 50,000 to 150,000 × g. Centrifugation can be performed at approximately 50,000×g, 75,000×g, 100,000×g, 125,000×g, or 150,000×g. In another embodiment, EVs (e.g., HMC-EVs) are separated from non-membranous particles by utilizing their relatively low buoyant density (Raposo et al., 1996; Escola et al., 1998; van Niel et al., 2003; Wubbolts et al., 2003). Kits for such isolation are commercially available, for example, from Qiagen, InVitrogen, and SBI. Methods for loading EVs with therapeutic agents are known in the art and include lipofection, electroporation, and any standard transfection method.

[0146]

[0249] In some embodiments, the present invention provides methods for isolating HMC-EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells. The methods include providing HMCs obtained by in vitro differentiation of pluripotent stem cells and isolating extracellular vesicles. HMC-EVs can be isolated by any method known in the art or described herein. In some embodiments, HMC-EVs are isolated by tangential flow filtration. In some embodiments, HMC-EVs are isolated by ultracentrifugation. In some embodiments, HMC-EVs are isolated by cation exchange chromatography. In some embodiments, HMC-EVs are isolated by anion exchange chromatography. Characteristics and composition of HMCs and / HMC-EVs

[0250] The present invention further provides compositions comprising HMCs obtained by in vitro differentiation of pluripotent stem cells and / or extracellular vesicles secreted from the HMCs (HMC-EVs) of the present invention. In one embodiment, HMCs are obtained by in vitro differentiation of hemangioblasts. The expression level of a particular phenotypic marker can be determined by any method known in the art, such as immunohistochemistry. The expression of a particular gene can be determined by any method known in the art, such as RT-PCR or RNA-Seq.

[0147]

[0251] In one embodiment, HMCs of the invention express at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight markers selected from the group comprising CD9, CD13, CD29, CD44, CD73, CD90, CD105, CD166, and HLA-ABC. In yet a further embodiment, HMCs of the invention express at least two, at least three, at least four, at least five, or at least six markers selected from the group consisting of CD9, CD13, CD29, CD44, CD73, CD90, and CD105, with the proviso that said HMCs do not express CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD61, CD62E, and CD133. In another embodiment, the HMCs of the present invention express at least one, at least two, at least three, at least four, at least five, or at least six markers selected from the group consisting of AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1.

[0148]

[0252] In one embodiment, the composition comprises HMCs, and about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the HMCs express CD9, CD13, CD29, CD44, CD73, CD90, CD105, CD166, and HLA-abc after about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days in culture. In one embodiment of the invention, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the HMCs in a composition of the invention express, in culture, at least two, at least three, at least four, at least five, or at least a few genes selected from the group consisting of CD9, CD13, CD29, CD44, CD73, CD90, CD105, CD166, and HLA-ABC. HMCs express at least six, at least seven, or at least eight markers and lack expression of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD61, CD62E, CD133, and Stro-1 after about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. HMCs included in the compositions of the invention may further express at least one, at least two, at least three, at least four, at least five, or at least six markers selected from the group consisting of AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1.

[0149]

[0253] In one embodiment, the composition comprises HMCs, and at least 30% of the HMCs are positive for CD10. Additionally, at least 60% of the HMCs may be positive for the following markers: CD73, CD90, CD105, CD13, CD29, CD44, and CD166, and HLA-ABC. In an exemplary embodiment, less than 30% of the HMCs may be positive for the following markers: CD31, CD34, CD45, CD133, FGFR2, CD271, Stro-1, CXCR4, and TLR3.

[0150]

[0254] In another embodiment, the composition comprises HMCs, and at least 50% of the HMCs become positive for CD105 or CD73 within about 7-20 days (e.g., 15 days) of culture. In a preferred embodiment of the invention, at least 50% of the HMCs become positive for CD105 or CD73 after about 7-15 days of culture. In a further embodiment of the invention, at least 80% of the HMCs become positive for CD105 and CD73 within about 20 days of culture. In yet a further embodiment of the invention, at least 80% of a composition of HMCs become positive for CD105 and CD73 within about 20 days of culture.

[0151]

[0255] In one embodiment, the composition comprises HMCs, and at least 20%, 30%, 40%, or 50% of the HMCs may be positive for (i) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90, (ii) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC, (iii) CD105, CD73, and / or CD90, or (iv) any combination thereof. At least 20%, 30%, 40%, or 50% of the HMCs may be positive for (i) at least two of CD105, CD73, and / or CD90, (ii) at least two of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90, or (iii) all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC. At least 20%, 30%, 40%, or 50% of the HMCs may be (i) positive for CD105, CD73, and CD90, (ii) positive for CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC, and / or (ii) negative for CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, and / or TLR3, or express them on less than 5% or less than 10% of the cells.At least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the HMCs may be positive for one or more of: (i) CD105, CD73, and CD90; (ii) one or more of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90; or (iii) one or more of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC.

[0152]

[0256] In another embodiment, the composition comprises HM, wherein at least 20%, 30%, 40%, or 50% of said HMCs (i) may be positive for all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC, and (ii) may be negative for CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, and / or TLR3, or express them in less than 5% or less than 10% of the cells.

[0153]

[0257] In further embodiments, the composition comprises HMCs, and at least 20%, 30%, 40%, or 50% of said HMCs may be positive for (i) all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90, or (ii) all of CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC.

[0154]

[0258] In yet another embodiment, the composition comprises HMCs, and at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of said HMCs may be positive for (i) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90, or (ii) at least one of CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC.

[0155]

[0259] In another embodiment, HMCs may not express CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, or TLR3, or less than 5% or less than 10% of HMCs may express at least one of them.

[0156]

[0260] In addition to the above characteristics, HMCs of the present invention may have a more youthful cellular phenotype compared to adult-derived MSCs. In one embodiment, HMCs have the capacity to undergo at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more population doublings in culture. In contrast, adult-derived MSCs typically undergo only 2-3 population doublings in culture. In another embodiment, HMCs of the present invention have longer telomere lengths, more immunosuppressive effects, fewer vacuoles, divide rapidly, divide easily in culture, express more CD90, show less lineage commitment, or a combination thereof, compared to adult-derived MSCs. In another embodiment, HMCs of the present invention have increased expression of transcripts that promote cell proliferation (i.e., have higher reproductive potential) and decreased expression of transcripts involved in terminal cell differentiation compared to adult-derived MSCs.

[0157]

[0261] In one embodiment, the HMCs are "early passage" HMCs and may be passaged no more than 1, 2, 3, 4, 5, 6, 7, or 8 times. In one embodiment, the early passage HMCs are passaged no more than 4 times. In another embodiment, the early passage HMCs are passaged no more than 5 times. In another embodiment, the early passage HMCs are passaged no more than 6 times. In addition to the HMC characteristics described above, the early passage HMCs may express mRNA encoding interleukin-6 (IL-6) in a resting or basal state at a level that may be less than 10 percent of the IL-6 mRNA level expressed by BM-MSCs or AD-MSCs in a resting or basal state. VEGF mRNA levels may also be downregulated in early passage HMCs in a resting or basal state compared to BM-MSCs in a resting or basal state. In another embodiment, HMCs may express mRNA encoding CD24 in a resting or basal state at levels greater than the CD24 mRNA levels expressed by resting or basal BM-MSC or AD-MSC preparations. Other mRNAs that may be upregulated in early-passage HMCs in a resting or basal state compared to resting or basal BM-MSCs include AIRE, ANGPT1 (ANG-1), CXCL1, CD10, and IL-11. Additionally, early-passage HMCs in a resting or basal state may be negative for one or more of the mRNAs encoding ANGPT2, CD31, CD34, CD45, HLA-G, IL2RA, IL3, and IL12B.

[0158]

[0262] In a further embodiment, the early passage HMCs express one or more markers selected from the group consisting of CD13, CD29, CD44, CD73, CD90, CD105, CD166, and HLA-ABC as determined by immunohistochemistry. In another embodiment, the early passage HMCs are negative for one or more markers selected from the group consisting of CD31, CD34, CD45, CXCR4, HLA-DR, FGFR2, TLR3, CD106, CD133, and CD271 as determined by immunohistochemistry.

[0159]

[0263] In one embodiment, the expression level of CD10 is upregulated in early-passage HMCs compared to the expression level of CD10 in BM-MSCs, as determined by immunohistochemistry. In another embodiment, the expression level of CD10 in early-passage HMCs can be approximately the same as the expression level of CD10 in BM-MSCs. In another embodiment, the expression level of Stro-1 is downregulated in early-passage HMCs of the present invention compared to the expression level of Stro-1 in BM-MSCs, as determined by immunohistochemistry. In a specific embodiment, the composition comprises early-passage HMCs, and approximately 5-10% of the early-passage HMCs express Stro-1.

[0160]

[0264] In further embodiments, HMCs of the invention express higher levels of certain genes compared to BM-MSCs, UCB-MSCs, or AD-MSCs. For example, HMCs of the invention may express higher levels of any of the genes listed in Table 3 compared to BM-MSCs, and / or any of the genes listed in Table 5 compared to UCB-MSCs, and / or any of the genes listed in Table 7 compared to AD-MSCs. In another embodiment, HMCs of the invention may express lower levels of any of the genes listed in Table 4 compared to BM-MSCs, and / or any of the genes listed in Table 6 compared to UCB-MSCs, and / or any of the genes listed in Table 8 compared to AD-MSCs.

[0161]

[0265] In one embodiment, genes associated with increased migration and chemotaxis, such as MMP9, are expressed at higher levels in HMCs of the present invention compared to BM-MSCs or UCB-MSCs. In another embodiment, Lgr5, a marker of multipotent stem cells, is expressed at higher levels in HMCs of the present invention compared to BM-MSCs or UCB-MSCs. In a further embodiment, CD24 is expressed at higher levels in HMCs of the present invention compared to BM-MSCs, and IL-6 is expressed at lower levels in MSCs of the present invention compared to BM-MSCs. In yet another embodiment, neural-related genes, such as NGF, NTF-4, NTRK-2, NTRK-3, and DCC (netrin-1), are expressed at higher levels in HMCs of the present invention compared to BM-MSCs or UCB-MSCs. MSCs of the present invention can be selected or purified based on differentially expressed genes.

[0162]

[0266] In some embodiments, HMCs of the invention may express lower levels of any of the miRNAs listed in Table 21 compared to HMC-EVs. In some embodiments, HMCs of the invention may express higher levels of any of the miRNAs listed in Table 22 compared to HMC-EVs.

[0163]

[0267] In a further embodiment, the HMC-EVs of the present invention express a higher level of a particular miRNA, gene, or protein compared to BM-MSC-EVs, UCB-MSC-EVs, or AD-MSC-EVs.

[0164]

[0268] In some embodiments, HMC-EVs of the present invention may express higher levels of any miRNA listed in Table 9 compared to UCB-MSC-EVs, and / or any miRNA listed in Table 11 compared to BM-MSC-EVs, and / or any miRNA listed in Table 13 compared to AD-MSC-EVs. In other embodiments, HMC-EVs of the present invention may express lower levels of any miRNA listed in Table 10 compared to UCB-MSC-EVs, and / or any miRNA listed in Table 12 compared to BM-MSC-EVs, and / or any miRNA listed in Table 13 compared to AD-MSC-EVs. In some embodiments, HMC-EVs of the present invention may express higher levels of any protein listed in Table 15 compared to UCB-MSC-EVs, and / or any protein listed in Table 17 compared to BM-MSC-EVs, and / or any miRNA listed in Table 19 compared to AD-MSC-EVs. In another embodiment, HMC-EVs of the present invention may express lower levels of any of the proteins listed in Table 16 compared to UCB-MSC-EVs, and / or any of the proteins listed in Table 18 compared to BM-MSC-EVs, and / or any of the proteins listed in Table 20 compared to AD-MSC-EVs.

[0165]

[0269] In some embodiments, HMC-EVs express at least one miRNA selected from the group consisting of hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-199b-5p, hsa-miR-21-5p, hsa-miR-23a-3p, hsa-miR-125a-5p, hsa-miR-106a-5p+hsa-miR-17-5p, and hsa-miR-221-3p at a higher level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0166]

[0270] In some embodiments, the HMC-EVs are selected from the group consisting of ALDOC, ANXA5, APBB2, BASP1, CAV1, CD81, CD99, CKM, EPB41L3, FDPS, GNAQ, GNG12, GP9, H2AC20, H2AC21, H3-3A, H3-7, H4-16, HLA-A, ITGA2, KPNA2, KRAS, KRT4, LRRC59, MAMDC2, MARCKSL1, MDGA1, MERTK, MFGE8, MMP14, MVP, PCDH1 , PDGFRB, PDIA3, RPL13, RPS18, RPS3A, RPS4X, SDCBP, SLC2A1, SLC3A2, TAGLN2, TNC, TSPAN14, TSPAN33, TSPAN9, TTYH3, UCHL1, VAT1, YWHAB, and YWHAQ at a higher level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0167]

[0271] In some embodiments, the HMC-EVs are selected from the group consisting of ADGRG6, AGRN, ANXA6, APOC4, ARHGAP1, ARGHDIA, ARL8A, ARPC5, B2M, BBS1, BLVRA, BST1, CA2, CCN2, CCNB3, CD34, CD36, CD47, CORO1A, DTD1, EEF1D, EEF1G, ENG, ESD, GNAI2, GNB1, H1-3, H2BC15, HIP1, KIF11, LAMP1, LAP3, LGALS1, LTBP3, MAPK3, MARCKS, MBTD1, MDH1, MOB1B, MYL12B, MYO1F, MYO3A, NIBAN2, PEBP1 , PF4, PGAP1, PLOD1, PPP2R1A, PRSS23, PXDN, RALA, RAP2A, RPS13, RPS3, RPSA, S100A11, SLC44A1, SLC44A2, SLTM, SMG1, SPARC, SRSF8, STRADB, STX11, STXBP2, TGM2, TPP1, TPTE2, TRIM5, TRPM2, TUBA8, TUBB3, VCAN, YWHAE, and ZFN607 at a higher level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0168]

[0272] In some embodiments, HMC-EVs express at least one protein selected from the group consisting of ADIPOQ, CAT, CEP290, IGLV6-57, TAS2R33, and TMEM198 at a lower level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0169]

[0273] In some embodiments, HMC-EVs express at least one protein selected from the group consisting of AKAP9, ALB, ALOX5, APLP2, CD109, CDSN, CHST9, ERC1, F11, ARMCX5, LAMB4, LRRTM2, LTF, MSH6, OAF, OLFML3, PAK6, RGS14, SEMA7A, SURF1, and TRIM4 at a lower level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0170]

[0274] In some embodiments, HMC-EVs of the invention may express higher levels of any of the miRNAs listed in Table 21 compared to HMCs of the invention. In some embodiments, HMC-EVs of the invention may express lower levels of any of the miRNAs listed in Table 22 compared to HMCs of the invention.

[0171]

[0275] In one embodiment, genes associated with or involved in the development of neuronal lineages, including axon guidance, CREB signaling in neurons, synaptogenesis signaling, or neuroinflammatory signaling, are expressed at higher levels in HMCs of the present invention compared to AD-MSCs or BM-MSCs.

[0172]

[0276] In another embodiment, the HMCs of the present invention have a different expression profile compared to mature MSCs, such as AD-MSCs, BM-MSCs, or UCB-MSCs. Specifically, the HMCs of the present invention can confer neuroprotective effects and provide neurotrophic factors (i.e., factors involved in supporting neuronal survival, growth, health, and recovery). Similarly, the HMC-EVs of the present invention share a similar profile to the HMCs from which they are derived. Similar signaling pathways that are enriched in HMCs compared to MSCs and EVs derived from other tissues are also enriched in HMC-EVs.

[0173]

[0277] In one embodiment, the compositions comprising HMCs of the present invention are substantially purified with respect to pluripotent stem cells. In a further embodiment, the compositions of HMCs of the present invention are substantially purified with respect to pluripotent stem cells such that the compositions comprise at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% HMCs. The pluripotent stem cells can be any pluripotent stem cell described herein.

[0174]

[0278] The composition may be about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%. %, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% pluripotent stem cells. The composition may lack pluripotent stem cells.

[0175]

[0279] In some embodiments, compositions comprising HMC-EVs of the invention are substantially purified with respect to HMCs. In further embodiments, compositions of HMC-EVs of the invention are substantially purified with respect to HMCs, such that the compositions comprise at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% HMC-EVs.

[0176]

[0280] The compositions may be approximately 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0. 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% HMC.

[0177]

[0281] In another embodiment of the invention, a composition comprising HMCs and / or HMC-EVs produced by any one or more of the processes of the invention does not form teratomas when introduced into a host.

[0178]

[0282] In an exemplary embodiment, the present disclosure provides at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 pieces or 10 9 In a particular embodiment, the composition comprises 10 6HMCs and less than 1 percent of any other cell type, where the mesenchymal stem cells have the replicative capacity to undergo at least 10 population doublings in cell culture, and where less than 25 percent of the cells undergo cell death, senescence, or differentiation into non-HMC cells by the 10th population doubling.

[0179]

[0283] The HMCs may have a replication rate that undergoes at least 10 population doublings in cell culture in less than 25 days. The HMCs may have an average terminal restriction fragment length (TRF) greater than 8 kb. The HMCs may have a statistically significant greater decrease in protein content and / or enzyme activity involved in one or more of: (i) cell cycle regulation and cellular senescence, (ii) cellular energy and / or lipid metabolism, and (iii) apoptosis compared to bone marrow-derived mesenchymal stem cell preparations that have undergone five population doublings. The HMCs may have a statistically significant greater increase in protein content and / or enzyme activity involved in cytoskeletal structure and associated cellular dynamics compared to bone marrow-derived mesenchymal stem cell preparations. The HMCs do not experience a greater than 75 percent increase in cells with a forward scatter value greater than 5,000,000 (as measured by flow cytometry) over 10 population doublings in culture.

[0180]

[0284] In one embodiment of the invention, a preparation of subject HMCs (e.g., produced by culturing hemangioblasts) is provided, wherein the preparation contains substantially similar levels of p53 and p21 protein, or wherein the level of p53 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher compared to p21. In one embodiment of the invention, a pharmaceutical preparation of subject HMCs (e.g., produced by culturing hemangioblasts) is provided, wherein the pharmaceutical preparation contains substantially similar levels of p53 and p21 protein, or wherein the level of p53 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher compared to p21.

[0181]

[0285] In one embodiment, the invention provides a composition comprising HMC, wherein the composition comprises a substantially similar percentage of HMC positive for p53 and p21 protein, or the percentage of HMC positive for p53 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher compared to p21.

[0182]

[0286] In one embodiment, the present disclosure provides a method for treating a pulmonary artery disease comprising administering to a subject a subject a pulmonary artery disease (PAGE) comprising administering to ... 3 ~about 10 13 In another embodiment, the present disclosure provides a composition comprising at least 10 HMC-EVs. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , or 10 13 A composition containing HMC-EVs is presented. Methods for determining neurite outgrowth of HMC and / or HMC-EV populations

[0287] The present invention also provides methods for determining the effects of HMCs and / or HMC-EVs on neurons, such as neurite outgrowth. In one aspect, the present invention provides a method for determining neurite outgrowth of an HMC and / or HMC-EV population. In one embodiment, the method includes the steps of: (a) preparing a mixed neuronal culture from isolated cerebral cortex; (b) seeding the HMC and / or HMC-EV population onto a permeable membrane; (c) applying strain to the mixed neuronal culture; (d) overlaying the strained mixed neuronal culture with the permeable membrane of step (b); and (e) measuring neurite outgrowth of the mixed neuronal culture. In one embodiment, the method further includes determining gene expression in the mixed neuronal culture in the presence and absence of the HMC and / or HMC-EV population. In another embodiment, the strain is physical scratching of the mixed neuronal culture. In another embodiment, strain is applied to the mixed neuronal culture by vacuum pressure and positive air pressure. In yet another embodiment, strain can be applied as a 15% to 0% stretching oscillation. In one embodiment, the stretching oscillation can be applied at a cycle of 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 0%. Pharmaceutical preparations containing HMC and HMC-EVs

[0288] Pharmaceutical preparations of the present invention may include any of the HMC or HMC compositions and / or HMC-EVs described herein. Pharmaceutical preparations containing the HMC and / or HMC-EV of the present invention may be formulated with a pharmaceutically acceptable carrier. For example, the HMC and / or HMC-EV of the present invention may be administered alone or as a component of a pharmaceutical preparation, in which case the HMC and / or HMC-EV may be formulated for administration in any convenient manner for use in medical care. One embodiment provides a pharmaceutical preparation comprising the HMC and / or HMC-EV in combination with one or more pharmaceutically acceptable, isotonic, sterile, aqueous or non-aqueous solutions selected from the group consisting of dispersions, suspensions, emulsions, sterile powders, optionally reconstituted into sterile injectable solutions or dispersions immediately prior to use, antioxidants, buffers, bactericides, solutes, or suspension thickeners.

[0183]

[0289] Exemplary pharmaceutical preparations of the present disclosure can be any formulation suitable for use in treating human patients, such as pyrogen-free or substantially pyrogen-free, and pathogen-free.

[0184]

[0290] The preparations containing HMC and / or HMC-EVs used in the methods described herein can be implanted into suspensions, gels, colloids, slurries, or mixtures. Also, at the time of injection, cryopreserved HMC and / or HMC-EVs can be resuspended using commercially available balanced salt solutions to achieve the desired osmolality and concentration for administration by injection (i.e., bolus or intravenous).

[0185]

[0291] One aspect of the present invention is to 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , or 10 13Yet another aspect of the present invention relates to a pharmaceutical preparation suitable for use in a mammalian patient, comprising at least 10 HMC and / or HMC-EVs, and a pharmaceutically acceptable carrier. 8 , 10 9 , 10 10 , 10 11 , 10 12 pieces or 10 13 Yet another aspect of the present invention provides a frozen cell bank that is free or substantially free of non-human cells and / or non-human animal products and that contains at least 10 HMCs and / or HMC-EVs. 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , or 10 13 The present invention provides pharmaceutical preparations comprising HMCs and / or HMC-EVs and containing less than 1% of any other cell type, more preferably less than 0.1%, 0.01%, or 0.001% of any other cell type.

[0186]

[0292] Pharmaceutical preparations comprising HMC and / or HMC-EVs may be administered at any effective concentration, and may be substantially free of, for example, PSCs. For example, pharmaceutical preparations may contain multiple numbers and types of HMC and / or HMC-EVs described herein. In certain embodiments, pharmaceutical preparations comprising HMC and / or HMC-EVs, when administered systemically to a host in need thereof, may be administered at concentrations of about 1 x 10 6 ~Approx. 1×10 7 , about 1×10 7 ~Approx. 1×10 8 , about 1×10 8 ~Approx. 1×10 9 , about 1×10 9 ~Approx. 1×10 10 , about 1×10 10 ~Approx. 1×10 11 , about 1×10 11 ~Approx. 1×10 12, or approximately 1 × 10 12 ~Approx. 1×10 13 When administered locally to a host containing or in need of HMCs and / or HMC-EVs, approximately 1 × 10 4 ~Approx. 1×10 5 , about 1×10 5 ~Approx. 1×10 6 , 1×10 6 ~Approx. 1×10 7 , about 1×10 7 ~Approx. 1×10 8 , about 1×10 8 ~Approx. 1×10 9 , about 1×10 9 ~Approx. 1×10 10 , about 1×10 10 ~Approx. 1×10 11 , about 1×10 11 ~Approx. 1×10 12 , or approximately 1 × 10 12 ~Approx. 1×10 13 The HMC and / or HMC-EV are included. Methods for Treating Brain Injury

[0293] The HMCs and / or HMC-EVs described herein, as well as pharmaceutical preparations containing HMCs and / or HMC-EVs, can be used to treat brain injuries, such as stroke or optic neuropathy. In particular, the present invention provides a method for treating or preventing the brain injuries described herein, comprising administering an effective amount of HMCs and / or HMC-EVs, wherein the HMCs are obtained by in vitro differentiation of pluripotent stem cells. In another embodiment, the HMCs are obtained by in vitro differentiation of hemangioblasts.

[0187]

[0294] In one embodiment, the brain injury is selected from traumatic brain injury, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, cerebral aneurysm, coma, stroke, optic neuropathy, and cerebral palsy.In a specific embodiment, the brain injury is traumatic brain injury.In another embodiment, the brain injury is cerebral palsy.In yet another embodiment, the brain injury is stroke.In another embodiment, the brain injury is optic neuropathy.

[0188]

[0295] The HMC and / or HMC-EV of the present invention can be administered systemically or locally. Depending on the specific pathology being treated, the HMC and / or HMC-EV can be administered using methods known in the art, including, but not limited to, injection via intravenous, intracranial, intrathecal, intracerebral, intracisternal, intramuscular, intraperitoneal, intravitreal, or other administration routes, or by local implantation.

[0189]

[0296] The HMC and / or HMC-EV of the present invention can be administered via local implantation, such as intracranial implantation, using a delivery device. The delivery device of the present invention is biocompatible and biodegradable. The delivery device of the present invention can be made of a material selected from the group consisting of biocompatible fibers, biocompatible threads, biocompatible foams, aliphatic polyesters, poly(amino acids), copoly(ether esters), polyalkylene oxalates, polyamides, tyrosine-derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamide esters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, biopolymers; homopolymers and copolymers of lactide, glycolide, epsilon-caprolactone, para-dioxanone, trimethylene carbonate; homopolymers and copolymers of lactide, glycolide, hydroxybenzoates ... The HMC and / or HMC-EVs can be manufactured using materials selected from the group including: methacrylate, epsilon-caprolactone, para-dioxanone, trimethylene carbonate, fibrillar collagen, non-fibrillar collagen, pepsin-treated collagen, collagen combined with other polymers, growth factors, extracellular matrix proteins, biologically relevant peptide fragments, hepatocyte growth factor, platelet-derived growth factor, platelet-rich plasma, insulin growth factor, growth differentiation factor, vascular endothelial cell-derived growth factor, nicotinamide, glucagon-like peptide, tenascin-C, laminin, anti-rejection agents, analgesics, antioxidants, anti-apoptotic agents, anti-inflammatory agents, and cytostatic agents. In some embodiments, the HMC and / or HMC-EVs are delivered via a sustained-release device, e.g., a transdermal microneedle patch.

[0190]

[0297] The specific treatment regimen, route of administration, and adjunctive therapy can be tailored based on the specific pathology, the severity of the pathology, and the patient's overall health. Administration of HMC and / or HMC-EV can be effective in reducing the severity of the symptoms of the pathology and / or preventing further deterioration of the symptoms of the pathology.

[0191]

[0298] In some embodiments, administration of HMC causes preservation of myelin. In some embodiments, administration of HMC causes suppression of neuroinflammatory responses in a subject. In some embodiments, administration of HMC causes reduced microglial activation and reduced astrocyte activation in the brain. In some embodiments, administration of HMC causes stimulation and / or activation of pathways involved in cell survival. In some embodiments, administration of HMC causes stimulation of neuroprotective gene expression in the brain. In some embodiments, the neuroprotective gene is selected from the group consisting of heat shock protein family B member 1 (HSPB1), insulin-like growth factor 1 (IGF2), and secreted phosphoprotein 1 (SPP1). In some embodiments, administration of HMC causes stimulation and / or activation of pathways involved in synaptic transmission in the brain. In some embodiments, administration of HMC causes reduced apoptosis. In some embodiments, administration of HMC causes stimulation and / or activation of pathways involved in neuronal lineage development, such as axon guidance, BREB signaling in neurons, or synaptogenesis signaling.

[0192]

[0299] In some embodiments, administration of HMC-EVs causes an increase in oligodendrocytes and progenitor cells in the brain. In some embodiments, administration of HMC-EVs causes preservation of myelin in the brain. In some embodiments, administration of HMC-EVs causes suppression of neuroinflammatory responses in a subject. In some embodiments, administration of HMC-EVs causes a decrease in microglial activation and astrocyte activation in the brain. In some embodiments, administration of HMC-EVs prevents or reduces oxidative damage in neurons. In some embodiments, administration of extracellular HMC-EVs prevents or reduces neuronal death caused by glutamate excitotoxic injury.

[0193]

[0300] The treatment methods of the present invention may include administration of a single dose of HMC and / or HMC-EV. Alternatively, the treatment methods described herein may include a course of therapy in which HMC and / or HMC-EV are administered multiple times over a period of time. Exemplary treatment courses may include treatment once per week, once every two weeks, once per month, once per quarter, once every two years, or once per year. Alternatively, treatment may progress in stages, whereby multiple administrations are required initially (e.g., daily administration for the first week), followed by less frequent administrations.

[0194]

[0301] HMC and / or HMC-EV can be administered individually or in combination. In some embodiments, the method includes administering an effective amount of HMC to a subject. In other embodiments, the method includes administering an effective amount of HMC-EV to a subject. In another embodiment, the method includes administering an effective amount of HMC and an effective amount of HMC-EV to a subject.

[0195]

[0302] HMC and HMC-EV can be administered simultaneously or sequentially. In one embodiment, HMC and HMC-EV are mixed together before being administered to a subject. In another embodiment, a subject is administered an effective amount of HMC, followed by an effective amount of HMC-EV. Alternatively, a subject is administered an effective amount of HMC-EV, followed by an effective amount of HMC.

[0196]

[0303] In one embodiment, HMC and / or HMC-EV are administered to a patient one or more times periodically throughout the patient's lifetime. In further embodiments of the present invention, HMC and / or HMC-EV are administered once a year, once every 6 to 12 months, once every 3 to 6 months, once every 1 to 3 months, or once every 1 to 4 weeks. Alternatively, more frequent administration may be desirable for certain conditions or disorders. In one embodiment of the present invention, HMC and / or HMC-EV are administered via a device once, twice or more periodically throughout the patient's lifetime, or as needed for the specific patient and the patient's pathology being treated. Treatment regimens that change over time are also contemplated. For example, more frequent treatments may be required at the onset of the disease (e.g., daily or weekly treatments). Over time, as the patient's condition improves, less frequent treatments may be required, or even no further treatments may be necessary.

[0197]

[0304] In some embodiments, about 20 million, about 40 million, about 60 million, about 80 million, about 100 million, about 120 million, about 140 million, about 160 million, about 180 million, about 200 million, about 220 million, about 240 million, about 260 million, about 280 million, about 300 million, about 320 million, about 340 million, about 360 million, about 380 million, about 400 million, about 420 million, about 440 million, about 460 million, about 480 million, about 500 million, about 520 million, About 540 million, about 560 million, about 580 million, about 600 million, about 620 million, about 640 million, about 660 million, about 680 million, about 700 million, about 720 million, about 740 million, about 760 million, about 780 million, about 800 million, about 820 million, about 840 million, about 860 million, about 880 million, about 900 million, about 920 million, about 940 million, about 960 million, or about 980 million MSCs and / or MSC-EVs are administered into a subject. In some embodiments, about 1 billion, about 2 billion, about 3 billion, about 4 billion, or about 5 billion or more HMCs and / or HMC-EVs are administered. In some embodiments, the number of HMCs and / or HMC-EVs ranges from about 20 million to about 4 billion, from about 40 million to about 1 billion, from about 60 million to about 750 million, from about 80 million to about 400 million, from about 100 million to about 350 million, and from about 175 million to about 250 million.

[0198]

[0305] The methods described herein may further include monitoring the effectiveness of the treatment or prevention using methods known in the art. [Example]

[0199]

[0306] The following examples are not intended to limit the invention in any way. Example 1 - Preparation of HMC from hemangioblasts

[0307] Hemangioblasts were generated from a single-blastomere-derived human ESC line, MA09 (Klimanskaya et al., Nature, 444 (2006) 481-485). First, 10 cm plates were coated with 0.1% gelatin and irradiated MEFs were added the day before ESCs were added to the plates. Approximately 25,000 cells / cm were cultured in MEF medium (high-glucose DMEM + 10% FCS). 2 The MEF medium was then aspirated, rinsed with PBS, and replaced with Reprocell Primate medium (Reprocell) supplemented with 10 ng / mL bFGF. Aliquots of MA09 cells were added to culture dishes and fed daily with fresh medium. MA09 cells were cultured in Reprocell Primate Media + 10 ng / mL bFGF until approximately 90% confluent. MA09 cells were then harvested using 0.05% trypsin / EDTA or Reprocell dissociation buffer (Reprocell). After cell detachment, the cells were washed and harvested. The cells were spun down at 300 × g for 10 minutes. The supernatant was aspirated, and the cell pellet was resuspended in Stemline II (Sigma) (plus pen / strep and L-glutamine) + 50 ng / mL VEGF and 50 ng / mL BMP4. MA09 ESCs were plated in 2 × 10 cm ultra-low attachment plates (Corning) in 15 ml of Stemline II medium (Sigma) supplemented with 50 ng / ml VEGF and 50 ng / ml BMP-4 (R&D or Peprotech) and incubated at 37°C, 5% CO 2 After 40–48 hours, half of the medium (1.5 ml) was replaced with fresh Stemline II medium supplemented with 50 ng / ml VEGF, 50 ng / ml BMP-4, and 40–45 ng / ml bFGF to a final bFGF concentration of 20–22.5 ng / ml bFGF, and incubation was continued for another 40–48 hours (i.e., a total of 3.5–4 days).

[0200]

[0308] Clusters of cells (embryoid bodies; EBs) were dissociated and plated as single cells in serum-free semisolid blast colony growth medium (BGM). Specifically, clusters of cells were dissociated with trypsin for 2–5 minutes, or until clumps began to break up. The cell suspension was pipetted up and down, followed by the addition of DMEM + 10% FCS to inactivate the trypsin. The cells were then passed through a 40 μm or 70 μm strainer to obtain a single-cell suspension. Cells were then counted and plated at 1–1.5 × 10 6 The cells were resuspended in Stemline II medium at 100 cells / ml.

[0201]

[0309] The single-cell suspension was mixed with hemangioblast (HB) growth medium (H4536-based medium formulation: basal medium methylcellulose product H4536 (StemCell Technologies) + penicillin / streptomycin (pen / strp), Excyte growth supplement (Millipore), and cytokines, 50 ng / ml Flt3-ligand (FL), 50 ng / ml vascular endothelial growth factor (VEGF), 50 ng / ml thrombopoietin (TPO), and 20–30 ng / ml basic fibroblast growth factor (bFGF)) to obtain approximately 1 × 10 cells. 5 The mixture was adjusted to a final concentration of 1000 cells / ml, vortexed briefly, and allowed to settle. The cell mixture was then transferred to a 4 x 10 cm ultra-low attachment plate using a syringe (30 ml) fitted with an 18G needle and incubated at 37°C and 5% CO2 for 8-12 days. HBs began to appear within 3 or 4 days, continued to adhere to the plate, and could be harvested between days 7 and 12 of culture. HBs were harvested on day 9 of culture and frozen.

[0202]

[0310] Frozen HBs were thawed and replated onto Matrigel-coated tissue culture plates in MSC medium [nucleoside-free α-MEM (Hyclone), 20% Defined FBS-Heat Inactivated (Hyclone), 1x Glutamax (Gibco), 1x MEM Non-Essential Amino Acids (Gibco), and 1x penicillin / streptomycin]. Cells were cultured for approximately 4–5 days and subsequently passaged and repeated up to passage 3 (P3) to generate HMCs. These P3 HMCs ("MARP12" cells) were frozen for further use.

[0203] Example 2 - In vivo testing of traumatic brain injury (TBI)

[0311] The HMCs obtained according to Example 1 were thawed and cultured in a T225 culture flask at 37°C and 5% CO2 for about 4 days at a density of about 4500 cells / cm. 2 The cells were cultured in the MSC medium described above at 4°C for 10 min. To harvest the cells for administration, they were washed with PBS, dissociated from the flask with trypsin, and then inactivated by adding MSC medium. The cells were collected in a 50 ml conical tube and centrifuged at 300 × g for 10 min. The supernatant was aspirated, and 1 ml of GS2 buffer (for 552.2 ml of GS2: 0.9% Sodium Chloride Wash Solution USP (408.6 ml); 5% Dextrose / 0.9% Sodium Chloride Injection USP (33.2 ml), and BSS Wash Solution (110.4 ml)) was added to each tube. The cells were strained through a 100 μm cell strainer and centrifuged at 300 × g for 5 min. The supernatant was aspirated and resuspended in GS2. The resulting cells are passage 4 (P4) HMCs.

[0204]

[0312] Mild to moderate experimental traumatic brain injury (TBI) was induced in 56 Sprague Dawley rats by controlled cortical impact (CCI) (Lee et al., Theranostics, 9:1029-1046 (2019)). Cells were injected either locally by intracerebral (IC) implantation or systemically (iv) into the rats, who were then sacrificed at early or late time points according to Table 1.

[0205]

[0313]

[0206] [Table 1]

[0207]

[0314] Rats were tested according to the following schedule:

[0315] early

[0316] Day -1: Swing test and Bederson test for baseline

[0317] Day 0: Controlled cortical impact is administered to all groups.

[0208]

[0318] Day 7: All groups are treated topically or intravenously with cells or vehicle. Post-treatment swing test and Bederson test for all groups.

[0319] Day 14: Swing test and Bederson test for all groups; all groups were sacrificed; H&E staining, CA3 neuron counting, DCX, OX6, IBA-1 staining, IHC for human cells in all groups.

[0209]

[0320] Late period

[0321] Day -1: Swing test and Bederson test for all groups relative to baseline

[0322] Day 0: Controlled cortical impact administered to all groups

[0323] Day 7: All groups are treated topically or intravenously with cells or vehicle. Post-treatment swing test and Bederson test for all groups.

[0324] Day 14: Swing test and Bederson test for all groups

[0325] Day 28: Swing test and Bederson test for all groups

[0326] Day 35: Swing test and Bederson test for all groups

[0327] Day 42: Swing test and Bederson test for all groups

[0328] Day 49: Swing test and Bederson test for all groups

[0329] Day 56: Swing test and Bederson test for all groups; all groups were sacrificed; H&E staining, CA3 neuron counting, DCX, OX6, IBA-1 staining, IHC for human cells in all groups. Behavioral test results

[0330] The CCI in vivo TBI model induces significant behavioral deficits in rats by 56 days after injury. Intracerebral (IC) transplantation of HMC significantly rescued these behavioral deficits compared with their respective vehicles, including the lifted body swing test (EBST) from days 14 to 42 after transplantation (Figure 1), forelimb akinesia from days 28 to 56 after transplantation (Figure 2), and paw grasping from days 14 to 56 after transplantation (Figure 3). Intravenous (IV) transplantation of HMC also significantly rescued these behavioral deficits compared with their respective vehicles, including the EBST from days 14 to 56 after transplantation (Figure 1), forelimb akinesia from days 42 to 56 after transplantation (Figure 2), and paw grasping from day 28 after transplantation (Figure 3). These findings support the use of HMC for the treatment of TBI. Histology results

[0331] The CCI in vivo model causes significant histopathological effects in rats after injury. IV and IC transplantation of HMC demonstrated neuroprotective effects compared to their respective vehicles. For example, H&E staining showed reduced tissue loss compared to vehicle (Figure 4A-B), Nissl staining demonstrated the neuroprotective effect of HMC administration by reducing cell death (Figure 5A-F), and doublecortin (DCX) staining showed a slight increase in neurogenesis after administration of HMC after injury (Figure 6A-F).

[0210]

[0332] IV and IC transplantation of HMCs also significantly reduced microglia and macrophage activation compared to their respective vehicles. Iba1 (Figure 7A-D) and OX6 (Figure 8A-D) staining demonstrated that HMCs reduced the abundance of microglia and macrophages, respectively, in the cortex and striatum after injury.

[0211]

[0333] Furthermore, IV and IC transplantation of HMC significantly reduced inflammatory markers in the spleen compared to their respective vehicles. The reduction in IL6 (Figures 9A-B) and TNF-alpha (Figures 10A-B) staining in the spleen demonstrates that HMC reduced inflammation after injury.

[0212]

[0334] IV and IC transplantation of HMCs also resulted in migration of HMCs across the blood-brain barrier (BBB) into the cortex, striatum, and hippocampus, as shown by HuNu staining (Figure 11A-F).

[0213]

[0335] These findings support the use of HMC for the treatment of TBI. Example 3 - In vitro migration assay of HMCs

[0336] HMCs were generated from the same bank of frozen hemangioblasts described in Example 1. Three separate lots of HMCs were generated, frozen at P4, thawed, and cultured for 4 days, and passage 5 (P5) cells were harvested according to the method described in Example 1. MSCs isolated from bone marrow (BM-MSCs) and MSCs isolated from umbilical cord blood (UCB-MSCs) were used as controls. HMCs, BM-MSCs, and UCB-MSCs were each seeded into two wells of an ibidi insert with a defined gap between them and allowed to adhere overnight. The insert was removed, leaving a 500 μm gap. The cells were washed, and MSC medium (described in Example 1) was added to the chamber with or without stimulation with 25 ng / mL TNF-α + 50 ng / mL IFN-γ. The cells were incubated at 37°C for 6 hours. Next, photographs of unstimulated cells were taken (Figure 12A), and the cells that migrated into the center of the gap (approximately 250 μm from the center) were visually counted using the open-source image processing program ImageJ (Schneider et al., Nature Methods, 9:671-675 (2012)) (Figure 12B). As can be seen from Figures 12A-B, HMCs (hESC-MSCs) had higher cell migration ability than BM-MSCs or UCB-MSCs.

[0214] Example 4 - In vitro neurite outgrowth / neuron migration in the presence of HMC

[0337] Primary rat mixed-type neuronal cultures were prepared from the whole brains of E18 Sprague-Dawley rat pups obtained from BrainBits, LLC (Springfield, IL). The midbrain, cerebellum, and hippocampus were removed to isolate the cerebral cortex. Cells were dissociated from the tissue and cultured for 14 days for maturation. Although the tissue was from fetal rat pups, neurons have been shown to exhibit mature receptor and electrophysiological profiles after 14 days in culture. Mixed neuronal cultures have been used in adapted migration assays to test neuronal regeneration and as an in vitro TBI model (Darbinyan et al., Methods Mol. Biol. 1078:45-54 (2013); Ali et al., High Content Screening with Primary Neurons. October 15, 2013; in Sittampalam GS, Coussens NP, Brimacombe K, et al., eds., Assay Guidance Manual. Bethesda, MD: Eli Lilly & Company and the National Center for Advancing Translational Sciences (2004)).

[0215]

[0338] On day 0, mixed neuronal cultures were plated. On day 9, MARP12 cells, frozen and thawed as described in Example 1, were plated into flasks for expansion. On day 13, MARP12 cells were harvested and plated onto transwell inserts at a ratio of neurons to MARP12 cells of approximately 10:1 in MSC medium. On day 14, two scratches were made per well in mixed neuronal cultures prepared as described above (Liang et al., Nat. Protoc. 2:329-333 (2007)). The MSC medium in the transwells was replaced with neuronal medium (Neurobasal™ Plus (ThermoFisher); 1x gentamicin; 1x GlutaMAX™ (ThermoFisher); 1x B27™ Plus (ThermoFisher)), serum was removed, and transwell inserts containing MARP12 cells were added to the wells containing the mixed neuronal cultures. As shown in Figure 13, coculture with MARP12 (hESC-MSC or HMC) promoted neurite outgrowth and increased migration.

[0216]

[0339] RNA-seq data may also indicate that the presence of co-cultured HMCs and / or HMC-EVs may affect gene expression in neurons. Neurons were dissociated from the cortex of E18 Sprague-Dawley rat brains and plated at 1.2 × 10 per well onto poly-D-lysine (Sigma)-coated 6-well BioFlex culture plates (FlexCell). 6Cells were plated at a density of 1.2 × 10 cells per insert onto transwell inserts (Corning). Neurons were supplemented with Neurobasal Plus / B27 Plus medium (Gibco) and maintained in a humidified CO2 incubator at 37°C for 14 days in vitro (DIV). Half of the medium was replaced every 3 days. For HMC treatment, HMCs were harvested after 4 days of culture in α-MEM medium (α-MEM (Hyclone) containing 1x GlutaMAX (Gibco), 1x MEM-NEAA (Gibco), and Pen-strep (Gibco)). 5 After 1 day of culture, the α-MEM medium was replaced with Neurobasal Plus / B27 Plus medium for 1 hour, followed by the addition of inserts to 6-well plates containing neurons at DIV14. For EV treatment, EVs were purified from HMCs (HMC-EVs) by tangential flow filtration. HMC-EVs were added to the plates containing neurons, followed by the addition of TNF-α at a concentration of 100 ng / mL, where appropriate, and then the plates were placed on a FlexCell FX-6000. The cultures were subjected to 15% to 0% strain oscillation (15%, 12.5%, 10%, 7.5%, 5%, 2.5%, and 0% cycles) overnight. The neurons were then removed from the BioFlex plates, pelleted, washed with PBS, and subjected to RNA isolation via the RNeasy Mini Kit (Qiagen). Subsequently, RNA (300 ng) was submitted to BGI Americas for RNA-seq analysis, and data were analyzed using Rosalind software (https: / / rosalind.onramp.bio / ). Reads were trimmed using Cutadapt and quality scores were assessed using FastQC. Reads were aligned to the rat (Rattus norvegicus) genome build rn5 using STAR. Individual sample reads were quantified using HTseq and normalized by relative log expression (RLE) using the DESeq2 R library.

[0217] Example 5 - In vivo neonatal hypoxia-ischemia model of cerebral palsy

[0340] The HMCs of the present invention were tested in an in vivo neonatal hypoxia-ischemia (HI) model of cerebral palsy. The HMCs used were MARP12 cells, as described in Example 1, which were thawed and passaged for 4 days as passage 5 (P5) cells, at which point they were harvested, rinsed, and formulated for injection. To establish an in vivo model of cerebral palsy, ischemia was induced by ligation of the common carotid artery in male Sprague Dawley rat pups on postnatal day (PND) 7. After recovery, the pups were subjected to a hypoxic episode followed by an additional 25 minutes of normoxia. Sham control pups received the same exposure, except that they received normoxia instead of hypoxia. Seven days after surgery and hypoxic exposure (i.e., PND 14), the pups were humanely euthanized, and blood, cerebrospinal fluid (CSF), and brain tissue were collected for further testing. The pups were treated according to Table 2.

[0218]

[0341]

[0219] [Table 2]

[0220] Endpoints assessed

[0342] Depending on the volume of sample, CSF and blood will be used for ELISA for inflammation panels and others.

[0221]

[0343] Brain tissue analyzed for:

[0344] Cell death-TUNEL,

[0345] Infarct volume-H&E,

[0346] Iba-1 - Microglial activation in peri-infarct tissue,

[0347] GFAP-Astrocyte activation in peri-infarct tissue,

[0348] Olig2 - oligodendrocyte precursor cells in the hippocampus

[0349] MBP - Myelin basic protein of mature oligodendrocytes in the corpus callosum and hippocampus. result

[0350] TUNEL staining (Figures 14A-B) suggests a neuroprotective effect of MARPS12 (Lot B), accompanied by a reduction in cell death. Furthermore, H&E staining (Figure 15) suggests a neuroprotective effect of MARPS12 (Lot B), accompanied by a reduction in lesion size. The reduction in microglial activation via Iba-1 staining (Figures 16A-C) suggests an anti-inflammatory effect of MARPS12 (Lot B). The mild reduction in astrocyte activation via GFAP staining (Figures 17A-C) also suggests an anti-inflammatory effect of MARPS12 (Lot B). The preservation of myelin in the corpus callosum via MBP staining (Figures 18A-C) suggests a beneficial role of MARPS12 for oligodendrocytes. Furthermore, Figures 19A-C suggest that Olig2 expression is partially rescued by MARPS12 administration.

[0222]

[0351] These results support the use of HMC in the treatment of cerebral palsy. Example 6 - RNAseq analysis of HMCs vs. BM-MSCs vs. UCB-MSCs

[0352] HMCs were generated from the same bank of frozen hemangioblasts described in Example 1. Three separate lots of HMCs were generated and passaged to passage 5 (P5) according to the method described in Example 1. RNA-seq analysis was performed on the three lots of HMCs in the basal state. MSCs isolated from bone marrow (BM-MSCs) (9 lots) and MSCs isolated from umbilical cord blood (UCB-MSCs) (9 lots) in the basal state were used as controls.

[0223]

[0353] Table 3 shows genes that are more highly expressed in HMCs compared to BM-MSCs. Table 4 shows genes that are more highly expressed in BM-MSCs compared to HMCs. Table 5 shows genes that are more highly expressed in HMCs compared to UCB-MSCs. Table 6 shows genes that are more highly expressed in UCB-MSCs compared to HMCs. The HMCs of the present invention can be selected or purified based on any of the differentially expressed genes.

[0224] [Table 3-1]

[0225] [Table 3-2]

[0226] [Table 3-3]

[0227] [Table 3-4]

[0228] [Table 3-5]

[0229] [Table 3-6]

[0230] [Table 3-7]

[0231] [Table 3-8]

[0232] [Table 3-9]

[0233]

Table 3-10

[0234]

Table 3-11

[0235]

Table 3-12

[0236]

Table 3-13

[0237]

Table 3-14

[0238]

Table 3-15

[0239]

Table 3-16

[0240]

Table 3-17

[0241]

Table 3-18

[0242]

Table 3-19

[0243]

Table 3-20

[0244]

Table 3-21

[0245]

Table 3-22

[0246]

Table 3-23

[0247]

Table 3-24

[0248]

Table 3-25

[0249]

Table 3-26

[0250]

Table 3-27

[0251]

Table 3-28

[0252]

Table 3-29

[0253]

Table 4-1

[0254]

Table 4-2

[0255]

Table 4-3

[0256]

Table 4-4

[0257]

Table 4-5

[0258]

Table 4-6

[0259]

Table 4-7

[0260]

Table 4-8

[0261]

Table 4-9

[0262]

Table 4-10

[0263]

Table 4-11

[0264]

Table 4-12

[0265]

Table 4-13

[0266]

Table 4-14

[0267]

Table 4-15

[0268]

Table 4-16

[0269]

Table 4-17

[0270]

Table 4-18

[0271]

Table 4-19

[0272]

Table 4-20

[0273]

Table 4-21

[0274]

Table 4-22

[0275]

Table 4-23

[0276]

Table 4-24

[0277]

Table 4-25

[0278]

Table 4-26

[0279]

Table 4-27

[0280]

Table 4-28

[0281]

Table 5-1

[0282]

Table 5-2

[0283]

Table 5-3

[0284]

Table 5-4

[0285]

Table 5-5

[0286]

Table 5-6

[0287]

Table 5-7

[0288]

Table 5-8

[0289]

Table 5-9

[0290]

Table 5-10

[0291]

Table 5-11

[0292]

Table 5-12

[0293]

Table 5-13

[0294]

Table 5-14

[0295]

Table 5-15

[0296]

Table 5-16

[0297]

Table 5-17

[0298]

Table 5-18

[0299]

Table 5-19

[0300]

Table 5-20

[0301]

Table 5-21

[0302]

Table 6-1

[0303]

Table 6-2

[0304]

Table 6-3

[0305]

Table 6-4

[0306]

Table 6-5

[0307]

Table 6-6

[0308]

Table 6-7

[0309]

Table 6-8

[0310] [Table 6-9]

[0311] [Table 6-10]

[0312] [Table 6-11]

[0313] [Table 6-12]

[0314] [Table 6-13]

[0315] [Table 6-14]

[0316] [Table 6-15]

[0317] [Table 6-16]

[0318] Example 7 - In vivo middle cerebral artery occlusion (MCAO) stroke model

[0354] The HMC and HMC-EV of the present invention were tested in an in vivo model of middle cerebral artery occlusion (MCAO) stroke.

[0319]

[0355] HMCs were generated from the same bank of frozen hemangioblasts described in Example 1.

[0356] For HMC-EVs, early passage (passage 4) HMCs were thawed, washed, counted, and cultured at 5,000 cells / cm in RoosterBio RoosterNourish-MSC-XF medium. 2 Cells were plated into Corning CellBIND flasks at a cell density of 1000 cells / cm. Cells were grown for 96 hours to approximately 70-90% confluence for medium acclimation and cell expansion. At 96 hours, cells were removed from the flask using TripLE dissociation, viable cells were counted, and at passage 5, cells were plated at 5,000 cells / cm in new flasks and fresh medium. 2 At this passage, the medium can be collected after 96 hours for EV isolation. For larger volume collections, cells can be passaged again up to passage 7. After collecting the medium for EV isolation, it was clarified to remove cells and debris using differential low-speed centrifugation at 300 x g for 10 minutes and 2,000 x g for 20 minutes, followed by 0.2 µm vacuum filtration. EVs were isolated from the clarified medium using tangential flow filtration (TFF) on a Repligen KR2i system equipped with a hollow-fiber, 300 kDa pore, mPES membrane filter. The filter, with a pore size of approximately 100 nm, removed small impurities and retained EVs. Overall, the clarification and TFF parameters were such that particles between 100 nm and 200 nm in size were isolated. The medium was first concentrated approximately 10-fold and then diafiltered using DPBS to improve sample purity and remove non-EV-associated proteins during the TFF process. The diafiltered medium was further concentrated so that the final product was approximately 100-fold concentrated. The resulting isolated and concentrated EVs in DPBS were ready for downstream analysis or could be further purified using chromatographic techniques. In vivo effects of HMC and HMC-EV on locomotor skills

[0357] The MCAO animal model was prepared as described herein. Briefly, the day before surgical injury, male Sprague-Dawley rats (300–400 g) were used to perform the body swing test to establish baseline performance. Each rat was held approximately 1 inch from the base of its tail. It was then elevated 1 inch above the table surface. The rat was held on a vertical axis, defined as 10° or less to either the left or right side. A swing was recorded if the rat moved its head to either side of the vertical axis. The next swing was counted after the rat was returned to a vertical position. A total of 30 swings were counted. In normal rats, the number of swings on both sides is typically equal. After focal ischemia, rats tend to swing to the contralateral (left) side. One day after testing, a focal cerebral infarction was produced by permanent occlusion of the proximal right middle cerebral artery (MCA) using a modified method from Tamura et al. Rats were anesthetized with 1–3% isoflurane in a 2:1 NO:O mixture and maintained with 1.5–2% isoflurane in a 2:1 NO:O mixture. The temporalis muscle was bisected and reflected through an incision midway between the eye and the tympanic membrane. The proximal MCA was exposed by subtemporal craniectomy without removing the zygomatic arch or transecting the facial nerve. The artery was then occluded 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. Cefazolin (40 mg / kg) was administered intraperitoneally (ip) before MCAO to prevent infection. Buprenorphine, sc (approximately 0.1 mg / kg Simbadol), was administered as an analgesic before the MCAO surgery. For sham conditions, rats underwent the same procedure as above, but without coagulating the middle cerebral artery.

[0320]

[0358] Treatments were administered on days 1 and 7 after MCAO surgery (24 hours and 7 days + / - 10%). For HMC treatments, cells were stored in liquid nitrogen until the day of use. Cells were thawed in a 37°C water bath, counted, and diluted in the vehicle Plasma-Lyte A. For HMC-EV treatments, EV aliquots were stored at -80°C until the day of use. EVs were thawed on ice and diluted in the vehicle DPBS or used as prepared.

[0321]

[0359] On days 1 and 7 (24 hours and 7 days + / - 10%) after MCAO, rats were anesthetized with 1-3% isoflurane in O2 and maintained at 1.5-2% isoflurane in O2. Jugular vein injections were performed using a 1 ml syringe fitted with a 25 G (¾") needle. 0.5 ml of vehicle or cells was injected into the jugular vein. Compression was applied to the injection site for approximately 1 minute to ensure no bleeding. Local injections were performed using a 50-microliter Hamilton syringe fitted with a 26 G needle. 10 microliters of vehicle, cells, or EVs were injected into three peri-infarct areas at 3-4 microliters per site. Intrathecal injections were performed using a 25 G hypodermic needle and an insulin syringe (0.5 mL). 40 microliters of vehicle, cells, or EVs were injected between the last lumbar and first sacral vertebrae (L6-S1).

[0322]

[0360] Body swing tests were performed on days 1, 7, 14, 21, and 28 after injury, and rats were sacrificed after the test on day 28 after injury. Twenty-eight days after MCAO (day 28), rats were deeply anesthetized with ketamine / xylazine (91 mg / kg ketamine and 9 mg / kg xylazine, respectively). Once the rats were deeply anesthetized, they were transcardially perfused with saline (containing 2 units / ml heparin) followed by 4% paraformaldehyde. Brains were removed and stored in 4% paraformaldehyde for 24 hours, then replaced with 1x PBS and stored at 0-4°C. All data are expressed as mean ± SEM. Data from the body swing test were analyzed by two-way ANOVA and Tukey's multiple comparison test. Significance was assessed. *p<0.05, ** p<0.01, *** p<0.001, **** Expressed as p<0.0001.

[0323]

[0361] The effects of HMC and HMC-EV of the present invention on locomotion were evaluated in an MCAO model.

[0362] HMC cells were injected via three routes of administration, including intravenous (IV), intracerebral (IC), and intrathecal (IT) administration. Cells were dosed at 4 million cells in 0.5 mL per IV injection, 400,000 cells in 10 microliters per IC injection, and 500,000 or 1 million cells in 40 microliters per IT injection. As shown in Figure 20, all treatment groups showed improved recovery of deficits in the body swing test, with the IV and IC treatments being the most significant.

[0324]

[0363] In another study, rats were subjected to MCAO injury as described above. Cell treatment was administered on days 1 and 7 (24 hours and 7 days + / - 10%) after MCAO surgery using HMCs, specifically HMCs derived from C-GS1 cells (C-GS1-HMCs) and N-lot QR57 cells (N-HMCs). The cell dose was 4 million cells in 0.5 mL per IV injection. Extracellular vesicles (EVs) treatment was administered on days 1 and 7 (24 hours and 7 days + / - 10%) after MCAO surgery using EVs derived from N-HMCs (N-HMC-EVs). The EV dose was 10 x 10 for intracerebral and intracisternal administration. 10 All treatment groups showed significant improvement in the limb-placing test (Figure 21). In the body-swing test, all treatment groups showed recovery, and C-GS1-HMC, N-HMC, and N-HMC-EV administered via intracerebral injection showed significant increases.

[0325]

[0364] In another study, N-HMC-EVs (N-lot p6 and p7 treated with 50 ng / ml IFN-gamma for 96 hours) were used to administer treatments on days 1 and 7 (24 hours and 7 days + / - 10%) after MCAO surgery. The dose of EVs was a total of 10 x 10 for N-HMC-EVs (primed N-lot) via intracisternal injection. 10 pieces or 30 x 10 10 All groups showed significant improvements in all three behavioral tests, with the most significant improvements shown in the forelimb placing test and body swing test (Figure 22).

[0326]

[0365] In a further study, HMC-EV (N-lot) or HE-VPC-EV were administered on days 1 and 7 (24 hours and 7 days + / - 10%) after MCAO surgery. The exosome dose was 10 x 10 for HMC-EV via intrathecal injection. 10 pieces, 30×10 10 pieces, and 10x10 11 For VPC-EV, it is 10 x 0 10 Injections without HMC-EVs were administered as a negative control. All groups showed significant improvements in all three behavioral tests, with the most significant improvements observed in the forelimb placing test and body swing test (Figure 23).

[0327]

[0366] Thus, the HMC and HMC-EVs of the present invention are effective in the MCAO stroke model via intravenous, intrathecal, intracerebral, and / or intracisternal administration, and both HMC and EV treatments resulted in improved locomotor recovery in behavioral tests. In vivo effects of HMC on histopathological outcomes

[0367] The effect of the HMC of the present invention on histopathological results was evaluated.Specifically, rats were subjected to MCAO injury as described above.HMC, specifically HMC derived from C-GS1 cells (C-GS1-HMC) and N-lot QR57 cells (N-HMC), was used for cell treatment on the 1st and 7th days (24 hours and 7 days + / - 10%) after MCAO surgery.The cell dose was 4 million cells in 0.5mL per IV injection.

[0328]

[0368] Sham, vehicle, and cell-treated groups were prepared for histopathological analysis for white matter loss (MBP) and markers of neuroinflammation, such as microglial activation (Iba-1) and astrocyte activation (GFAP).

[0329]

[0369] Figure 24 shows myelin preservation in the striatum with HMC cell treatment. Specifically, a statistically significant difference was observed between the sham and vehicle groups for MBP, but no statistically significant difference was observed between the vehicle and treatment groups in the ipsilateral portion of the cortex. A statistically significant difference was observed between the vehicle and N-lineage cell-treated groups in the contralateral cortex, but no statistically significant difference was observed between the groups in the ipsilateral portion of the cortex, and no statistically significant difference was observed between the sham and vehicle groups in the contralateral portion of the cortex. A statistically significant difference was observed between the sham and vehicle groups in both the ipsilateral and contralateral portions of the striatum, but a statistically significant difference was observed between the vehicle and both cell-treated groups only in the ipsilateral portion of the striatum. No statistically significant difference was observed between the groups in the contralateral portion of the striatum.

[0330]

[0370] Figure 25 shows the reduction in microglial activation after HMC administration. Specifically, for Iba-1, statistically significant differences were observed between the sham group and the vehicle group in both the ipsilateral and contralateral portions of the cortex, while statistically significant differences were observed between the vehicle group and the cell-treated group only in the ipsilateral portion of the cortex. No statistically significant differences were observed between the vehicle group and the treatment group in the contralateral portion of the cortex. Statistically significant differences were observed between the sham group and the vehicle group in both the ipsilateral and contralateral portions of the striatum, and statistically significant differences were observed between the vehicle group and the C-GS1 cell-treated group in the ipsilateral portion of the striatum. No statistically significant differences were observed between the vehicle group and the treatment group in the contralateral portion of the striatum.

[0331]

[0371] Figure 26 shows that HMC treatment reduces astrocyte reactivity. Specifically, statistically significant differences were found between the sham group and the vehicle group, and between the vehicle group and the cell-treated group, for GFAP in both the ipsilateral and contralateral parts of the cortex. Statistically significant differences were found between the sham group and the vehicle group, and between the vehicle group and the cell-treated group, for both the ipsilateral and contralateral parts of the striatum.

[0332]

[0372] These results therefore demonstrate that the MSCs of the present invention not only increased the preservation of myelin and therefore white matter, but also resulted in a robust reduction in neuroinflammatory markers by reducing the number of reactive astrocytes and microglia. In vivo effects of HMC-EVs on histopathological outcomes

[0373] The effect of HMC-EVs on histopathological outcomes was also evaluated. Specifically, rats were subjected to MCAO injury as described above. HMC-EVs (N-lot p6 and p7 treated with 50 ng / ml IFN-gamma for 96 hours) were used for treatment on days 1 and 7 (24 hours and 7 days + / - 10%) after MCAO surgery. The EV dose was a total of 10 × 10 HMC-EVs (stimulated N-lot) via intracisternal injection. 10 pieces or 30 x 10 10 The number was set to 1.

[0333]

[0374] Sham, vehicle, and cell-treated groups were prepared for histopathological analysis of MBP, Iba-1, GFAP, Olig-2, and NG2. Figure 27 shows myelin preservation with intracisternal delivery of EVs. Specifically, MBP IF staining showed stable staining areas in all treatment groups, ranging from 0.81 to 0.88. The vehicle group had the lowest mean ratio (0.64). The differences between the vehicle group and all treatment groups were significant.

[0334]

[0375] Figure 28 shows the effect of HMC-EV treatment on microglial activation. Specifically, Iba-1 IF staining revealed that the vehicle group and HMC-EV 10 10 Individual treatment groups and HMC-EV 30 10 Each treatment group showed the same mean ratio (R / L) of the number of positive cells (approximately 2.5).

[0335]

[0376] Figure 29 shows the effect of intracisternal HMC-EV delivery on astrocyte reactivity. Specifically, GFAP IF staining did not reveal any differences between the control group and all treatment groups, demonstrating a stable mean ratio (R / L) for the number of positively stained cells.

[0336]

[0377] Figure 30 shows that intracisternal delivery of HMC-EVs increased oligodendrocytes. Specifically, Olig-2 IF staining revealed the highest mean ratio of positively stained cells (R / L) in all exosome-treated groups (compared to the vehicle group). The vehicle group and HMC-EV 10 10 Swarm and HMC-EV 30 10 The differences between the groups were significant.

[0337]

[0378] Figure 31 shows that intracisternal delivery of HMC-EVs increased oligodendrocyte precursor cells. Specifically, NG2 IF staining revealed that HMC-EVs increased the number of oligodendrocyte precursor cells compared to the vehicle group. 10 Swarm and HMC-EV 30 10A statistically significant increase in the mean ratio of positive staining areas (R / L) was observed in the group.

[0338]

[0379] Therefore, these results demonstrated that HMC-EVs increased myelin preservation. In addition, EV treatment also increased oligodendrocytes and oligodendrocyte progenitor cells.

[0339] Example 8 - In vitro oxygen-glucose deprivation stroke model

[0380] The neuroprotective effects of the MSCs of the present invention were investigated in vitro. An oxygen glucose deprivation (OGD) assay, which combines hypoxic conditions with a glucose-deprived medium, was used to model stroke in vitro.

[0340]

[0381] An outline of the assay is shown in Figure 32. For primary neuronal culture, embryonic day 18 (E18) rat cortical samples (#SDECX) sourced from Sprague-Dawley rats were ordered from Brain Bits, LLC (Springfield, IL). Cortices were washed three times in dissociation medium (DM). DM consisted of 50 mL of 10x HBSS (Ca- and Mg-free; Gibco 14185-052), 500 μL of gentamicin, 5 mL of pyruvate (Gibco 11360070), 5 mL of Hepes (Gibco 15630080) 10 mM final, 15 mL of glucose 30 mM final (1M stock), and 425 mL of water. After washing, the DM was aspirated, and the tissue was subsequently minced into equally sized pieces with a scalpel. While washing the tissue, DM, papain, and DNase I solutions were prepared by measuring 1 mL of DM, 40 μL of papain (Worthington LS003126), and 2.5 μL of DNase I (Sigma #DN-25) per brain, incubating in a 37°C water bath for 30 minutes to activate the papain, and sterile filtering using a 0.22 micron filter. The DM, activated papain, and DNase I solutions were added to the cortical samples and incubated at 37°C for 30 minutes to dissociate the tissue.

[0341]

[0382] During this time, neuronal medium (NM0) was also prepared and incubated at 37°C. NM0 consisted of Neurobasal Plus medium containing 1x B27 + fresh supplements (Neurobasal Plus and B27; Life Tech Corp A3653401), 1x Glutamax (Gibco #35050-061), and gentamicin sulfate (MP Biomedical #0916760-CF). Dissociation pipettes were prepared by heat-treating Pasteur pipettes with successively smaller tip diameters (1 = heat-treating only, 2 = ¾ of the original diameter, 3 = ½ of the original diameter). After a 30-minute incubation, the tissue was removed from the water bath. DM / papain / DNase A was added. The I solution was gently aspirated and 5 mL of pre-warmed N-methyl-2-hydroxybenzoates (NM0) supplemented with B27 was added. The tissue was allowed to settle, and the N-methyl-2-hydroxybenzoates (NM0) was gently removed with a pipette. The tissue was washed again with 5 mL of fresh N-methyl-2-hydroxybenzoates (NM0) (with B27), a total of three times. The N-methyl-2-hydroxybenzoates (NM0) were removed after the final wash. Starting with the largest pipette, the brain tissue was dissociated by gently triturating it through a fire-polished Pasteur pipette. This was done by adding 3 mL of N-methyl-2-hydroxybenzoates (NM0) and gently triturating 4-5 times, dispensing the tissue against the wall of the tube to prevent bubble formation, as neurons trapped in bubbles will die. After the remaining tissue had settled, the supernatant was removed and fresh N-methyl-2-hydroxybenzoates (NM0) was added. The cells were added to a 50 mL Falcon tube. This was repeated for all pipette sizes, and the cell mixture was then passed through a 70-micron cell strainer. The cells were counted and diluted to 600,000 cells / mL. The cells were plated onto tissue culture plates pre-coated with poly-D-lysine (PDL). For 6-well plates, 2 mL was added to achieve a total of 1.2 million cells per well. For 24-well plates, 0.5 mL was added to achieve a total of 300,000 cells per well. The cultures were then fed and replaced with half the medium every three days to prevent the accumulation of metabolic byproducts. After one week, the cells were subjected to the OGD assay.

[0342]

[0383] Five days prior to neuronal endpoint treatment, N-lot HMCs were thawed in a 37°C water bath with gentle swirling. Once thawed, cells were pipetted into prewarmed MSC medium (nucleoside-free alpha MEM (Hyclone, #SH30568.01), 20% Defined Fibre Sheat Inactivated (Hyclone, #SH30070.03 HI), 1X Glutamax (Gibco #35050-061), 1X MEM NEAA (Gibco #11140-050), and 1X Pen / Strep (Gibco #15140-120)). Cells were then centrifuged at 300 x g for 5 minutes, resuspended, and counted. One million MSCs were plated into a T225 flask using 50 mL of MSC medium and maintained in culture for 4 days. HMCs were then harvested by first aspirating the medium. The flask was washed with 10 mL of PBS, the PBS was aspirated, 3 mL of TrypLE Express (Gibco, #12604021) was added, and the cells were incubated at 37°C for 4–6 minutes. After incubation, the cells were washed with MSC medium and collected in a 50 mL conical tube. The plate was washed with MSC medium to remove remaining cells, and the cells were centrifuged at 300 × g for 5 minutes. The cells were then resuspended in MSC medium and counted. HMCs were then plated in MSC medium onto transwell inserts to achieve a 1:10 ratio of HMCs to neurons (120,000 HMCs per well for 6-well transwell inserts and 30,000 HMCs per well for 24-well transwell inserts). HMCs were allowed to recover for 24 hours, and the MSC medium was replaced with NMO, removing traces of FBS. HMCs were incubated in NMO medium for 24 hours before being used for recovery in the oxygen glucose deprivation (OGD) assay.

[0343]

[0384] For the OGD assay, OGD medium was used to deplete glucose from neurons. The OGD medium consisted of 1 mM CaCl2, 5 mM KCl, 137 mM NaCl, 0.4 mM KH2PO4, 0.3 mM Na3HPO4, 0.5 mM MgCl2, 0.4 mM MgSO4, 25 mM HEPES, 4 mM NaHCO3, and 1x Pen / Strep diluted in 450 mL of DI water. The pH was adjusted to 7.3, and water was added to a final volume of 500 mL. The medium was then sterile filtered using a 0.2 μm filter. One day before the OGD experiment began, the OGD medium was placed in a T75 vented flask and incubated overnight in a hypoxic chamber (C-Chamber with a ProOx C21 Oxygen CO2 Single Chamber Controller, BioSpherix, Parish, NY) to allow oxygen to diffuse out of the medium. The following day, the OGD medium was removed from the hypoxic chamber, and the neurons were washed once with OGD medium to remove traces of NM0. The OGD medium was removed, and a complete medium exchange with OGD medium was performed immediately before adding the cells to the chamber. That is, the medium for the 3-hour OGD period was changed, but the medium at the 2-hour point was not changed until immediately before adding the cells to the chamber. This ensured that the recovery time was the same for all conditions. Neurons were incubated in the hypoxic chamber containing OGD medium for 1, 2, or 3 hours. At the end of the incubation, neurons were removed and a complete medium exchange with NM0 medium (+B27) was performed. For the non-injured control, NM0 was replaced with OGD medium, but the neurons were not incubated in the hypoxic chamber. In the non-injured control, the OGD medium was replaced with NM0 at the same time as the injured cells. HMC coculture conditions were performed for both the non-injured control and the injured cells. Immediately after replacing the OGD medium with NM0, the transwell insert with HMC was added to the coculture conditions. Recovery from the OGD injury was allowed to continue for 24 h in an incubator under normal cell culture conditions. Neurons were either collected for RNA isolation or fixed and subjected to TUNEL staining. In vitro OGD assay TUNEL analysis

[0385] Primary neuronal cultures were generated from embryonic day 18 (E18) rat cortical samples sourced from Sprague-Dawley rats ordered from Brain Bits (Springfield, IL) as described above. HMC co-culture conditions (no direct contact) using transwell inserts at a 1:10 HMC-to-neuron ratio were performed with N-lot cells, starting immediately after the OGD insult and continuing for a total of 24 hours.

[0344]

[0386] To evaluate the effect of HMC coculture on preventing neuronal cell death induced by the OGD assay, TUNEL staining, imaging, and quantification were performed. After the OGD assay, transwells were removed from the coculture conditions, and neurons were first fixed with 4% paraformaldehyde. To fix the cells, NMO was removed, and 4% paraformaldehyde was applied to each well and incubated at room temperature for 10 minutes. After fixation, cells were washed three times with PBS and permeabilized with 0.02% Triton-X in PBS for 10 minutes at room temperature. Subsequently, cells were washed three times with PBS. A positive control was designated and treated with DNase I (Sigma #4536282001) in DNase I reaction buffer (20 mM Tris-HCl, pH 8.4, 2 mM MgCl2, 50 mM KCl) for 30 minutes at room temperature and 30 minutes at 37°C. The positive control was then washed three times with PBS.

[0345]

[0387] To achieve TUNEL staining, we used TUNEL Label Mix (Sigma #11767291910) and TUNEL Enzyme Kit (Sigma #11767305001) according to the manufacturer's protocol with minor modifications. Generally, two kits were used per experiment, diluted with PBS to accommodate the larger volume required for 24-well plates. While the instructions suggest using the kit directly at a volume of 50 μL per well, we diluted the samples to 150 μL per well using PBS to ensure coverage of the 24-well plate. As a negative control, we used TUNEL labeling reagent without TUNEL enzyme diluted in PBS. For all samples, 200 μL of DAPI staining solution (VWR #10791-650) was added to the combined solution. The TUNEL labeling reagent with TUNEL enzyme dilution was added to the desired wells, and the samples were incubated at 37°C for 1 hour. The samples were washed three times with PBS. Imaging was performed with a Leica DMi8 microscope, and quantification was performed using Leica LAS X Navigation software. Three wells were stained for each condition, and nine images per well were taken and quantified, resulting in 27 images per analyzed condition. TUNEL staining and analysis demonstrated a significant increase in cell death with increasing duration of OGD insult.

[0346]

[0388] As shown in Figure 33, HMC co-culture prevented cell death in primary rat neurons after OGD injury. The neuroprotective effect of HMC cells in ischemic injury does not require direct contact with neurons but acts through a paracrine effect on target neurons.

[0347]

[0389] Thus, this in vitro analysis demonstrated that the HMCs of the present invention can confer protection against ischemic injury (i.e., oxygen-glucose deprivation) in isolated neuronal culture preparations, demonstrating the benefit of direct access to the central nervous system in stroke. RNAseq analysis of oxygen-glucose deprived rat neurons

[0390] Primary rat neuronal cultures were subjected to oxygen glucose deprivation (OGD) for various periods (e.g., 0, 1, 2, and 3 h injury duration). Then, 24 h after OGD treatment, neurons were co-cultured with HMCs. RNA samples were collected 24 h after OGD treatment. RNA-seq analysis was performed to examine transcriptome and pathway enrichment after OGD in vivo injury with or without subsequent HMC co-culture.

[0348]

[0391] For RNA isolation, neurons were washed with PBS, detached, and collected by centrifugation at 500g for 5 minutes in a microfuge. The PBS was aspirated, and the cell pellet was either flash-frozen and placed at -80°C or immediately processed using the RNeasy RNA Isolation Kit (Qiagen #74104) according to the manufacturer's protocol. RNA was quantified using Nano Drop, and all samples were normalized to 50ng / µL. A total of 1µg was submitted to GeneWiz for RNAseq analysis to analyze changes in gene expression in response to OGD insult and HMC coculture. The conditions were: control, control with HMC, 1h OGD, 1h OGD with MSC, 2h OGD, 2h OGD with MSC, 3h OGD, and 3h OGD with HMC. Triplicate biological replicates were provided for each condition.

[0349]

[0392] Library preparation was performed using the NEB Ultra II RNA Library Prep Kit, followed by Illumina sequencing. 20-30 million reads were achieved for each sample. Bioinformatics analysis was performed to analyze the RNAseq data. Reads were trimmed using cutadapt1. Quality scores were assessed using FastQC2. Reads were aligned to the rat (Rattus norvegicus) genome build rn6 using STAR3. Individual sample reads were quantified using HTseq4 and normalized via relative log expression (RLE) using the DESeq2 R library5. Read distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step using RSeQC6. DEseq2 was also used to calculate fold changes and p-values, with optional covariate correction. Clustering of genes for the final heatmap of differentially expressed genes was performed using the PAM (Partitioning Around Medoids) method using the fpc R library. Hypergeometric distribution was used to analyze enrichment of pathways, gene ontologies, domain structures, and other ontologies. The topGO R library was used to determine local similarities and dependencies between GO terms to perform Elim pruning correction. Several database sources were consulted for enrichment analysis, including Interpro, NCBI, MSigDB REACTOME, and WikiPathways. Enrichment was calculated against a set of background genes relevant to the experiment. Although numerous gene expression changes were observed, we focused on genes involved in neuroprotection.

[0350]

[0393] The therapeutic effect of HMC-enriched cultures on OGD neuronal growth was observed for neurons subjected to a 3-hour OGD insult. Pathway enrichment analysis of differential expression between neurons subjected to a 3-hour OGD insult and grown on HMC-enriched and control media was performed using the Qiagen Ingenuity Pathway Analysis framework. As shown in Figures 34A-C, pathways enriched by this differential expression include: (a) the STAT3 pathway, which is inactivated in OGD neurons cultured with HMCs (p-value: 4x10); -11 ), (b) CREB signaling in neurons (p-value: 4.4 × 10 -8 ), and (c) numerous inflammatory activation pathways (e.g., IL-6 signaling, IL-10 signaling, Th1 / 2 activation pathway) that are downregulated in OGD neurons cultured with HMCs.

[0351]

[0394] The enrichment of differential expression between OGD neurons grown on HMC-enriched and control media for gene ontology terms (Figures 34C-F) subsequently indicates increased cell viability of OGD neurons grown on HMC-enriched cultures (Figure 34C), a direct neuroprotective effect (Figure 34C; genes involved in upregulation of neuroprotection are shown in Figure 34D), and upregulation of pathways involved in synaptic transmission (Figure 34C). Concurrently, pathways involved in apoptosis (Figure 34E; genes downregulated by the effect of HMC-enriched growth media are shown in Figure 34F) and general responses to cell death are strongly downregulated. This reflects the relationship between the complete differential expression and displacement of molecular markers of OGD injury induced by the presence of HMC-enriched growth media.

[0352]

[0395] To verify these increases in gene expression, the same RNA samples used for RNAseq analysis were used for qPCR analysis. To perform qPCR analysis, Taqman probes (ThermoFisher Scientific) were designed and used with Taqman Fast Advanced Master Mix (ThermoFisher Scientific #4444556) to analyze samples on a QuantStudio Flex 7 RT-PCR system (Applied Biosystems #4485698). Three biological replicates were performed in duplicate for each sample, and the analysis demonstrates similar increases in gene expression due to the presence of HMC. Statistical significance was achieved by two-way ANOVA and Sidak's multiple comparison test ( * p<0.05, ** p<0.01, **** p<0.0001).

[0353]

[0396] qPCR analysis validated the RNAseq results for genes involved in cell viability and neuroprotection, as shown in Figure 35. Specifically, HMC cells stimulated the expression of neuroprotective genes in neurons undergoing ischemic injury, such as heat shock protein family B member 1 (HSPB1), insulin-like growth factor 1 (IGF2), and secreted phosphoprotein 1 (SPP1), also known as osteopontin.

[0354] Example 9 - In vitro oxidative damage model

[0397] The HMC-EVs of the present invention were tested in an in vitro oxidative damage model. Briefly, neurons were subjected to H2O2 oxidative damage and treated with HMC-EVs at doses of approximately 10,000, 30,000, or 100,000 EVs / cell. The percentage of cell death was determined as the number of propidium iodide (PI)-positive cells among the total number of cells.

[0355]

[0398] As shown in Figure 36, HMC-EV treatment resulted in a dose-dependent attenuation of cell death. Significant rescue from cell death by HMC-EV was observed at doses of 30K and 100K. The overall cell death rate was approximately 44% lower than that of the control group without EV treatment.

[0356]

[0399] Therefore, these results demonstrated that HMC-EVs can prevent oxidative injury in neurons. Example 10 - In vitro glutamate excitotoxicity model

[0400] The HMC-EVs of the present invention were tested in an in vitro glutamate excitotoxicity (high-dose L-glutamate) model. Briefly, neurons were exposed to various concentrations of L-glutamate (approximately 0, 30, 300, and 3000 μM) and treated with HMC-EVs at a dose of approximately 50,000 EVs / cell. The percentage of cell death was determined as the number of propidium iodide (PI)+ cells out of the total number of cells.

[0357]

[0401] HMC-EV treatment maintained cells at the nuclear swelling stage and maintained viability after glutamate-induced injury, as shown in Figure 37. Staining with TMRM, a cell-permeant dye that accumulates in active mitochondria with intact membrane potential, demonstrated that HMC-EV treatment also maintained mitochondrial activity in injured cells.

[0358]

[0402] Therefore, these results demonstrated that HMC-EVs prevent neuronal death due to glutamate excitotoxic injury. Example 11 - RNAseq analysis of HMCs vs. bone marrow-MSCs vs. adipose tissue-MSCs

[0403] RNAseq analysis was performed on HMCs of the present invention under both basal and stimulated conditions. HMCs were generated from both N-line (N-HMC) and GMP-1 (GMP-HMC) cell lines, and triplicate technical replicates were prepared for each condition. MSCs isolated from adipose tissue and bone marrow were also analyzed and compared with HMCs of the present invention. AD-MSCs were collected from three different adult donors, and duplicate technical replicates were prepared for each biological replicate. BM-MSCs were also collected from three different adult donors. HMCs vs. adipose tissue-derived MSCs

[0404] Principal component analysis of the transcriptomes of HMCs (obtained from the N cell line) and AD-MSCs shows that HMCs differ from the latter in both the basal and interferon-gamma stimulated states (Figure 38). The first principal component primarily explains the effect of stimulation with gamma interferon, while the second principal component explains the differences between HMCs and AD-MSCs.

[0359]

[0405] The weights of different genes contributing to the second principal component, which determines the variance between HMCs and AD-MSCs, are particularly noteworthy. Compared with AD-MSCs, HMCs show a downregulation of collagen genes (e.g., COL1A1, COL3A1), mitochondrial function genes, and TGF-beta 1 (one of the main factors promoting angiogenesis), demonstrating that HMCs are somewhat immature (Figure 39).

[0360]

[0406] Hierarchical clustering demonstrates similarities between biological / technical replicates of the same biological type, as well as clear differences between HMCs and AD-MSCs, in both basal and gamma interferon-stimulated cell states (Figure 40).

[0361]

[0407] As shown in Figure 41, genes in this cluster were upregulated in HMCs (both basal and INFN gamma stimulated) compared to AD-MSCs. Genes included: CALR, UBB, PKM, CXCL8, C15orf48, PSME2, TPM3, ANKRD1, PFN1, SRGN, ACTB, MDK, TAGLN2, CFL1, HSP90AA1, HSPA8, CXCL12, UCHL1, HMGA2, HMGA1, HN1, PTMA, SP90AB1, PRDX1, GSTP1, KRT18, IGFBP4, CALD1, COL4A1, COL4A2, and GAPDH. The differential expression of these genes between HMCs and AD-MSCs was consistent across biological and technical replicate samples according to the hierarchical clustering map.

[0362]

[0408] Functional annotation of the biological pathways enriched in the clusters in Figure 41 was performed using Reactome (https: / / reactome.org / ). The top pathways enriched by the corresponding genes were related to axon guidance. Other pathways that were significantly enriched included cellular stress response and developmental biology (related to the relative immaturity of HMCs).

[0363]

[0409] As shown in Figure 42, genes in this cluster were downregulated in HMCs (both basal and INFN-gamma stimulated) compared to AD-MSCs. Genes included: SERPINE1, ACTA2, TPM2, CTGF, SERPINE2, CRYAB, ELN, MFGE8, ANXA2, POSTN, VIM, MFAP5, ISLR, THBS1, TIMP3, DKK1, COL6A3, COL6A1, TPT1, BCYRN1, COL1A1, SPARC, TPM1, BGN, COL1A2, COL3A1, TGFBI, CRLF1, COMP, NEAT1, MT-CO3, MT-CO2, MT-ATP8, MT-CYB, MT-CO1, MT-ATP6, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-ND3, MT-ND1, MT-ND2, GREM1, TMSB4X, ITGB1, LMNA, H2AFZ, FTL, EEF1G, NPM1, EEF1A1, RACK1, ACTG1, and TPM4. The differential expression of these genes between HMCs and AD-MSCs was consistent across biological and technical replicate samples according to hierarchical clustering maps.

[0364]

[0410] Functional annotation of the biological pathways enriched in the clusters in Figure 42 was performed using Reactome (https: / / reactome.org / ). The top pathways enriched by the corresponding genes were related to respiratory electron transport and mitochondrial function in general, as well as collagen biosynthesis.

[0365]

[0411] Canonical pathway enrichment of differential gene expression signatures between HMCs and AD-MSCs shows significant HMC-specific upregulation of several pathways (indicated by red arrows) involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, and synaptogenesis signaling (Figure 43). These results suggest that HMCs have distinct expression profiles compared to AD-MSCs and may contribute neurotrophic factors, factors involved in conferring neuroprotective effects and supporting neuronal health and recovery.

[0366]

[0412] A list of genes that contribute to the activated pathways that establish this difference is shown in Figures 44-47.

[0413] Figure 44 shows the top 15 most strongly differentially expressed genes that contribute to the activation of neuronal CREB signaling in HMCs. The expression log ratio refers to the base 10 logarithm of the fold change between the average TPM expression of a gene in HMCs and its average TPM expression in adipose tissue-derived MSCs; that is, an expression log ratio greater than 2 represents an increase in gene expression by a factor greater than 100.

[0367]

[0414] Figure 45 shows the top 15 most strongly upregulated genes that contribute to the enrichment of axon guidance pathways in HMCs. Although the activation pattern of axon guidance signaling pathways was not determined by Qiagen Ingenuity Pathway Analysis, this pathway was enriched in HMCs compared with AD-MSCs with a p-value of about 1.38e-4.

[0368]

[0415] Figure 46 shows the top 15 most highly expressed genes that contribute to the activation of synaptogenesis signaling pathways in HMCs, with an enrichment p-value of 1.14e-3 and an activation pattern z-score of 3.578, the highest among all differentially upregulated pathways in HMCs.

[0369]

[0416] Figure 47 shows the top 15 most upregulated genes that do not contribute to the activation of neuroinflammatory signaling pathways in HMCs. Pathway enrichment p-value 4.97e-3, activation z-score 1.508.

[0370]

[0417] HMCs were also generated from a different pluripotent stem cell, i.e., GMP1 cells. Principal component analysis of the transcriptome of GMP1-HMCs was also performed and compared with HMCs derived from N-lineage cells (N-HMCs) and AD-MSCs under both basal and stimulated conditions (Figure 48).

[0371]

[0418] Hierarchical clustering analysis showed that GMP1-HMC had a similar profile to N-HMC (Figure 49). As shown in Figure 50, the genes in this cluster were upregulated in N-HMC and GMP1-HMC (both basal and INFN gamma stimulated) compared to AD-MSC. The genes included: TMSB4X, ACTG1, GSTP1, KRT18, IGFBP5, NPY, KRT8, PRDX6, MDK, DKK3, UCHL1, TUBB3, HN1, PTMA, HSP90AB1, HMGA1, HSPA8, TAGLN2, ANKRD1, PFN1, CYBA, and UBB. The differential expression of these genes between N-HMC, GMP1-HMC, and adipose tissue-derived MSC lines was consistent across biological and technical replicate samples according to the hierarchical clustering map.

[0372]

[0419] Functional annotation of the biological pathways enriched in the clusters in Figure 50 was performed using Reactome (https: / / reactome.org / ). The top pathways enriched by the corresponding genes were related to axon guidance. Other significantly enriched pathways included cellular stress response and developmental biology.

[0373]

[0420] As shown in Figure 51, genes in these clusters were down-regulated in basal N-HMC and GMP1-HMC compared to AD-MSC. Genes included: SERPINE1, S100A6, CD59, POSTN, VIM, MFAP5, ISLR, THBS1, COL6A3, TIMP3, ELN, ANXA2, COL1A1, BYRNN1, CCDC80, COL6A1, COL6A2, BGN, COL1A2, COL3A1, TGFB1, CRLF1, COMP, and GREM1. The differential expression of these genes between N-HMC, GMP1-HMC, and AD-MSC lines was consistent across biological and technical replicates according to the hierarchical clustering map.

[0374]

[0421] As shown in Figure 52, genes in these clusters were down-regulated in N-HMCs and GMP1-HMCs stimulated with INFN gamma compared to AD-MSCs. The genes included: MT1X, MT1G, TMSB10, CCL8, INHBA, CTSB, SERPINB2, ADM, APOL1, FTH1, CCL2, CCL5, CSF1, IL1B, IGFBP3, P4HB, DCN, FSTL1, ANXA5, LOX, CD63, CTSZ, FN1, LGALS1, LDHA, RCN3, MMP2, and TIMP1. The differential expression of these genes between N-HMC, GMP1-HMC, and AD-MSC lines was consistent across biological and technical replicates according to the hierarchical clustering map.

[0375]

[0422] Reactome (https: / / reactome.org / ) was used to perform functional annotation of the biological pathways enriched in the clusters in Figures 51 and 52. The top pathways enriched by the corresponding genes were related to general extracellular matrix organization as well as collagen biosynthesis.

[0376]

[0423] Similarly, canonical pathway enrichment of differential gene expression signatures between N-HMCs, GMP1-HMCs, and AD-MSCs reveals a significant HMC-specific upregulation of several pathways (indicated by red arrows) involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, synaptogenesis signaling, etc. (Figures 53A-C and 54A-C). Thus, N-HMCs and GMP1-HMCs have similar profiles, and both showed enrichment for axon guidance.

[0377]

[0424] Therefore, it can be concluded that the HMCs of the present invention are different from AD-MSCs. Specifically, the MSCs of the present invention have a different expression profile compared to AD-MSCs, and may confer neuroprotective effects and provide neurotrophic factors, i.e., factors involved in supporting neuronal survival, growth, health, and recovery. HMCs vs. bone marrow-derived MSCs

[0425] Principal component analysis of the transcriptomes of HMCs (obtained from the N cell line) and BM-MSCs shows that HMCs differ from the latter in both basal and INFN-gamma stimulated states. The first principal component primarily explains the effect of stimulation with gamma interferon, while the second principal component explains the differences between HMCs and BM-MSCs (Figure 55).

[0378]

[0426] The weights of different genes contributing to the second principal component, which determines the variance between HMCs and BM-MSCs, are particularly noteworthy. Compared to BM-MSCs, HMCs show a downregulation of collagen genes (COL1A1, COL1A2, COL3A1, COL6A2, etc.), mitochondrial function genes, and TGF-beta1 (one of the main factors promoting angiogenesis), demonstrating a certain degree of immaturity in HMCs compared to the latter (Figure 56).

[0379]

[0427] Hierarchical clustering demonstrates similarities between biological replicate samples of the same type, as well as clear differences between HMCs and BM-MSCs, in both basal and gamma interferon-stimulated cell states (Figure 57).

[0380]

[0428] Genes in this cluster were upregulated in HMCs (both basal and INFN-gamma stimulated) compared to BM-MSCs (Figure 58). Genes included: PPIA, NPM1, HNRNPA1, IGFBP5, KRT19, KRT18, GSTP1, TUBB, TUBA1B, KRT8, HN1, PTMA, TUBA1C, HSPA8, HMGA1, CFL1, MYL6, ACTB, UCHL1, TAGLN2, MDK, GREM1, MMP1, and CTSC. The differential expression of these genes between HMCs and BM-MSCs was consistent across biological and technical replicates according to the hierarchical clustering map.

[0381]

[0429] Functional annotation of the biological pathways enriched in the clusters in Figure 58 was performed using Reactome (https: / / reactome.org / ). Among the top pathways enriched for corresponding genes is axon guidance. Other pathways that were significantly enriched included cellular stress response and developmental biology (related to the relative immaturity of HMCs).

[0382]

[0430] Genes in this cluster were downregulated in HMCs (both basal and INFN-gamma stimulated) compared to BM-MSCs (Figure 59). Genes included: ANXA2, TPT1, VIM, COL6A1, BGN, COL6A2, CTGF, TIMP3, ACTA2, COL3A1, SPARC, ITGB1, SERPINH1, TPM2, TGFBI, COL1A1, TPM1, COL6A3, TPM4, SERPINE2, CALD1, COL1A2, TAGLN, MYL9, MT-RNR2, and POSTN. The differential expression of these genes between HMCs and BM-MSCs was consistent across biological and technical replicates according to the hierarchical clustering map.

[0383]

[0431] Functional annotation of the biological pathways enriched in the clusters in Figure 59 was performed using Reactome (https: / / reactome.org / ). The top pathways enriched by the corresponding genes were related to collagen biosynthesis / assembly, demonstrating the similarity between BM-MSCs and AD-MSCs.

[0384]

[0432] Canonical pathway enrichment of differential gene expression signatures between HMCs and BM-MSCs reiterates HMC-specific upregulation of pathways involved in neuronal lineage development, such as CREB signaling in neurons (Figure 60).

[0385]

[0433] Figure 61 shows the top 15 most strongly differentially expressed genes that contribute to the activation of neuronal CREB signaling in HMCs compared to BM-MSCs. Figure 62 shows the top 15 most strongly upregulated genes that contribute to the activation of synaptogenesis signaling in HMCs compared to BM-MSCs.

[0386]

[0434] Therefore, it is concluded that the HMCs of the present invention are distinct from BM-MSCs, specifically, they have a distinct expression profile and confer neuroprotective effects when compared to BM-MSCs.

[0387]

Table 7-1

[0388]

Table 7-2

[0389]

Table 7-3

[0390]

Table 7-4

[0391]

Table 7-5

[0392]

Table 7-6

[0393]

Table 7-7

[0394]

Table 7-8

[0395]

Table 7-9

[0396]

Table 7-10

[0397]

Table 7-11

[0398]

Table 7-12

[0399]

Table 7-13

[0400]

Table 7-14

[0401]

Table 7-15

[0402]

Table 7-16

[0403]

Table 8-1

[0404]

Table 8-2

[0405]

Table 8-3

[0406]

Table 8-4

[0407]

Table 8-5

[0408]

Table 8-6

[0409]

Table 8-7

[0410]

Table 8-8

[0411]

Table 8-9

[0412]

Table 8-10

[0413]

Table 8-11

[0414]

Table 8-12

[0415]

Table 8-13

[0416]

Table 8-14

[0417]

Table 8-15

[0418]

Table 8-16

[0419]

Table 8-17

[0420] [Table 8-18]

[0421] [Table 8-19]

[0422] [Table 8-20]

[0423] [Table 8-21]

[0424] [Table 8-22]

[0425] [Table 8-23]

[0426] [Table 8-24]

[0427] [Table 8-25]

[0428] [Table 8-26]

[0429] Example 12 - miRNA Nanostring nCounter analysis of HMC-EVs vs. BM-MSC-EVs vs. UCB-MSC-EVs vs. AD-MSC-EVs

[0435] HMCs were generated from the same bank of frozen hemangioblasts described in Example 1. HMCs were generated according to the method described in Example 1 and passaged up to passage 6 (P6). Extracellular vesicles (EVs) were purified from HMCs (HMC-EVs) by tangential flow filtration (TFF). miRNA profiling was performed on three lots of HMC-EVs under basal conditions using the Nanostring nCounter Analysis system. EVs isolated from bone marrow (BM-MSC-EVs) (3 lots), umbilical cord blood (UCB-MSC-EVs) (3 lots), and adipose tissue (AD-MSC-EVs) under basal conditions were used as controls.

[0430]

[0436] Table 9 shows miRNAs that are more highly expressed in HMC-EVs compared to UCB-MSC-EVs. Table 10 shows miRNAs that are more highly expressed in UCB-MSC-EVs compared to HMC-EVs. Table 11 shows miRNAs that are more highly expressed in HMC-EVs compared to BM-MSC-EVs. Table 12 shows miRNAs that are more highly expressed in BM-MSC-EVs compared to HMC-EVs. Table 13 shows miRNAs that are more highly expressed in HMC-EVs compared to AD-MSC-EVs. Table 14 shows miRNAs that are more highly expressed in AD-MSC-EVs compared to HMC-EVs. The HMC-EVs of the present invention can be selected or purified based on any of the differentially expressed miRNAs.

[0431] [Table 9]

[0432] [Table 10]

[0433] [Table 11]

[0434] [Table 12-1]

[0435] [Table 12-2]

[0436] [Table 13]

[0437] [Table 14]

[0438] Example 13 - Proteomic profiling of HMC-EVs vs. BM-MSC-EVs vs. UCB-MSC-EVs vs. AD-MSC-EVs.

[0439] HMCs were generated from the same bank of frozen hemangioblasts described in Example 1. HMCs were generated according to the method described in Example 1 and passaged up to passage 6 (P6). Extracellular vesicles (EVs) were purified from HMCs (HMC-EVs) by tangential flow filtration (TFF). Proteome profiling by standard mass spectrometry was performed on three lots of HMC-EVs under basal conditions. EVs isolated from bone marrow (BM-MSC-EVs) (3 lots), umbilical cord blood (UCB-MSC-EVs) (3 lots), and adipose tissue (AD-MSC-EVs) under basal conditions were used as controls.

[0440] T-test statistical analysis was used to identify proteins with significant differences in abundance between EV types. Table 15 shows proteins that were more highly abundant in HMC-EVs compared to UCB-MSC-EVs. Table 16 shows proteins that were more highly abundant in UCB-MSC-EVs compared to HMC-EVs. Table 17 shows proteins that were more highly abundant in HMC-EVs compared to BM-MSC-EVs. Table 18 shows proteins that were more highly abundant in BM-MSC-EVs compared to HMC-EVs. Table 19 shows proteins that were more highly abundant in HMC-EVs compared to AD-MSC-EVs. Table 20 shows proteins that were more highly abundant in AD-MSC-EVs compared to HMC-EVs. HMC-EVs of the present invention can be selected or purified based on any of the differentially abundant proteins.

[0441] The proteomic data were then analyzed to determine how the overall protein expression profile might affect different signaling pathways. Figure 63A shows pathway enrichment of the differential expression patterns between HMC-EVs and BM-MSC-EVs. Figure 64A shows pathway enrichment of the differential expression patterns between HMC-EVs and AD-MSC-EVs. Figure 65A shows pathway enrichment of the differential expression patterns between HMC-EVs and EVs secreted from umbilical cord blood-derived MSCs (UCB-MSC-EVs). As shown in Figures 63A, 64A, and 65A, specific pathways, such as LXR / RXR activation, acute phase response signaling, B cell receptor signaling, and pathways involved in systemic lupus erythematosus in the B cell signaling pathway, are upregulated in HMC-EVs compared with EVs secreted from other tissue-derived MSCs (see orange bars). In addition, proteins that contribute to certain pathways, such as IL-15 signaling, claritin-mediated endocytosis signaling, and FXR / RXR activation, are also enriched (see white and gray bars), etc.

[0442] The disease or function annotation of differentially expressed proteins in HMC-EVs and EVs secreted from tissue-derived MSCs was also analyzed. Figure 63B shows the functional annotation of proteins upregulated in HMC-EVs compared to BM-MSC-EVs. Figure 63C shows the functional annotation of proteins downregulated in HMC-EVs compared to BM-MSC-EVs. Figure 64B shows the functional annotation of proteins upregulated in HMC-EVs compared to AD-MSC-EVs. Figure 64C shows the functional annotation of proteins downregulated in HMC-EVs compared to AD-MSC-EVs. Figure 65B shows the functional annotation of proteins upregulated in HMC-EVs compared to UCB-MSC-EVs. Figure 65C shows the functional annotation of proteins downregulated in HMC-EVs compared to UCB-MSC-EVs. An activation z-score greater than 2 or less than -2 is considered the threshold. This analysis suggests that proteins involved in cell viability / survival, cell migration, intercellular signaling and interaction pathways are upregulated in HMC-EVs, whereas proteins involved in cell death or apoptosis are downregulated in HMC-EVs.

[0443] [Table 15-1]

[0444] [Table 15-2]

[0445] [Table 15-3]

[0446] [Table 15-4]

[0447]

Table 15-5

[0448]

Table 16-1

[0449]

Table 16-2

[0450]

Table 16-3

[0451]

Table 17-1

[0452]

Table 17-2

[0453]

Table 17-3

[0454]

Table 17-4

[0455]

Table 17-5

[0456]

Table 17-6

[0457]

Table 17-7

[0458]

Table 17-8

[0459]

Table 18-1

[0460]

Table 18-2

[0461]

Table 19-1

[0462]

Table 19-2

[0463]

Table 19-3

[0464]

Table 19-4

[0465]

Table 19-5

[0466]

Table 19-6

[0467] [Table 19-7]

[0468] [Table 19-8]

[0469] [Table 19-9]

[0470] [Table 20-1]

[0471] [Table 20-2]

[0472] [Table 20-3]

[0473] [Table 20-4]

[0474] Example 14 - smRNAseq profiling of HMC cells versus HMC-EVs HMCs were generated from the same bank of frozen hemangioblasts described in Example 1. HMCs were generated according to the method described in Example 1 and passaged to passage 6 (P6). Extracellular vesicles (EVs) were purified from HMCs (HMC-EVs) by tangential flow filtration (TFF). smRNAseq profiling was performed on HMC-EVs (n=3) and HMCs (n=3).

[0475] Table 21 shows the smRNAs that were more highly abundant in HMC-EVs compared to HMCs. Table 22 shows the smRNAs that were more highly abundant in HMCs compared to HMC-EVs.

[0476] [Table 21-1]

[0477] [Table 21-2]

[0478] [Table 21-3]

[0479] [Table 21-4]

[0480] [Table 22-1]

[0481] [Table 22-2]

[0482] [Table 22-3]

[0483] [Table 22-4]

[0484]

[0443] While the above description and figures represent exemplary embodiments of the present disclosure, it will be understood that various additions, modifications, and substitutions can be made therein without departing from the spirit and scope of the appended claims and their equivalents. In particular, it will be apparent to those skilled in the art that the present invention can be embodied in other forms, structures, arrangements, and with other elements, materials, and components without departing from its spirit or essential characteristics. In addition, numerous variations in the methods / processes described herein may be made within the scope of the present disclosure without departing from the principles described herein. Accordingly, the embodiments disclosed herein should be considered in all respects as illustrative and not restrictive. The appended claims should be interpreted broadly to include other variations and embodiments of the present disclosure that may be made by those skilled in the art without departing from the scope and scope of equivalents.

Claims

1. A method for treating brain injury in a subject suffering from or suspected of suffering from a brain injury, comprising administering to the subject an effective amount of extracellular vesicles (EVs) secreted from mesenchymal stem cells (HMCs) obtained by in vitro differentiation of pluripotent stem cells, thereby treating the brain injury in the subject.

2. 2. The method of claim 1, wherein the brain injury is selected from the group consisting of stroke, optic neuropathy, traumatic brain injury, cerebral palsy, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, cerebral aneurysm, and coma.

3. 3. The method of claim 2, wherein the brain injury is a stroke.

4. The method according to any one of claims 1 to 3, comprising a step of increasing oligodendrocytes and progenitor cells in the brain after administering EVs (HMC-EVs) secreted from the HMC to the subject.

5. The method of any one of claims 1 to 3, comprising a step of preserving myelin in the brain after administering the HMC-EV to the subject.

6. The method of any one of claims 1 to 3, comprising a step of preventing oxidative damage in neurons after administering the HMC-EV to the subject.

7. The method of any one of claims 1 to 3, comprising a step of preventing neuronal death caused by glutamate excitotoxic injury after administering the HMC-EV to the subject.

8. The method of any one of claims 1 to 3, comprising a step of reducing tissue loss in the brain after administering the HMC-EV to the subject.

9. The method of any one of claims 1 to 3, comprising a step of reducing cell death in the brain after administering the HMC-EV to the subject.

10. The method of any one of claims 1 to 3, comprising stimulating pathways involved in the development of neuronal lineages after administering the HMC-EV to the subject.

11. The method of any one of claims 1 to 10, wherein the HMC-EV is administered systemically.

12. The method of any one of claims 1 to 10, wherein the HMC-EV is administered intracerebrally.

13. The method of any one of claims 1 to 10, wherein the HMC-EV is administered intrathecally.

14. The method of any one of claims 1 to 10, wherein the HMC-EVs are administered intracisternally.

15. The method of any one of claims 1 to 10, wherein the HMC-EV is administered intraperitoneally.

16. 16. The method of any one of claims 1 to 15, wherein the subject is a human.

17. 17. The method of any one of claims 1 to 16, wherein the HMCs are obtained by in vitro differentiation of human pluripotent stem cells.

18. 18. The method of any one of claims 1 to 17, wherein the pluripotent stem cells are further differentiated into hemangioblasts.

19. 19. The method of any one of claims 1 to 18, wherein the pluripotent stem cells are embryonic stem cells.

20. 19. The method of any one of claims 1 to 18, wherein the pluripotent stem cells are induced pluripotent stem cells.

21. 21. The method of claim 20, wherein the induced pluripotent stem cells are generated by contacting cells with one or more reprogramming factors.

22. 22. The method of any one of claims 1 to 21, wherein the HMC-EVs express at least one miRNA in Table 9 at a higher level compared to EVs secreted from umbilical cord blood-derived mesenchymal stem cells (UCB-MSC-EVs).

23. 23. The method of any one of claims 1 to 22, wherein the HMC-EVs express at least one miRNA in Table 10 at a lower level compared to UCB-MSC-EVs.

24. 24. The method of any one of claims 1 to 23, wherein the HMC-EVs express at least one miRNA in Table 11 at a higher level compared to EVs secreted from bone marrow-derived mesenchymal stem cells (BM-MSC-EVs).

25. 25. The method of any one of claims 1 to 24, wherein the HMC-EVs express at least one miRNA in Table 12 at a lower level compared to BM-MSC-EVs.

26. 26. The method of any one of claims 1 to 25, wherein the HMC-EVs express at least one miRNA in Table 13 at a higher level compared to EVs secreted from adipose tissue-derived mesenchymal stem cells (AD-MSC-EVs).

27. 27. The method of any one of claims 1 to 26, wherein the HMC-EVs express at least one of the miRNAs in Table 14 at a lower level compared to AD-MSC-EVs.

28. 28. The method of any one of claims 1 to 27, wherein the HMC-EVs express at least one of the proteins in Table 15 at a higher level compared to UCB-MSC-EVs.

29. 29. The method of any one of claims 1 to 28, wherein the HMC-EVs express at least one of the proteins in Table 16 at a lower level compared to UCB-MSC-EVs.

30. 30. The method of any one of claims 1 to 29, wherein the HMC-EVs express at least one of the proteins in Table 17 at a higher level compared to BM-MSC-EVs.

31. 31. The method of any one of claims 1 to 30, wherein the HMC-EVs express at least one of the proteins in Table 18 at a lower level compared to BM-MSC-EVs.

32. 32. The method of any one of claims 1 to 31, wherein the HMC-EVs express at least one of the proteins in Table 19 at a higher level compared to AD-MSC-EVs.

33. 33. The method of any one of claims 1 to 32, wherein the HMC-EVs express at least one of the proteins in Table 20 at a lower level compared to AD-MSC-EVs.

34. 34. The method of any one of claims 1 to 33, wherein the HMC-EVs express at least one miRNA in Table 21 at a higher level compared to the HMCs.

35. 35. The method of any one of claims 1 to 34, wherein the HMC-EVs express at least one miRNA in Table 22 at a lower level compared to the HMCs.

36. 36. The method of any one of claims 1 to 35, wherein the HMC-EVs express at least one miRNA selected from the group consisting of hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-199b-5p, hsa-miR-21-5p, hsa-miR-23a-3p, hsa-miR-125a-5p, hsa-miR-106a-5p+hsa-miR-17-5p, and hsa-miR-221-3p at a higher level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

37. The HMC-EVs express at higher levels ALDOC, ANXA5, APBB2, BASP1, CAV1, CD81, CD99, CKM, EPB41L3, FDPS, GNAQ, GNG12, GP9, H2AC20, H2AC21, H3-3A, H3-7, H4-16, HLA-A, ITGA2, KPNA2, KRAS, KRT4, LRRC59, MAMDC2, MARCKS, and the like, as compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

37. The method of any one of claims 1 to 36, wherein the cell expresses at least one protein selected from the group consisting of L1, MDGA1, MERTK, MFGE8, MMP14, MVP, PCDH1, PDGFRB, PDIA3, RPL13, RPS18, RPS3A, RPS4X, SDCBP, SLC2A1, SLC3A2, TAGLN2, TNC, TSPAN14, TSPAN33, TSPAN9, TTYH3, UCHL1, VAT1, YWHAB, and YWHAQ.

38. The HMC-EVs express at higher levels ADGRG6, AGRN, ANXA6, APOC4, ARHGAP1, ARGHDIA, ARL8A, ARPC5, B2M, BBS1, BLVRA, BST1, CA2, CC, IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-59 ... N2, CCNB3, CD34, CD36, CD47, CORO1A, DTD1, EEF1D, EEF1G, ENG, ESD, GNAI2, GNB1, H1 -3, H2BC15, HIP1, KIF11, LAMP1, LAP3, LGALS1, LTBP3, MAPK3, MARCKS, MBTD1, MDH1, M 38. The method of any one of claims 1 to 37, wherein the cell expresses at least one protein selected from the group consisting of OB1B, MYL12B, MYO1F, MYO3A, NIBAN2, PEBP1, PF4, PGAP1, PLOD1, PPP2R1A, PRSS23, PXDN, RALA, RAP2A, RPS13, RPS3, RPSA, S100A11, SLC44A1, SLC44A2, SLTM, SMG1, SPARC, SRSF8, STRADB, STX11, STXBP2, TGM2, TPP1, TPTE2, TRIM5, TRPM2, TUBA8, TUBB3, VCAN, YWHAE, and ZFN607.

39. 39. The method of any one of claims 1 to 38, wherein the HMC-EVs express at least one protein selected from the group consisting of ADIPOQ, CAT, CEP290, IGLV6-57, TAS2R33, and TMEM198 at a lower level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

40. 40. The method of any one of claims 1 to 39, wherein the HMC-EVs express at least one protein selected from the group consisting of AKAP9, ALB, ALOX5, APLP2, CD109, CDSN, CHST9, ERC1, F11, ARMCX5, LAMB4, LRRTM2, LTF, MSH6, OAF, OLFML3, PAK6, RGS14, SEMA7A, SURF1, and TRIM4 at a lower level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

41. Approximately 1×10 ６ ~Approx. 1×10 １３ 41. The method of any one of claims 1 to 40, wherein HMC-EVs are administered to the subject.

42. Approximately 10×10 １０ pieces or about 30 x 10 １０ 42. The method of any one of claims 1 to 41, wherein HMC-EVs are administered to the subject.

43. The method of any one of claims 1 to 42, wherein the HMC-EV is administered in the form of a pharmaceutical composition.

44. The pharmaceutical composition comprises: (a) a buffer that maintains the solution at physiological pH; (b) at least 2 mM or at least 0.05% (w / v) glucose; (c) an osmotically active agent that maintains the solution at physiological osmolality; 44. The method of claim 43, comprising:

45. 45. The method of claim 44, wherein the glucose is D-glucose (dextrose).

46. 45. The method of claim 44, wherein the osmotically active agent is a salt.

47. 47. The method of claim 46, wherein the salt is sodium chloride.

48. 48. The method of any one of claims 1 to 47, further comprising administering to the subject an effective amount of HMCs obtained by in vitro differentiation of pluripotent stem cells.

49. 1. A method for treating brain injury in a subject suffering from or suspected of suffering from a brain injury, comprising administering to the subject an effective amount of mesenchymal stem cells (HMCs) obtained by in vitro differentiation of pluripotent stem cells, thereby treating the brain injury in the subject.

50. 50. The method of claim 49, wherein the brain injury is selected from the group consisting of stroke, optic neuropathy, traumatic brain injury, cerebral palsy, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, cerebral aneurysm, and coma.

51. 51. The method of claim 50, wherein the brain injury is a stroke.

52. 52. The method of any one of claims 49 to 51, comprising preserving myelin in the brain after administering the HMC to the subject.

53. 52. The method of any one of claims 49 to 51, comprising a step of suppressing a neuroinflammatory response after administering the HMC to the subject.

54. 52. The method of any one of claims 49 to 51, comprising reducing microglial and astrocytic activation in the brain after administering the HMC to the subject.

55. 52. The method of any one of claims 49 to 51, comprising stimulating a pathway involved in cell survival after administering the HMC to the subject.

56. 52. The method of any one of claims 49 to 51, comprising stimulating expression of neuroprotective genes in the brain after administering the HMC to the subject.

57. 57. The method of claim 56, wherein the neuroprotective gene is selected from the group consisting of heat shock protein family B member 1 (HSPB1), insulin-like growth factor 1 (IGF2), and secreted phosphoprotein 1 (SPP1).

58. 52. The method of any one of claims 49 to 51, comprising stimulating pathways involved in synaptic transmission in the brain after administering the HMC to the subject.

59. 52. The method of any one of claims 49 to 51, comprising stimulating pathways involved in the development of neuronal lineages after administering the HMC to the subject.

60. 52. The method of any one of claims 49 to 51, comprising a step of reducing apoptosis after administering the HMC to the subject.

61. 51. The method of claim 50, wherein the brain injury is a traumatic brain injury.

62. 62. The method of claim 61, comprising a step of reducing tissue loss in the brain after administering the HMC to the subject.

63. 63. The method of claim 61 or 62, comprising a step of reducing cell death in the brain after administering the HMC to the subject.

64. 64. The method of any one of claims 61 to 63, comprising increasing neurogenesis after administering the HMC to the subject.

65. 65. The method of any one of claims 61 to 64, comprising a step of reducing the abundance of microglia and macrophages in the cortex and striatum after administering the HMC to the subject.

66. 66. The method of any one of claims 61 to 65, comprising a step of reducing inflammation in the spleen after administering the HMC to the subject.

67. 67. The method of any one of claims 61 to 66, comprising the movement of HMCs across the blood-brain barrier to the cortex, striatum, and / or hippocampus.

68. 51. The method of claim 50, wherein the brain injury is cerebral palsy.

69. 69. The method of claim 68, comprising a step of reducing apoptosis in the brain after administering the HMC to the subject.

70. 70. The method of claim 68 or 69, comprising a step of reducing lesion size in the brain after administering the HMC to the subject.

71. 71. The method of any one of claims 68 to 70, comprising a step of reducing microglial and astrocytic activation in the brain after administering the HMC to the subject.

72. 72. The method of any one of claims 68 to 71, comprising preserving myelin in the corpus callosum after administering the HMC to the subject.

73. 73. The method of any one of claims 68 to 72, comprising at least partial rescue of Olig2 in the brain after administering the HMC to the subject.

74. 74. The method of any one of claims 49 to 73, wherein the HMC is administered systemically.

75. 74. The method of any one of claims 49 to 73, wherein the HMC is administered intracerebrally.

76. 74. The method of any one of claims 49 to 73, wherein the HMC is administered intrathecally.

77. 74. The method of any one of claims 49 to 73, wherein the HMC is administered intracisternally.

78. 74. The method of any one of claims 49 to 73, wherein the HMC is administered intraperitoneally.

79. 79. The method of any one of claims 49 to 78, wherein the mesenchymal stem cells are human cells.

80. 80. The method of any one of claims 49 to 79, wherein the subject is a human.

81. 81. The method of any one of claims 49 to 80, wherein the pluripotent stem cells are further differentiated into hemangioblasts.

82. 82. The method of any one of claims 49 to 81, wherein the pluripotent stem cells are embryonic stem cells.

83. 83. The method of any one of claims 49 to 82, wherein the pluripotent stem cells are induced pluripotent stem cells.

84. 84. The method of any one of claims 49 to 83, wherein the pluripotent stem cells are human pluripotent stem cells.

85. 85. The method of any one of claims 49 to 84, wherein the HMCs have been passaged in vitro no more than five times prior to administration to the subject.

86. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one of the genes in Table 3 at a higher level compared to bone marrow-derived MSCs (BM-MSCs).

87. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one of the genes in Table 4 at a lower level compared to BM-MSCs.

88. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one of the genes in Table 5 at a higher level compared to umbilical cord blood-derived MSCs (UCB-MSCs).

89. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one of the genes in Table 6 at a lower level compared to UCB-MSCs.

90. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one of the genes in Table 7 at a higher level compared to adipose tissue-derived MSCs (AD-MSCs).

91. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one of the genes in Table 8 at a lower level compared to AD-MSCs.

92. 86. The method of any one of claims 49 to 85, wherein the HMCs basally express mRNA encoding interleukin-6 (IL-6) at a level that is less than 10 percent of the IL-6 mRNA level expressed by BM-MSCs basally, and the HMCs basally express mRNA encoding CD24 at a level that is greater than the CD24 mRNA level expressed by BM-MSCs basally.

93. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one gene selected from the group consisting of CALR, UBB, PKM, CXCL8, C15orf48, PSME2, TPM3, ANKRD1, PFN1, SRGN, ACTB, MDK, TAGLN2, CFL1, HSP90AA1, HSPA8, CXCL12, UCHL1, HMGA2, HMGA1, HN1, PTMA, SP90AB1, PRDX1, GSTP1, KRT18, IGFBP4, CALD1, COL4A1, COL4A2, and GAPDH at a higher level compared to AD-MSCs.

94. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one gene selected from the group consisting of TMSB4X, ACTG1, GSTP1, KRT18, IGFBP5, NPY, KRT8, PRDX6, MDK, DKK3, UCHL1, TUBB3, HN1, PTMA, HSP90AB1, HMGA1, HSPA8, TAGLN2, ANKRD1, PFN1, CYBA, and UBB at a higher level compared to AD-MSCs.

95. The HMCs express lower levels of SERPINE1, ACTA2, TPM2, CTGF, SERPINE2, CRYAB, ELN, MFGE8, ANXA2, POSTN, VIM, MFAP5, ISLR, THBS1, TIMP3, DKK1, COL6A3, COL6A1, TPT1, BCYRN1, COL1A1, SPARC, TPM1, BGN, COL1A2, COL3A1, TGFBI, CRLF1, COMP, NEAT1, MT-CO, and MT-CO2 compared to AD-MSCs. 3, MT-CO2, MT-ATP8, MT-CYB, MT-CO1, MT-ATP6, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-ND3, MT-ND1, MT-ND2, GREM1, TMSB4X, ITGB1, LMNA, H2AFZ, FTL, EEF1G, NPM1, EEF1A1, RACK1, ACTG1, and TPM4.

96. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one gene selected from the group consisting of SERPINE1, S100A6, CD59, POSTN, VIM, MFAP5, ISLR, THBS1, COL6A3, TIMP3, ELN, ANXA2, COL1A1, BCYRN1, CCDC80, COL6A1, COL6A2, BGN, COL1A2, COL3A1, TGFB1, CRLF1, COMP, and GREM1 at a lower level compared to AD-MSCs.

97. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one gene selected from the group consisting of MT1X, MT1G, TMSB10, CCL8, INHBA, CTSB, SERPINB2, ADM, APOL1, FTH1, CCL2, CCL5, CSF1, IL1B, IGFBP3, P4HB, DCN, FSTL1, ANXA5, LOX, CD63, CTSZ, FN1, LGALS1, LDHA, RCN3, MMP2, and TIMP1 at a lower level compared to AD-MSCs.

98. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one gene selected from the group consisting of PPIA, NPM1, HNRNPA1, IGFBP5, KRT19, KRT18, GSTP1, TUBB, TUBA1B, KRT8, HN1, PTMA, TUBA1C, HSPA8, HMGA1, CFL1, MYL6, ACTB, UCHL1, TAGLN2, MDK, GREM1, MMP1, and CTSC at a higher level compared to BM-MSCs.

99. 86. The method of any one of claims 49 to 85, wherein the HMCs express at least one gene selected from the group consisting of ANXA2, TPT1, VIM, COL6A1, BGN, COL6A2, CTGF, TIMP3, ACTA2, COL3A1, SPARC, ITGB1, SERPINH1, TPM2, TGFBI, COL1A1, TPM1, COL6A3, TPM4, SERPINE2, CALD1, COL1A2, TAGLN, MYL9, MT-RNR2, POSTN at a lower level compared to BM-MSCs.

100. 86. The method of any one of claims 49-85, wherein the HMC express at least one miRNA in Table 21 at a lower level compared to HMC-EV.

101. 86. The method of any one of claims 49-85, wherein the HMC expresses at least one miRNA in Table 22 at a higher level compared to HMC-EV.

102. Approximately 1×10 ６ ~Approx. 1×10 １３ 102. The method of any one of claims 49 to 101, wherein HMC is administered to the subject.

103. 103. The method of any one of claims 49 to 102, wherein the HMC is administered in a pharmaceutical composition.

104. The pharmaceutical composition comprises: (a) a buffer that maintains the solution at physiological pH; (b) at least 2 mM or at least 0.05% (w / v) glucose; (c) an osmotically active agent that maintains the solution at physiological osmolality; 104. The method of claim 103, comprising:

105. 105. The method of claim 104, wherein the glucose is D-glucose (dextrose).

106. 105. The method of claim 104, wherein the osmotically active agent is a salt.

107. 107. The method of claim 106, wherein the salt is sodium chloride.

108. 1. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of CALR, UBB, PKM, CXCL8, C15orf48, PSME2, TPM3, ANKRD1, PFN1, SRGN, ACTB, MDK, TAGLN2, CFL1, HSP90AA1, HSPA8, CXCL12, UCHL1, HMGA2, HMGA1, HN1, PTMA, SP90AB1, PRDX1, GSTP1, KRT18, IGFBP4, CALD1, COL4A1, COL4A2, and GAPDH at a higher level compared to AD-MSCs.

109. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of TMSB4X, ACTG1, GSTP1, KRT18, IGFBP5, NPY, KRT8, PRDX6, MDK, DKK3, UCHL1, TUBB3, HN1, PTMA, HSP90AB1, HMGA1, HSPA8, TAGLN2, ANKRD1, PFN1, CYBA, and UBB at a higher level compared to AD-MSCs.

110. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of PPIA, NPM1, HNRNPA1, IGFBP5, KRT19, KRT18, GSTP1, TUBB, TUBA1B, KRT8, HN1, PTMA, TUBA1C, HSPA8, HMGA1, CFL1, MYL6, ACTB, UCHL1, TAGLN2, MDK, GREM1, MMP1, and CTSC at a higher level compared to BM-MSCs.

111. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express lower levels of SERPINE1, ACTA2, TPM2, CTGF, SERPINE2, CRYAB, ELN, MFGE8, ANXA2, POSTN, VIM, MFAP5, ISLR, THBS1, TIMP3, DKK1, COL6A3, COL6A1, TPT1, BCYRN1, COL1A1, SPARC, TPM1, BGN, COL1A2, COL3A1, TGF-β, and IFN-γ at the same time as AD-MSCs. A composition expressing at least one gene selected from the group consisting of BI, CRLF1, COMP, NEAT1, MT-CO3, MT-CO2, MT-ATP8, MT-CYB, MT-CO1, MT-ATP6, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-ND3, MT-ND1, MT-ND2, GREM1, TMSB4X, ITGB1, LMNA, H2AFZ, FTL, EEF1G, NPM1, EEF1A1, RACK1, ACTG1, and TPM4.

112. 1. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of SERPINE1, S100A6, CD59, POSTN, VIM, MFAP5, ISLR, THBS1, COL6A3, TIMP3, ELN, ANXA2, COL1A1, BCYRN1, CCDC80, COL6A1, COL6A2, BGN, COL1A2, COL3A1, TGFB1, CRLF1, COMP, and GREM1 at a lower level compared to AD-MSCs.

113. 1. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of MT1X, MT1G, TMSB10, CCL8, INHBA, CTSB, SERPINB2, ADM, APOL1, FTH1, CCL2, CCL5, CSF1, IL1B, IGFBP3, P4HB, DCN, FSTL1, ANXA5, LOX, CD63, CTSZ, FN1, LGALS1, LDHA, RCN3, MMP2, and TIMP1 at a lower level compared to AD-MSCs.

114. 1. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at least one gene selected from the group consisting of ANXA2, TPT1, VIM, COL6A1, BGN, COL6A2, CTGF, TIMP3, ACTA2, COL3A1, SPARC, ITGB1, SERPINH1, TPM2, TGFBI, COL1A1, TPM1, COL6A3, TPM4, SERPINE2, CALD1, COL1A2, TAGLN, MYL9, MT-RNR2, and POSTN at a lower level compared to BM-MSCs.

115. 115. The composition of any one of claims 108 to 114, wherein the HMCs further express at least one of the genes in Table 3 at a higher level compared to BM-MSCs.

116. 115. The composition of any one of claims 108 to 114, wherein the HMCs further express at least one of the genes in Table 4 at a lower level compared to BM-MSCs.

117. 115. The composition of any one of claims 108 to 114, wherein the HMCs further express at least one of the genes in Table 5 at a higher level compared to UCB-MSCs.

118. 115. The composition of any one of claims 108 to 114, wherein the HMCs further express at least one of the genes in Table 6 at a lower level compared to UCB-MSCs.

119. 115. The composition of any one of claims 108 to 114, wherein the HMCs further express at least one of the genes in Table 7 at a higher level compared to AD-MSCs.

120. 115. The composition of any one of claims 108 to 114, wherein the HMCs further express at least one of the genes in Table 8 at a lower level compared to AD-MSCs.

121. 115. A pharmaceutical composition comprising a HMC according to any one of claims 108 to 114 and a pharmaceutically acceptable carrier.

122. A population of HMC-EVs described in any one of claims 108 to 114.

123. 123. The population of EVs of claim 122, wherein the HMC-EVs express at least one miRNA in Table 9 at a higher level compared to UCB-MSC-EVs.

124. 124. The population of EVs of claim 122 or 123, wherein the HMC-EVs express at least one miRNA in Table 10 at a lower level compared to UCB-MSC-EVs.

125. 125. The population of EVs of any one of claims 122 to 124, wherein the HMC-EVs express at least one miRNA in Table 11 at a higher level compared to BM-MSC-EVs.

126. 126. The population of EVs of any one of claims 122 to 125, wherein the HMC-EVs express at least one miRNA in Table 12 at a lower level compared to BM-MSC-EVs.

127. 127. The population of EVs of any one of claims 122 to 126, wherein the HMC-EVs express at least one miRNA in Table 13 at a higher level compared to AD-MSC-EVs.

128. 128. The population of EVs of any one of claims 122 to 127, wherein the HMC-EVs express at least one miRNA in Table 14 at a lower level compared to AD-MSC-EVs.

129. 129. The population of EVs of any one of claims 122 to 128, wherein the HMC-EVs express at least one of the proteins in Table 15 at a higher level compared to UCB-MSC-EVs.

130. 130. The population of EVs of any one of claims 122 to 129, wherein the HMC-EVs express at least one of the proteins in Table 16 at a lower level compared to UCB-MSC-EVs.

131. 131. The population of EVs of any one of claims 122 to 130, wherein the HMC-EVs express at least one of the proteins in Table 17 at a higher level compared to BM-MSC-EVs.

132. 132. The population of EVs of any one of claims 122-131, wherein the HMC-EVs express at least one of the proteins in Table 18 at a lower level compared to BM-MSC-EVs.

133. 133. The population of EVs of any one of claims 122 to 132, wherein the HMC-EVs express at least one of the proteins in Table 19 at a higher level compared to AD-MSC-EVs.

134. 134. The population of EVs of any one of claims 122 to 133, wherein the HMC-EVs express at least one of the proteins in Table 20 at a lower level compared to AD-MSC-EVs.

135. 135. The population of EVs of any one of claims 122 to 134, wherein the HMC-EVs express at least one miRNA in Table 21 at a higher level compared to HMCs.

136. 136. The population of EVs of any one of claims 122 to 135, wherein the HMC-EVs express at least one miRNA in Table 22 at a lower level compared to HMCs.

137. 137. The population of EVs of any one of claims 122 to 136, wherein the HMC-EVs express at least one miRNA selected from the group consisting of hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-199b-5p, hsa-miR-21-5p, hsa-miR-23a-3p, hsa-miR-125a-5p, hsa-miR-106a-5p+hsa-miR-17-5p, and hsa-miR-221-3p at higher levels compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

138. the HMC-EVs express at higher levels, as compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs, ALDOC, ANXA5, APBB2, BASP1, CAV1, CD81, CD99, CKM, EPB41L3, FDPS, GNAQ, GNG12, GP9, H2AC20, H2AC21, H3-3A, H3-7, H4-16, HLA-A, ITGA2, KPNA2, KRAS, KRT4, LRRC59, MAMDC2, MARCKSL1, 138. The population of EVs of any one of claims 122 to 137, wherein the EVs express at least one protein selected from the group consisting of MDGA1, MERTK, MFGE8, MMP14, MVP, PCDH1, PDGFRB, PDIA3, RPL13, RPS18, RPS3A, RPS4X, SDCBP, SLC2A1, SLC3A2, TAGLN2, TNC, TSPAN14, TSPAN33, TSPAN9, TTYH3, UCHL1, VAT1, YWHAB, and YWHAQ.

139. The HMC-EVs express at higher levels ADGRG6, AGRN, ANXA6, APOC4, ARHGAP1, ARGHDIA, ARL8A, ARPC5, B2M, BBS1, BLVRA, BST1, CA2, CCN, and / or BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs. 2, CCNB3, CD34, CD36, CD47, CORO1A, DTD1, EEF1D, EEF1G, ENG, ESD, GNAI2, GNB1, H1-3 , H2BC15, HIP1, KIF11, LAMP1, LAP3, LGALS1, LTBP3, MAPK3, MARCKS, MBTD1, MDH1, MOB1 139. The population of EVs of any one of claims 122 to 138, wherein the EVs express at least one protein selected from the group consisting of: B, MYL12B, MYO1F, MYO3A, NIBAN2, PEBP1, PF4, PGAP1, PLOD1, PPP2R1A, PRSS23, PXDN, RALA, RAP2A, RPS13, RPS3, RPSA, S100A11, SLC44A1, SLC44A2, SLTM, SMG1, SPARC, SRSF8, STRADB, STX11, STXBP2, TGM2, TPP1, TPTE2, TRIM5, TRPM2, TUBA8, TUBB3, VCAN, YWHAE, and ZFN607.

140. 140. The population of HMC-EVs of any one of claims 122 to 139, wherein the HMC-EVs express at least one protein selected from the group consisting of ADIPOQ, CAT, CEP290, IGLV6-57, TAS2R33, and TMEM198 at a lower level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

141. 141. The population of HMC-EVs of any one of claims 122 to 140, wherein the HMC-EVs express at least one protein selected from the group consisting of AKAP9, ALB, ALOX5, APLP2, CD109, CDSN, CHST9, ERC1, F11, ARMCX5, LAMB4, LRRTM2, LTF, MSH6, OAF, OLFML3, PAK6, RGS14, SEMA7A, SURF1, and TRIM4 at a lower level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

142. A pharmaceutical composition comprising the HMC-EV of any one of claims 122 to 141 and a pharmaceutically acceptable carrier.

143. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 9 at a higher level compared to UCB-MSC-EVs.

144. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 10 at a lower level compared to UCB-MSC-EVs.

145. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 11 at a higher level compared to BM-MSC-EVs.

146. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 12 at a lower level compared to BM-MSC-EVs.

147. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 13 at a higher level compared to AD-MSC-EVs.

148. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the miRNAs in Table 14 at a lower level compared to AD-MSC-EVs.

149. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 15 at a higher level compared to UCB-MSC-EVs.

150. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 16 at a lower level compared to UCB-MSC-EVs.

151. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 17 at a higher level compared to BM-MSC-EVs.

152. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 18 at a lower level compared to BM-MSC-EVs.

153. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 19 at a higher level compared to AD-MSC-EVs.

154. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one of the proteins in Table 20 at a lower level compared to AD-MSC-EVs.

155. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one miRNA selected from the group consisting of hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-199b-5p, hsa-miR-21-5p, hsa-miR-23a-3p, hsa-miR-125a-5p, hsa-miR-106a-5p+hsa-miR-17-5p, and hsa-miR-221-3p at a higher level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

156. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express higher levels of ALDOC, ANXA5, APBB2, BASP1, CAV1, CD81, CD99, CKM, EPB41L3, FDPS, GNAQ, GNG12, GP9, H2AC20, H2AC21, H3-3A, H3-7, H4-16, HLA-A, ITGA2, KPNA2, KRAS, and KRT4 compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs. , LRRC59, MAMDC2, MARCKSL1, MDGA1, MERTK, MFGE8, MMP14, MVP, PCDH1, PDGFRB, PDIA3, RPL13, RPS18, RPS3A, RPS4X, SDCBP, SLC2A1, SLC3A2, TAGLN2, TNC, TSPAN14, TSPAN33, TSPAN9, TTYH3, UCHL1, VAT1, YWHAB, and YWHAQ.

157. Pluripotent stem cells in A population of HMC-EVs obtained by in vitro differentiation, wherein the HMC-EVs exhibit higher levels of ADGRG6, AGRN, ANXA6, APOC4, ARHGAP1, ARGHDIA, ARL8A, ARPC5, B2M, BBS1, BLVRA, BST1, CA2, CCN2, CCNB3, CD34, CD36, CD47, CORO1A, DTD1, EEF1D, EEF1G, ENG, ESD, GNAI2, GNB1, H1-3, H2BC15, HIP1, KIF11, LAMP1, LAP3, LGALS1, LTBP3, MAPK, compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs. 3, MARCKS, MBTD1, MDH1, MOB1B, MYL12B, MYO1F, MYO3A, NIBAN2, PEBP1, PF4, PGAP1, PLOD 1, PPP2R1A, PRSS23, PXDN, RALA, RAP2A, RPS13, RPS3, RPSA, S100A11, SLC44A1, SLC44A2 A population of HMC-EVs expressing at least one protein selected from the group consisting of: SLTM, SMG1, SPARC, SRSF8, STRADB, STX11, STXBP2, TGM2, TPP1, TPTE2, TRIM5, TRPM2, TUBA8, TUBB3, VCAN, YWHAE, and ZFN607.

158. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one protein selected from the group consisting of ADIPOQ, CAT, CEP290, IGLV6-57, TAS2R33, and TMEM198 at a lower level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

159. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one protein selected from the group consisting of AKAP9, ALB, ALOX5, APLP2, CD109, CDSN, CHST9, ERC1, F11, ARMCX5, LAMB4, LRRTM2, LTF, MSH6, OAF, OLFML3, PAK6, RGS14, SEMA7A, SURF1, and TRIM4 at a lower level compared to BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

160. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein said HMC-EVs express at least one of the miRNAs in Table 21 at a higher level compared to HMCs.

161. A population of HMC-EVs obtained by in vitro differentiation of pluripotent stem cells, wherein said HMC-EVs express at least one of the miRNAs in Table 22 at a lower level compared to HMCs.

162. A pharmaceutical composition comprising the HMC-EV of any one of claims 143 to 161 and a pharmaceutically acceptable carrier.

163. 1. A method for determining neurite outgrowth of a population of HMCs, comprising: (a) preparing a mixed neuronal culture from the isolated cerebral cortex; (b) seeding the HMC population onto a permeable membrane; (c) straining the mixed neuronal culture; (d) overlaying the strained mixed neuronal culture with the permeable membrane of step (b); (e) measuring neurite outgrowth of the mixed neuronal cultures; A method comprising:

164. 164. The method of claim 163, further comprising determining gene expression in the mixed neuronal culture in the presence and absence of the HMC population.

165. 164. The method of claim 163, wherein the strain is a physical scratch performed on the mixed neuronal culture.

166. 164. The method of claim 163, wherein the strain is a vacuum pressure and a positive air pressure applied to the mixed neuronal culture.

167. 164. The method of claim 163, wherein the strain is applied as a stretching vibration of 15% to 0%.

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