Methods of 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 comprise administering to a subject HMC obtained by in vitro differentiation of pluripotent stem cells and / or extracellular vesicles (EV) derived from these HMCs (HMC-EV).

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 390,044, filed on July 18, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to a method for treating brain damage using mesenchymal stem cells and / or extracellular vesicles secreted from mesenchymal stem cells. Background Art

[0004] Brain injuries are complex and can have a variety of severe clinical outcomes. Acquired brain injury is brain injury that is non-genetic, congenital, degenerative, or caused by birth trauma. The injury results in altered brain neuronal activity, which affects the physical integrity, metabolic activity, or functional capacity of nerve cells in the brain. There are two main types of acquired brain injury: traumatic and non-traumatic.

[0005] Traumatic brain injury (TBI) is the leading cause of death and disability in the United States. More than 1.7 million people in the United States suffer from TBI each year. TBI is caused by an external force, such as a blow, hit, or jolt to the head that disrupts the normal function of the brain. The severity of TBI can range from "mild" (i.e., a brief change in mental state or consciousness) to "severe" (i.e., loss of consciousness or memory loss for a long time after the injury). TBI accounts for approximately 30% of all traumatic deaths. (Taylor et al. MMWR Surveill. Summ. 2017; 66(No.SS-9): 1-16). Approximately 153 people die every day in the United States from trauma, including TBI. Ibid. Those who survive TBI may face effects that last for days or for a lifetime. The effects of TBI can include impairment of thinking or memory, movement, sensory (e.g., vision or hearing), or emotional function (e.g., personality changes, depression).

[0006] About 20%-40% of TBI patients will experience related visual impairment (Houston KE et al., Am JPhys. Med. Rehabil. 2017, 96: e70-4). This can include blurred vision, visual field loss, and decreased visual acuity. Depending on the type, location, and severity of the injury, these symptoms can occur acutely or chronically. TBI can affect different parts of the visual system, including the optic nerve and optic tract, the lateral geniculate nucleus, and the optic radiation, leading to a variety of visual problems (Barnett BP et al., Curr Treat Options Neurol., 2015; 17: 329). One known site of afferent pathway damage is through the optic nerve and optic tract. Structurally, the optic nerve is susceptible to compression, traction, extrusion, tearing, and avulsion injuries. Rapid acceleration or deceleration of the head may indirectly cause traction or axonal shearing of the optic nerve, which can lead to optic neuropathy.

[0007] To date, some 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, paper ID 1582182, 6 pages). However, there is still a need for improved treatments for TBI.

[0008] Non-traumatic brain injury is usually caused by damage to the brain caused by internal factors, such as lack of oxygen, exposure to toxins, tumor compression, etc. Stroke is an example of non-traumatic brain injury. Stroke is the fifth leading cause of death in the United States, and nearly 800,000 people have a stroke each year. A stroke occurs when a blood vessel blockage or bleeding interrupts or reduces the blood supply to the brain. When this happens, the brain does not get enough oxygen or nutrients, and brain cells begin to die. People who experience a stroke need immediate emergency treatment, such as medicines to break up blood clots and prevent further clots from forming. Although strokes can be treatable, some can result in disability or death.

[0009] Cerebral palsy occurs as a result of brain damage sustained during fetal development or delivery. Cerebral palsy is caused by damage to the brain's motor cortex, which affects muscle control and coordination, including a person's ability to move, grasp objects and speak. It is the leading cause of disability in young children and affects approximately 500,000 children and adults. There is currently no known cure for cerebral palsy.

[0010] The nerves and brain cells damaged in a brain injury are usually irreparable because brain tissue cannot regenerate. Stem cell therapy has shown some promise in neuroregenerative treatment. However, there is still a need for improved treatments for brain injuries. Summary of the invention

[0011] The present invention provides mesenchymal stem cells (MSC, or also referred to herein as "HMC") obtained by in vitro differentiation of pluripotent stem cells, and extracellular vesicles ("EV") secreted from the HMC of the present invention (HMC-EV), and their use in methods for treating brain injury. Specifically, the inventors of the present invention have found that the HMC and HMC-EV of the present invention are different from MSCs and EVs derived from other sources, for example, adipose tissue-derived MSCs, bone marrow-derived MSCs, and / or umbilical cord blood-derived MSCs. Specifically, when compared with other MSCs, for example, adipose tissue-derived MSCs, bone marrow-derived MSCs, and / or umbilical cord blood-derived MSCs, the HMC of the present invention has a different expression profile. Proteins / genes involved in neuroprotection and cell viability / survival pathways are upregulated in the HMC of the present invention, indicating that the HMC of the present invention is able to confer neuroprotection and provide neurotrophic factors, i.e., factors involved in supporting neuronal survival, growth, health and / or recovery. Similarly, the HMC-EV of the present invention shares a similar profile with the HMC that secretes them. Similar signal transduction pathways enriched in HMCs are also enriched in HMC-EVs when compared to other tissue-derived MSCs and EVs. This different spectrum makes HMCs and HMC-EVs particularly useful and effective in treating diseases such as brain injury. Examples of brain injuries treatable with 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.

[0012] Therefore, in one aspect, the present invention provides a method for treating brain damage in a subject suffering from or suspected of suffering from brain damage, the method comprising administering to the subject an effective amount of EVs secreted from HMCs obtained by in vitro differentiation of pluripotent stem cells (HMC-EVs), thereby treating the brain damage in the subject.

[0013] 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.

[0014] In some embodiments, the method comprises increasing oligodendrocytes and precursor cells in the brain after administering HMC-EV to a subject. In some embodiments, the method comprises preserving myelin in the brain after administering HMC-EV to a subject. In some embodiments, the method comprises preventing oxidative damage in neurons after administering HMC-EV to a subject. In some embodiments, the method comprises preventing neuronal death due to glutamate excitotoxic injury after administering HMC-EV to a subject. In some embodiments, the method comprises reducing tissue loss in the brain after administering EV to a subject. In some embodiments, the method comprises reducing cell death in the brain after administering HMC-EV to a subject. In some embodiments, the method comprises stimulating pathways involved in neuronal lineage development after administering HMC-EV to a subject.

[0015] In some embodiments, the HMC-EV is administered systemically. In some embodiments, the HMC-EV is administered intracerebrally. In some embodiments, the HMC-EV is administered intrathecally. In some embodiments, the HMC-EV is administered intracisternal. In some embodiments, the HMC-EV is administered intraperitoneally.

[0016] In some embodiments, the subject is a human.

[0017] In some embodiments, HMC is obtained by in vitro differentiation of human pluripotent stem cells. In some embodiments, pluripotent stem cells are further differentiated into hemangioblasts. In some embodiments, pluripotent stem cells are embryonic stem cells. In some embodiments, pluripotent stem cells are induced pluripotent stem cells. In some embodiments, induced pluripotent stem cells are produced by contacting cells with one or more reprogramming factors.

[0018] In some embodiments, HMC-EVs express at least one miRNA in Table 9 at a higher level than EVs secreted from umbilical cord blood-derived MSCs (UCB-MSC-EVs).

[0019] In some embodiments, HMC-EVs express at least one miRNA in Table 10 at a lower level than UCB-MSC-EVs.

[0020] In some embodiments, HMC-EVs express at least one miRNA in Table 11 at a higher level than EVs secreted from bone marrow-derived MSCs (BM-MSC-EVs).

[0021] In some embodiments, HMC-EVs express at least one miRNA in Table 12 at a lower level than BM-MSC-EVs.

[0022] In some embodiments, HMC-EVs express at least one miRNA in Table 13 at a higher level than EVs secreted from adipose tissue-derived MSCs (AD-MSC-EVs).

[0023] In some embodiments, HMC-EVs express at least one miRNA in Table 14 at a lower level than AD-MSC-EVs.

[0024] In some embodiments, HMC-EVs express at least one protein in Table 15 at a higher level than UCB-MSC-EVs.

[0025] In some embodiments, HMC-EVs express at least one protein in Table 16 at a lower level than UCB-MSC-EVs.

[0026] In some embodiments, HMC-EVs express at least one protein in Table 17 at a higher level than BM-MSC-EVs.

[0027] In some embodiments, HMC-EVs express at least one protein in Table 18 at a lower level than BM-MSC-EVs.

[0028] In some embodiments, HMC-EVs express at least one protein in Table 19 at a higher level than AD-MSC-EVs.

[0029] In some embodiments, HMC-EVs express at least one protein in Table 20 at a lower level than AD-MSC-EVs.

[0030] 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 than BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs.

[0031] In some embodiments, HMC-EVs express at least one protein 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, K PNA2, 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.

[0032] In some embodiments, HMC-EVs express at least one protein 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, LGA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3 LS1, 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.

[0033] 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 than EVs secreted from BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0034] 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 than BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0035] In some embodiments, HMC-EV expresses at least one miRNA in Table 21 at a higher level than the HMC.

[0036] In some embodiments, HMC-EV expresses at least one miRNA in Table 22 at a lower level than the HMC.

[0037] In some embodiments, about 1×10 6 About 1×10 13 In some embodiments, about 10×10 10 or about 30×10 10 HMC-EV.

[0038] In some embodiments, the HMC-EV is administered in a pharmaceutical composition.

[0039] In some embodiments, the pharmaceutical composition comprises (a) a buffer that maintains the solution at a 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 a physiological osmotic pressure.

[0040] In some embodiments, glucose is D-glucose (dextrose). In some embodiments, the osmotic agent is a salt. In some embodiments, the osmotic agent is a magnesium salt, a phosphate, a sulfate, a chloride salt, a poorly absorbed disaccharide, such as lactulose, a sugar alcohol, such as mannitol and sorbitol and polyethylene glycol or a combination thereof. In some embodiments, the osmotic agent is CaCl2, KCl, NaCl, KH2PO4, Na3HPO4, MgCl2, MgSO4, HEPES, NaHCO3 or a combination thereof. In some embodiments, the salt is sodium chloride.

[0041] 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.

[0042] In one aspect, the present invention provides a method of treating brain damage in a subject having or suspected of having brain damage, 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 damage in the subject.

[0043] 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.

[0044] In some embodiments, the brain injury is a stroke.

[0045] In some embodiments, the brain injury is optic neuropathy.

[0046] In some embodiments, the method includes retaining myelin in the brain after administering HMC to the subject. In some embodiments, the method includes inhibiting neuroinflammatory response after administering HMC to the subject. In some embodiments, the method includes reducing microglia and astrocyte activation in the brain after administering HMC to the subject. In some embodiments, the method includes stimulating pathways involved in cell survival after administering HMC to the subject. In some embodiments, the method includes stimulating the expression of neuroprotective genes in the brain after administering HMC to the 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 secretory phosphoprotein 1 (SPP1). In some embodiments, the method includes stimulating pathways involved in synaptic transmission in the brain after administering HMC to the subject. In some embodiments, the method includes stimulating pathways involved in neuronal lineage development after administering HMC to the subject. In some embodiments, the method includes reducing apoptosis after administering HMC to the subject.

[0047] In some embodiments, the brain injury is traumatic brain injury.

[0048] In some embodiments, the method comprises reducing tissue loss in the brain after administering HMC to a subject. In some embodiments, the method comprises reducing cell death in the brain after administering HMC to a subject. In some embodiments, the method comprises increasing neurogenesis after administering HMC to a subject. In some embodiments, the method comprises reducing the presence of microglia and macrophages in the cortex and striatum after administering HMC to a subject. In some embodiments, the method comprises reducing spleen inflammation after administering HMC to a subject. In some embodiments, the method comprises migration of HMC across the blood-brain barrier to the cortex, striatum, and / or hippocampus.

[0049] In some embodiments, the brain injury is cerebral palsy.

[0050] In some embodiments, the method comprises reducing apoptosis in the brain after administering HMC to a subject. In some embodiments, the method comprises reducing lesion size in the brain after administering HMC to a subject. In some embodiments, the method comprises reducing microglia and astrocyte activation in the brain after administering HMC to a subject. In some embodiments, the method comprises preserving myelin in the corpus callosum after administering HMC to a subject. In some embodiments, the method comprises at least partially rescuing Olig2 in the brain after administering HMC to a subject.

[0051] 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 intracisternal. In some embodiments, the HMC is administered intraperitoneally. In some embodiments, the mesenchymal stem cells are human cells.

[0052] In some embodiments, the subject is a human.

[0053] 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.

[0054] In some embodiments, the HMCs have been passaged no more than 5 times in vitro prior to administration to a subject.

[0055] In some embodiments, HMCs express at least one gene in Table 3 at a higher level than bone marrow-derived MSCs (BM-MSCs).

[0056] In some embodiments, HMCs express at least one gene in Table 4 at a lower level than BM-MSCs.

[0057] In some embodiments, HMCs express at least one gene in Table 5 at a higher level than umbilical cord blood-derived MSCs (UCB-MSCs).

[0058] In some embodiments, HMCs express at least one gene in Table 6 at a lower level than UCB-MSCs.

[0059] In some embodiments, HMCs express at least one gene in Table 7 at a higher level than adipose tissue-derived MSCs (AD-MSCs).

[0060] In some embodiments, HMCs express at least one gene in Table 8 at a lower level than AD-MSCs.

[0061] In some embodiments, HMCs express mRNA encoding interleukin-6 (IL-6) at a level lower than 10% of the IL-6 mRNA level expressed by BM-MSCs at a basal state, and wherein HMCs express mRNA encoding CD24 at a level higher than the CD24 mRNA level expressed by BM-MSCs at a basal state.

[0062] 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 a higher level than adipose tissue-derived MSCs (AD-MSCs).

[0063] 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 a higher level than AD-MSCs.

[0064] In some embodiments, HMCs express at least one gene selected from the group consisting 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, COL1A 2. 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.

[0065] 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 a lower level than AD-MSCs.

[0066] 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 a lower level than AD-MSCs.

[0067] 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 a higher level than bone marrow-derived MSCs (BM-MSCs).

[0068] 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 a lower level than BM-MSCs.

[0069] In some embodiments, HMC expresses at least one miRNA in Table 21 at a lower level than HMC-EV secreted by the HMC.

[0070] In some embodiments, HMC expresses at least one miRNA in Table 22 at a higher level than HMC-EVs secreted by the HMC.

[0071] In some embodiments, about 1×10 6 About 1×10 13 HMCs.

[0072] In some embodiments, the HMC is administered as a pharmaceutical composition.

[0073] In some embodiments, the pharmaceutical composition comprises (a) a buffer that maintains the solution at a 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 a physiological osmotic pressure.

[0074] In some embodiments, the glucose is D-glucose (dextrose). In some embodiments, the osmotically active agent is salt. In some embodiments, the salt is sodium chloride.

[0075] In another aspect, the present invention provides a method for treating brain damage in a subject suffering from or suspected of suffering from brain damage, the method 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 damage in the subject.

[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 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 than AD-MSCs.

[0077] 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 than AD-MSCs.

[0078] 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 a higher level than BM-MSCs 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.

[0079] 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 SERPINE1, ACTA2, TPM2, CTGF, SERPINE2, CRYAB, ELN, MFGE8, ANXA2, POSTN, VIM, MFAP5, ISLR, THBS1, TIMP3, DKK1, COL6A3, COL6A1, TPT1, BCYRN1, COL1A1, SPAR C, TPM1, BGN, COL1A2, COL3A1, TGFBI, CRLF1, COMP, NEAT1, MT-CO3, MT-CO2, MT-ATP8, MT-CYB, MT-CO1, MT-ATP6, MT-ND4, MT-ND4 L, MT-ND5, MT-ND6, MT-ND3, MT-ND1, MT-ND2, GREM1, TMSB4X, ITGB1, LMNA, H2AFZ, FTL, EEF1G, NPM1, EEF1A1, RACK1, ACTG1 and TPM4.

[0080] 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 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 than AD-MSCs.

[0081] 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 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 than AD-MSCs.

[0082] 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 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 than BM-MSCs.

[0083] In some embodiments, HMCs also express at least one gene in Table 3 at a higher level than BM-MSCs.

[0084] In some embodiments, HMCs also express at least one gene in Table 4 at a lower level than BM-MSCs.

[0085] In some embodiments, HMCs also express at least one gene in Table 5 at a higher level than UCB-MSCs.

[0086] In some embodiments, HMCs also express at least one gene in Table 6 at a lower level than UCB-MSCs.

[0087] In some embodiments, HMCs also express at least one gene in Table 7 at a higher level than AD-MSCs.

[0088] In some embodiments, HMCs also express at least one gene in Table 8 at a lower level than AD-MSCs.

[0089] In one aspect, the present invention provides a pharmaceutical composition comprising the HMC of the present invention and a pharmaceutically acceptable carrier.

[0090] In one aspect, the present invention provides a population of HMC-EVs of the present invention.

[0091] In some embodiments, HMC-EVs express at least one miRNA in Table 9 at a higher level than UCB-MSC-EVs.

[0092] In some embodiments, HMC-EVs express at least one miRNA in Table 10 at a lower level than UCB-MSC-EVs.

[0093] In some embodiments, HMC-EVs express at least one miRNA in Table 11 at a higher level than BM-MSC-EVs.

[0094] In some embodiments, HMC-EVs express at least one miRNA in Table 12 at a lower level than BM-MSC-EVs.

[0095] In some embodiments, HMC-EVs express at least one miRNA in Table 13 at a higher level than AD-MSC-EVs.

[0096] In some embodiments, HMC-EVs express at least one miRNA in Table 14 at a lower level than AD-MSC-EVs.

[0097] In some embodiments, HMC-EVs express at least one protein in Table 15 at a higher level than UCB-MSC-EVs.

[0098] In some embodiments, HMC-EVs express at least one protein in Table 16 at a lower level than UCB-MSC-EVs.

[0099] In some embodiments, HMC-EVs express at least one protein in Table 17 at a higher level than BM-MSC-EVs.

[0100] In some embodiments, HMC-EVs express at least one protein in Table 18 at a lower level than BM-MSC-EVs.

[0101] In some embodiments, HMC-EVs express at least one protein in Table 19 at a higher level than AD-MSC-EVs.

[0102] In some embodiments, HMC-EVs express at least one protein in Table 20 at a lower level than AD-MSC-EVs.

[0103] In some embodiments, HMC-EV expresses at least one miRNA in Table 21 at a higher level than the HMC.

[0104] In some embodiments, HMC-EV expresses at least one miRNA in Table 22 at a lower level than the HMC.

[0105] 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 than EVs secreted from BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs.

[0106] In some embodiments, HMC-EVs express at least one protein 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, K PNA2, 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.

[0107] In some embodiments, HMC-EVs express at least one protein 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, LGA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3 LS1, 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.

[0108] 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 than BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0109] 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 than BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0110] In one aspect, the present invention provides a pharmaceutical composition comprising the HMC-EV of the present invention and a pharmaceutically acceptable carrier.

[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 9 at a higher level than UCB-MSC-EVs.

[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 miRNA in Table 10 at a lower level than UCB-MSC-EVs.

[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 miRNA in Table 11 at a higher level than BM-MSC-EVs.

[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 miRNA in Table 12 at a lower level than BM-MSC-EVs.

[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 miRNA in Table 13 at a higher level than AD-MSC-EVs.

[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 miRNA in Table 14 at a lower level than EVs secreted by AD-MSC-EVs.

[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 protein in Table 15 at a higher level than UCB-MSC-EVs.

[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 protein in Table 16 at a lower level than UCB-MSC-EVs.

[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 protein in Table 17 at a higher level than BM-MSC-EVs.

[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 protein in Table 18 at a lower level than BM-MSC-EVs.

[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 protein in Table 19 at a higher level than AD-MSC-EVs.

[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 protein in Table 20 at a lower level than AD-MSC-EVs.

[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 miRNA in Table 21 at a higher level than the HMCs.

[0124] 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 22 at a lower level than the HMCs.

[0125] 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 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 than BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs.

[0126] 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 ALDOC, ANXA5, APBB2, BASP1, CAV1, CD81, CD99, CKM, EPB41L3, FDPS, GNAQ, GNG12, GP9, H2AC20, H2AC21, H3-3A, H3-7 at a higher level than BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs. , H4-16, HLA-A, ITGA2, KPNA2, KRAS, KRT4, LRRC59, MAMDC2, MARCKSL1, MDGA1, MERTK, MFGE8, MMP14, MVP, PCDH1, PDGFRB, PDIA3, R PL13, RPS18, RPS3A, RPS4X, SDCBP, SLC2A1, SLC3A2, TAGLN2, TNC, TSPAN14, TSPAN33, TSPAN9, TTYH3, UCHL1, VAT1, YWHAB, and YWHAQ.

[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 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.

[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 ADIPOQ, CAT, CEP290, IGLV6-57, TAS2R33 and TMEM198 at a lower level than BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs.

[0129] 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 a lower level than BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs.

[0130] In one aspect, the present invention provides a pharmaceutical composition comprising the HMC-EV of the present invention and a pharmaceutically acceptable carrier.

[0131] The present invention also provides a method for determining the neurite growth of HMC and / or HMC-EV populations. The method includes (a) preparing a mixed neuron culture from an isolated cerebral cortex, (b) plating the HMC and / or HMC-EV population on a permeable membrane, (c) applying tension to the mixed neuron culture, (d) covering the tensioned mixed neuron culture with the permeable membrane of step (b), and (e) measuring the neurite growth of the mixed neuron culture. In one embodiment, step (d) is cultured in a culture medium substantially lacking serum. In another embodiment, the method further includes determining the gene expression of the mixed neuron culture in the presence and absence of HMC and / or HMC-EV populations. In another embodiment, the tension is a physical scratch produced in the mixed neuron culture. In another embodiment, the tension is a vacuum pressure and a positive air pressure applied to the mixed neuron culture. In another embodiment, tension can be applied under a tensile oscillation of 15% to 0%.

[0132] The present invention also provides a method for determining the neurite outgrowth of HMC and / or HMC-EV populations. The method includes preparing a mixed neuron culture from isolated cerebral cortex, (b) plating HMC and / or HMC-EV populations on a permeable membrane, (c) applying tension to the mixed neuron culture, (d) covering the tensioned mixed neuron culture with the permeable membrane of step (b), and (e) measuring the neurite outgrowth of the mixed neuron culture. In one embodiment, the method also includes determining the gene expression of the mixed neuron culture in the presence and absence of HMC and / or HMC-EV populations. In another embodiment, the tension is a physical scratch produced in the mixed neuron culture. In another embodiment, the tension is vacuum pressure and positive air pressure applied to the mixed neuron culture. In another embodiment, tension is applied under tensile oscillations of 15% to 0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 Shown are the results of the elevated body swing test (EBST) in rats with TBI induced by controlled cortical impact (CCI) and HMC or vehicle administered intracerebrally (IC) or intravenously (IV).

[0134] Figure 2 Forelimb akinesia in rats with controlled cortical impact (CCI)-induced TBI and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle is shown.

[0135] Figure 3 Shown is paw grasping in rats with controlled cortical impact (CCI) induced TBI and HMC or vehicle administered intracerebrally (IC) or intravenously (IV).

[0136] Figure 4A Shown are H&E staining of the brains of rats in which TBI was induced by controlled cortical impact (CCI) and HMC or vehicle was administered intracerebrally (IC) or intravenously (IV). Figure 4B Shown is a bar graph of TBI impact area in rats as measured by H&E staining.

[0137] Figure 5A Shown is Nissl staining of periaplasty cortex of rats in which TBI was induced by controlled cortical impact (CCI) and HMC or vehicle was administered intracerebrally (IC) or intravenously (IV). Figure 5B Shown is a bar graph of the percentage of live cells in the cortex surrounding rat impacts as determined by Nissl staining. Figure 5C Shown is Nissl staining of the striatum in rats with controlled cortical impact (CCI) induced TBI and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Figure 5D Shown is a bar graph of the percentage of live cells in rat striatum as determined by Nissl staining.

[0138] Figure 5E Shown are Nissl staining of the hippocampus of rats in which TBI was induced by controlled cortical impact (CCI) and HMC or vehicle was administered intracerebrally (IC) or intravenously (IV). Fig. 5F Shown is a bar graph of the percentage of live cells in the hippocampus of rats as determined by Nissl staining.

[0139] Fig. 6A Shown is doublecortin (DCX) staining of the cortex of rats in which TBI was induced by controlled cortical impact (CCI) and HMC or vehicle was administered intracerebrally (IC) or intravenously (IV). Figure 6B Shown is a bar graph of DCX cell counts in rat cortical areas. Figure 6C Shown are DCX staining of the striatum of rats with TBI induced by controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Fig.6D Shown is a bar graph of DCX cell counts in rat striatum regions. Fig. 6E Shown are DCX staining of the hippocampus of rats with TBI induced by controlled cortical impact (CCI) and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Fig. 6F Shown is a bar graph of DCX cell counts in the rat hippocampus region.

[0140] Fig. 7A Shown is Iba1 staining in the cortex of rats in which TBI was induced by controlled cortical impact (CCI) and HMC or vehicle was administered intracerebrally (IC) or intravenously (IV). Figure 7B Shown is a bar graph of Iba1 cell counts in rat cortex. Figure 7C Shown is Iba1 staining in the striatum of rats with controlled cortical impact (CCI) induced TBI and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Fig.7D Shown is a bar graph of Iba1 cell counts in rat striatum.

[0141] Fig. 8A Shown are OX6 staining of the cortex of rats in which TBI was induced by controlled cortical impact (CCI) and HMC or vehicle was administered intracerebrally (IC) or intravenously (IV). Figure 8B Shown is a bar graph of OX6 cell counts in rat cortex. Figure 8C Shown are OX6 staining in the striatum of rats with controlled cortical impact (CCI) induced TBI and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Fig.8D Shown is a bar graph of OX6 cell counts in rat striatum.

[0142] Fig. 9A Shown is IL6 staining in the spleen of rats with controlled cortical impact (CCI) induced TBI and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Fig. 9B A bar graph showing the staining intensity of IL6 in rat spleen is shown.

[0143] Fig. 10A Shown are TNF-α staining in spleens of rats with controlled cortical impact (CCI) induced TBI and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Fig. 10B A bar graph showing the intensity of TNF-α staining in rat spleen is shown.

[0144] Fig.11AShown is HuNu staining in the cortex of rats in which TBI was induced by controlled cortical impact (CCI) and HMC or vehicle was administered intracerebrally (IC) or intravenously (IV). Fig. 11B Shown is a bar graph of HuNu cell counts in rat cortex. Fig. 11C Shown is HuNu staining in the striatum of rats with controlled cortical impact (CCI) induced TBI and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Fig.11D Shown is a bar graph of HuNu cell counts in rat striatum. Fig.11E Shown is HuNu staining in the hippocampus of rats with controlled cortical impact (CCI) induced TBI and intracerebral (IC) or intravenous (IV) administration of HMC or vehicle. Fig.11F Shown is a bar graph of HuNu cell counts in rat hippocampus.

[0145] Fig. 12A Migration of unstimulated hESC-MSCs ("HMCs"), BM-MSCs, and UCB-MSCs into a gap approximately 500 μm wide at 0 hr and 6 hr is shown. Fig. 12B Shown are histograms of the number of unstimulated and stimulated cells that have migrated into the gap.

[0146] Fig.13 Shown are images of neurite outgrowth staining after scratching and co-culture of hESC-MSCs ("HMCs") with mixed neuronal cultures for 1 and 7 days.

[0147] Fig.14A Shown is the TUNEL grading of each rat tested in the in vivo neonatal hypoxia-ischemia model of cerebral palsy. Fig. 14B A bar graph showing the average TUNEL grading for each group of rats tested is shown. The TUNEL grading is 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; 5 = very strong damage / disappearance of tissue. Comparison of rats in the sham vs HI groups showed a t-test of 0.006284 and a Mann-Whitney test of 0.0256; sham vs batch B group showed a t-test of 0.148904 and a Mann-Whitney test of 0.2; and HI group vs batch B group showed a t-test of 0.101453 and a Mann-Whitney test of 0.1841.

[0148] Fig.15 Shown is H&E staining of the brains of rats tested in the in vivo neonatal hypoxia-ischemia model of cerebral palsy.

[0149] Fig.16AShown are images of Iba-1 staining in peri-infarct tissue of rats tested in the in vivo neonatal hypoxia-ischemia model of cerebral palsy. Fig. 16B Shown are the mean signal intensity of Iba-1 staining in each rat tested in the in vivo neonatal hypoxia-ischemia model of cerebral palsy. Fig. 16C The mean signal intensity of Iba-1 staining in each group of rats tested is shown. Comparison of rats in the sham vs HI groups showed a t-test of 0.039335 and a Mann-Whitney test of 0.065; sham vs batch B group showed a t-test of 0.129562 and a Mann-Whitney test of 0.1949; and HI group vs batch B group showed a t-test of 0.353204 and a Mann-Whitney test of 0.4418.

[0150] Fig.17A Shown are images of GFAP staining in peri-infarct tissue of rats tested in the in vivo neonatal hypoxia-ischemia model of cerebral palsy. Fig. 17B Shown are the mean signal intensity of GFAP staining in each rat tested in the in vivo neonatal hypoxia-ischemia model of cerebral palsy. Fig. 17C The mean signal intensity of GFAP staining in each group of rats tested is shown. Comparison of rats in the sham vs HI groups showed a t-test of 0.011749 and a Mann-Whitney test of 0.0047; sham vs batch B group showed a t-test of 0.070012 and a Mann-Whitney test of 0.0207; and HI group vs batch B group showed a t-test of 0.57941 and a Mann-Whitney test of 0.7984.

[0151] Fig.18A Shown are images of MBP staining in the corpus callosum in rats tested in the in vivo neonatal hypoxia-ischemia model of cerebral palsy. Fig.18B Shown are the mean signal intensity of MBP staining in each rat tested in the in vivo neonatal hypoxia-ischemia model of cerebral palsy. Fig. 18C The mean signal intensity of MBP staining in each group of rats tested is shown. Comparison of rats in the sham vs HI groups showed a t-test of 0.012963 and a Mann-Whitney test of 0.007; sham vs batch B group showed a t-test of 0.189251 and a Mann-Whitney test of 0.3282; and HI group vs batch B group showed a t-test of 0.172857 and a Mann-Whitney test of 0.2345.

[0152] Fig.19A Shown are images of Olig2 staining in the hippocampus of the ipsilateral lesioned cerebral hemisphere of rats tested in the in vivo neonatal hypoxia-ischemia model of cerebral palsy. Fig.19BShown are the mean signal intensity of Olig2 staining in the SVZ, cortex, hippocampus and regional means for each rat tested in the in vivo neonatal hypoxia-ischemia model of cerebral palsy. Fig.19C The average signal intensity of Olig2 staining in the SVZ, cortex, hippocampus and regional averages are shown for each group of rats tested. For Olig2 staining in the SVZ, the comparison of rats in batch B vs HI showed a t-test of 0.3962; in the cortex, a t-test of 0.4399 was shown; in the hippocampus, a t-test of 0.5435 was shown; and the regional average showed a t-test of 0.3597.

[0153] Fig. 20 Shown are the results of the body swing test in rats with middle cerebral artery occlusion (MCAO) stroke and receiving HMC via 3 routes of administration: intravenous (IV), intracerebral (IC), and intrathecal (IT). Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test (Tukey's MCT), *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0154] Fig.21 Shown are the results of 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. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0155] Fig. 22 Results of forelimb placing, hindlimb placing, and body swing tests in rats with middle cerebral artery occlusion (MCAO) stroke and receiving HMC-EV via intracisternal administration are shown. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0156] Fig.23 Results of forelimb placing, hindlimb placing, and body swing tests in rats with middle cerebral artery occlusion (MCAO) stroke and receiving HMC-EV via intrathecal administration are shown. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0157] Fig.24AShown are images of MBP staining in the cortex and striatum of rats with MCAO stroke and receiving HMCs (derived from C-GS1 and N-line cells) via IV administration. Fig. 24B Shown are the mean signal intensities of MBP staining in the cortex of rats tested in the in vivo MCAO stroke model. Fig.24C The mean signal intensity of MBP staining in rat striatum tested in the in vivo MCAO stroke model is shown. For sham treatment vs vehicle group: statistical analysis was performed using the Welch test, ***p<0.001. For vehicle group vs treatment group: statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test, *p<0.05, **p<0.01 and ***P<0.001.

[0158] Fig.25A Shown are images of Iba1 staining in the cortex and striatum of rats with MCAO stroke and receiving HMCs (derived from C-GS1 and N-line cells) via IV administration. Fig.25B Shown are the mean signal intensities of Iba1 staining in the cortex of rats tested in the in vivo MCAO stroke model. Fig.25C The mean signal intensity of Iba1 staining in rat striatum tested in the in vivo MCAO stroke model is shown. For sham vs vehicle group: statistical analysis was performed using the Welch test, ***p<0.001. For vehicle vs treatment group: statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test, *p<0.05, **p<0.01 and ***P<0.001.

[0159] Fig.26A Shown are images of GFAP staining in the cortex and striatum of rats with MCAO stroke and receiving HMCs (derived from C-GS1 and N-line cells) via IV administration. Fig.26B Shown are the mean signal intensities of GFAP staining in the cortex of rats tested in the in vivo MCAO stroke model. Fig.26C The mean signal intensity of GFAP staining in rat striatum tested in the in vivo MCAO stroke model is shown. For sham treatment vs vehicle group: statistical analysis was performed using the Welch test, ***p<0.001. For vehicle group vs treatment group: statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test, *p<0.05, **p<0.01 and ***P<0.001.

[0160] Fig.27A Shown are images of MBP staining in rats with MCAO stroke that received HMC-EVs (derived from N-lineage cells, treated with IFNγ at 50 ng / mL for 96 hours) via intracisternal administration. Fig.27B Mean signal intensity of MBP staining in rats tested in the in vivo MCAO stroke model is shown. cc: corpus callosum; ec: external capsule; cg: cingulate gyrus. For vehicle group vs. treatment group: Statistical analysis was performed using Bonferroni comparison, **p<0.01.

[0161] Fig.28A Shown are images of Iba1 staining in rats with MCAO stroke that received HMC-EVs (derived from N-lineage cells treated with gamma interferon at 50 ng / mL for 96 hours) via intracisternal administration. Fig.28B The mean signal intensity of Iba1 staining in rats tested in the in vivo MCAO stroke model is shown. cc: corpus callosum; ec: external capsule; cg: cingulate gyrus. For vehicle group vs. treatment group: statistical analysis was performed using Bonferroni comparison, **p<0.01.

[0162] Fig.29A Shown are images of GFAP staining in rats with MCAO stroke that received HMC-EVs (derived from N-lineage cells treated with gamma interferon at 50 ng / mL for 96 hours) via intracisternal administration. Fig.29B The mean signal intensity of GFAP staining in rats tested in the in vivo MCAO stroke model is shown. cc: corpus callosum; ec: external capsule; cg: cingulate gyrus. For vehicle group vs. treatment group: statistical analysis was performed using Bonferroni comparison, **p<0.01.

[0163] Fig. 30A Shown are images of Olig2 staining in rats with MCAO stroke that received HMC-EVs (derived from N-lineage cells treated with gamma interferon at 50 ng / mL for 96 hours) via intracisternal administration. Fig. 30B The mean signal intensity of Olig2 staining in rats tested in the in vivo MCAO stroke model is shown. cc: corpus callosum; ec: external capsule; cg: cingulate gyrus. For vehicle group vs. treatment group: statistical analysis was performed using Bonferroni comparison, **p<0.01.

[0164] Fig.31A Shown are images of NG2 staining in rats with MCAO stroke that received HMC-EVs (derived from N-lineage cells treated with gamma interferon at 50 ng / mL for 96 hours) via intracisternal administration. Fig. 30B The mean signal intensity of NG2 staining in rats tested in the in vivo MCAO stroke model is shown. cc: corpus callosum; ec: external capsule; cg: cingulate gyrus. For vehicle group vs. treatment group: statistical analysis was performed using Bonferroni comparison, **p<0.01.

[0165] Fig.32Schematic diagram of the study design of the in vitro oxygen glucose deprivation (OGD) assay used to model stroke.

[0166] Fig.33A Shown are TUNEL staining and imaging of primary rat neurons treated with or without HMC after 0hr, 1hr, 2hr, and 3hr oxygen glucose deprivation (OGD) insult. Fig.33B Shown are the average TUNEL quantifications of primary rat neurons treated with or without MSCs after 0 hr, 1 hr, 2 hr, and 3 hr OGD injury.

[0167] Fig.34A -F shows enrichment analysis of differentially expressed pathways between neurons subjected to 3 h glucose-oxygen deprivation insult and grown in HMC-enriched medium and control medium. Fig.34A -B shows pathways enriched by differential expression. Fig.34C -F shows the differential expression of Gene Oncology terms between OGD neurons grown in HMC-rich medium and control medium. Fig.34C Upregulation of pathways involved in cell viability, neuroprotection, and synaptic transmission in OGD neurons grown on HMC-enriched culture was shown. Fig.34D Shown is the upregulation of genes involved in neuroprotection in OGD neurons grown on HMC-enriched culture. Fig.34E Downregulation of pathways involved in apoptosis in OGD neurons grown on HMC-enriched culture is shown. Fig.34F Shown is the downregulation of genes involved in apoptosis or general response to cell death in OGD neurons grown on HMC-enriched culture.

[0168] Fig.35A Shown is RNAseq analysis of in vitro oxygen glucose deprivation (OGD) assay in primary rat neurons treated with or without HMC after 0hr, 1hr, 2hr and 3hr OGD insult. Fig.35B Shown is qPCR analysis of primary rat neurons treated with or without HMC after 0hr, 1hr, 2hr and 3hr oxygen glucose deprivation (OGD) insult. Statistical analysis was performed using two-way ANOVA with Sidak's multiple comparison test: *p<0.05, **p<0.01 and ****p<0.0001.

[0169] Fig.36A Attenuation of cell death by HMC-EV is shown. Percent cell death was determined as the number of PI+ cells among total Hoechst+ cells. Two-way ANOVA was used for statistical significance analysis. ****p<0.0001. Fig.36BDose-dependent attenuation of cell death by HMC-EV treatment is shown. Percent cell death was determined as the number of PI+ cells among total Hoechst+ cells.

[0170] Single Factor

[0171] Fig.37 Shown is the maintenance of mitochondrial membrane potential in HMC-EV treated cells undergoing nuclear swelling. Following glutamate-induced injury, HMC-EV treatment maintained cells in the nuclear swelling phase.

[0172] Fig.38 The principal component analysis of the transcriptome of HMCs (derived from N-lineage cells) and adipose tissue-derived MSCs is shown, which indicates that HMCs are different from adipose tissue-derived MSCs in the basal state and interferon-γ stimulated state. AMSC-B-1,2,3: Adipose tissue-derived MSCs collected from 3 different adult donors, 2 technical replicates were used for each biological replicate. AMSC-S-1,2,3: Adipose tissue-derived MSCs, but stimulated with interferon-γ. NHMC-B: 3 technical replicates of MSCs derived from N-lineage cells (basal state). NHMC-S: MSCs derived from N-lineage cells, but stimulated with interferon-γ.

[0173] Fig.39 The weights of the different genes contributing to the second principal component determining the differences between HMCs (derived from N-lineage cells) and adipose tissue-derived MSCs are shown.

[0174] Fig.40 Hierarchical clustering diagrams are shown, which show that HMC (derived from N-lineage cells) are different from adipose tissue-derived MSCs in the basal state and in the interferon-gamma-stimulated state. AB1, AB2, AB3—Adipose tissue-derived MSCs collected from 3 different adult donors, 2 technical replicates per donor; basal cell state. AS1, AS2, AS3—Adipose tissue-derived MSCs, stimulated with interferon-gamma. NB—MSCs derived from N-lineage cells, basal state, 3 technical replicates. NS—MSCs derived from N-lineage cells, stimulated with interferon-gamma.

[0175] Fig.41 The basal HMC-specific gene cluster is shown.

[0176] Fig.42 Adipose tissue-derived MSC-specific gene clusters are shown.

[0177] Fig.43Pathway enrichment of the differential expression pattern between HMCs (derived from N-lineage cells) and adipose tissue-derived MSCs is shown, showing 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.

[0178] Fig.44 The top 15 most strongly differentially expressed genes contributing to activation of neuronal CREB signaling in HMCs (derived from N-lineage cells) are shown.

[0179] Fig.45 The top 15 most strongly upregulated genes contributing to the enrichment of axon guidance pathways in HMCs (derived from N-lineage cells) are shown.

[0180] Fig.46 The top 15 most strongly expressed genes contributing to activation of synaptogenic signaling pathways in HMCs (derived from N-lineage cells) are shown.

[0181] Fig.47 The top 15 most strongly upregulated genes contributing to activation of neuroinflammatory signaling pathways in HMCs (derived from N-lineage cells) are shown.

[0182] Fig.48 Transcriptome principal component analysis of HMCs from N-lineage cells, HMCs from GMP1 cells, and adipose tissue-derived MSCs is shown. AMSC-B-1,2,3—Adipose tissue-derived MSCs collected from 3 different adult donors, basal state, 2 technical replicates per biological replicate. AMSC-S-1,2,3—Adipose tissue-derived MSCs collected from 3 different adult donors, but stimulated with interferon-γ. NHMC-B—HMCs derived from N-lineage cells, basal state. NHMC-S—HMCs derived from N-lineage cells, but stimulated with interferon-γ. GMP-B—HMCs derived from the GMP1 cell line, basal state. GMP-S—HMCs derived from the GMP1 cell line, but stimulated with interferon-γ.

[0183] Fig.49Hierarchical clustering diagrams are shown, which demonstrate that HMC (derived from N-lineage cells) and HMC (derived from GMP1 cells) are different from adipose tissue-derived MSCs in the basal state and in the cell state stimulated with interferon-gamma. AB1, AB2, AB3—Adipose tissue-derived MSCs collected from 3 different adult donors, 2 technical replicates per donor; basal cell state. AS1, AS2, AS3—Adipose tissue-derived MSCs collected from 3 different adult donors, stimulated with interferon-gamma. NB—HMCs derived from N-lineage cells, basal state, 3 technical replicates. NS—HMCs derived from N-lineage cells, stimulated with interferon-gamma. GB—HMCs derived from the GMP1 cell line, basal state, 3 technical replicates. GS—HMCs derived from the GMP1 cell line, stimulated with interferon-gamma.

[0184] Fig.50 HMC-specific gene clusters are shown.

[0185] Fig.51 Adipose tissue-derived MSC-specific gene clusters are shown.

[0186] Fig.52 Stimulated adipose tissue-derived MSC-specific gene clusters are shown.

[0187] Fig.53A Pathway enrichment of the differential expression pattern between HMCs (derived from GMP1 cells) and adipose tissue-derived MSCs was shown, showing significant HMC-specific upregulation of several pathways involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, and synaptogenic signaling. Fig.53B The first few canonical pathways that are differentially regulated in HMCs are shown. Fig.53C Exemplary regulators of activation and repression in HMC are shown.

[0188] Fig.54A Pathway enrichment of the differential expression pattern between HMCs (derived from N-lineage cells) and adipose tissue-derived MSCs was shown, showing significant HMC-specific upregulation of several pathways involved in neuronal lineage development, including axon guidance, CREB signaling in neurons, and synaptogenic signaling. Fig.54B The first few canonical pathways that are differentially regulated in HMCs are shown. Fig.54C Exemplary regulators of activation and repression in HMC are shown.

[0189] Fig.55The principal component analysis of the transcriptome of HMC (derived from N-lineage cells) and bone marrow-derived MSCs is shown, which shows that HMCs are different from bone marrow-derived MSCs in the basal state and interferon-γ stimulation state. BM-B—Bone marrow-derived MSCs collected from 3 different adult donors, basal state, 2 technical replicates per biological replicate. BM-S—Bone marrow-derived MSCs, but stimulated with interferon-γ. NB—3 technical replicates of HMCs derived from N-lineage cells, basal state. NS—HMCs derived from N-lineage cells, but stimulated with interferon-γ.

[0190] Fig.56 The weights of the different genes contributing to the second principal component determining the differences between HMCs and bone marrow-derived MSCs are shown.

[0191] Fig.57 Hierarchical clustering diagrams are shown, demonstrating that HMCs (derived from N-lineage cells) are distinct from bone marrow-derived MSCs in the basal state and in the interferon-gamma-stimulated cell state. BMB1, BMB2, BMB3—bone marrow-derived MSCs collected from 3 different adult donors, 2 technical replicates per donor; basal cell state. BMS1, BMS2, BMS3—bone marrow-derived MSCs, stimulated with interferon-gamma. NB—HMCs derived from N-lineage cells, basal state, 3 technical replicates. NS—HMCs derived from N-lineage cells, stimulated with interferon-gamma.

[0192] Fig.58 The basal HMC-specific gene cluster is shown.

[0193] Fig.59 The basal bone marrow-derived MSC-specific gene cluster is shown.

[0194] Fig.60 Pathway enrichment of the differential expression pattern between HMCs (derived from N-lineage cells) and bone marrow-derived MSCs is shown, showing several pathways involved in neuronal lineage development (indicated by arrows), such as a significant HMC-specific upregulation of CREB signaling in neurons.

[0195] Fig.61 The top 15 most strongly differentially expressed genes contributing to activation of neuronal CREB signaling in HMCs (derived from N-lineage cells) are shown.

[0196] Fig.62 The top 15 most strongly upregulated genes contributing to activation of synaptogenic signaling in HMCs (derived from N-lineage cells) are shown.

[0197] Fig.63APathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from bone marrow-derived MSCs (BM-MSC-EVs) is shown. Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange columns. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue columns. White / grey columns represent pathways enriched in HMC-EVs, i.e., enriched for proteins contributing to these pathways. Fig.63B Disease or functional annotations of proteins with higher expression levels in HMC-EVs when compared to BM-MSC-EVs are shown. Fig.63C Disease or functional annotations for proteins with lower expression levels in HMC-EVs when compared to BM-MSC-EVs are shown. Activation z scores greater than 2 or less than -2 were considered as thresholds.

[0198] Fig.64A Pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from adipose tissue-derived MSCs (AD-MSC-EVs) is shown. Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange columns. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue columns. White / grey columns represent pathways enriched in HMC-EVs, i.e., proteins contributing to these pathways are enriched. Fig.64B Disease or functional annotations of proteins with higher expression levels in HMC-EVs when compared to AD-MSC-EVs are shown. Fig.64C Disease or functional annotations for proteins with lower expression levels in HMC-EVs when compared to AD-MSC-EVs are shown. Activation z scores greater than 2 or less than -2 were considered as thresholds.

[0199] Fig.65A Pathway enrichment of differential expression patterns between HMC-EVs and EVs secreted from umbilical cord blood-derived MSCs (UCB-MSC-EVs) is shown. Pathways upregulated in HMC-EVs have positive z-scores and are represented by orange columns. Pathways downregulated in HMC-EVs have negative z-scores and are represented by blue columns. White / grey columns represent pathways enriched in HMC-EVs, i.e., enriched for proteins that contribute to these pathways. Fig.65B Disease or functional annotations of proteins with higher expression levels in HMC-EVs when compared to UCB-MSC-EVs are shown. Fig.65C Disease or functional annotations for proteins with lower expression levels in HMC-EVs when compared to UCB-MSC-EVs are shown. Activation z scores greater than 2 or less than -2 were considered as thresholds. DETAILED DESCRIPTION

[0200] definition

[0201] As used herein, "pluripotent cells", "pluripotent stem cells" and "PSCs" broadly refer to cells that can proliferate in vitro for a long time or almost indefinitely while maintaining their undifferentiated state, these cells 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. Typically, pluripotent cells (a) are able to induce teratomas when implanted into immunodeficient (SCID) mice; (b) are able to differentiate into cell types with all three germ layers (e.g., ectoderm, mesoderm and endoderm 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, TRA181, SOX2, REX1). Exemplary pluripotent cells may express Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA 4 surface antigen, TRA 1 60 and / or TRA 1 81. Other exemplary pluripotent cells include, but are not limited to, embryonic stem cells, induced pluripotent cells (iPS), cells of embryonic origin, pluripotent cells produced by embryonic germ (EG) cells (for example, by cultivating in the presence of FGF-2, LIF and SCF), parthenogenetic ES cells, ES cells produced by the inner cell mass cells (ICM) of culture, ES cells produced by schizonts and ES cells produced by nuclear transfer (for example, transferred to the somatic cell nucleus of the recipient oocyte). Exemplary pluripotent cells can be produced without destroying the embryo. For example, induced pluripotent cells can be produced from cells obtained without embryonic destruction. As another example, pluripotent cells can be produced by schizonts of biopsy (which are realized without damaging the remaining embryo); alternatively, the remaining embryo can be cryopreserved, cultivated and / or implanted in a suitable host. Genetic modification or other modifications can be carried out to improve life span, effectiveness, homing to the pluripotent cells (from any source), or in the cells (for example, MSC and angioblasts) differentiated from these pluripotent cells, required factors are delivered.As non-limiting examples thereof, pluripotent cells can be genetically modified to express Sirt1 (thereby extending lifespan), express one or more telomerase subunit genes, optionally under the control of an inducible or repressible promoter, incorporate a fluorescent marker, incorporate iron oxide particles or other such agents (which can 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 can improve lifespan (see Go et al., J. Biochem. 142, 741-748 (2007)), express CXCR4 for homing (see Shi et al., Haematologica. 2007 Jul; 92(7):897-904), express recombinant TRAIL to induce caspase-mediated apoptosis in cancer cells, such as gliomas (see Sasportas et al., Proc Natl Acad Sci USA. 2009 Mar 24;106(12):4822-7) etc.

[0202] As used herein, "embryo" or "embryonic" broadly refers to a developing mass of cells that has not yet implanted into the uterine membrane of a maternal host. An "embryonic cell" is a cell isolated from or contained in an embryo. This also includes schizonts and aggregated schizonts that can be obtained as early as the two-cell stage.

[0203] "Embryonic stem cells" (ES cells or ESCs) encompass pluripotent cells produced by embryonic cells (such as from the inner cell mass cells of culture or the schizonts of culture). Typically, these cells are or have been used as cell lines for continuous passage. Embryonic stem cells can be used as pluripotent stem cells in the process of producing hemangioblasts as described herein. For example, ES cells can be produced by methods known in the art, including obtaining from the embryo produced by any method (including by sexual or asexual means), such as fertilization, nuclear transfer (including somatic cell nuclear transfer) or parthenogenesis of egg cells and sperm or sperm DNA. As another example, embryonic stem cells also include cells produced 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 blastocyst stage embryos, and embryonic stem cells derived from one or more schizonts. These embryonic stem cells can be produced from embryonic materials produced by fertilization or by asexual means, including somatic cell nuclear transplantation (SCNT), parthenogenesis and androgenesis. As discussed further above (see "Pluripotent Cells"), ES cells can be genetically or otherwise modified to increase lifespan, potency, homing, or to deliver desired factors in cells differentiated from these pluripotent cells (eg, MSCs and hemangioblasts).

[0204] ES cells can be produced in a homozygous or hemizygous state in one or more HLA genes, for example, by genetic manipulation, screening for spontaneous loss of heterozygosity, etc. DayES cells can be genetically modified or otherwise modified to improve lifespan, potency, homing, or to deliver desired factors in cells differentiated from these pluripotent cells (e.g., MSCs and angioblasts). Regardless of their source or the specific method used to produce them, embryonic stem cells generally have one or more of the following properties: (i) the ability to differentiate into cells with all three germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and (iii) the ability to produce teratomas when implanted in immunocompromised animals. Embryonic stem cells that can be used in embodiments of the present invention include, but are not limited to, human ES cells ("hESC" or "hES cells"), such as CT2, MA01, MA09, ACT-4, No.3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Other exemplary cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. See also NIH Human Embryonic Stem Cell Registry. An exemplary human embryonic stem cell line that can be used is MA09 cells. The separation and preparation of MA09 cells were previously described in Klimanskaya et al. (2006) "Human Embryonic Stem Cell lines Derived from Single Blastomeres." Nature 444: 481-485. The human ES cells used according to an exemplary embodiment of the present invention can be obtained and maintained according to GMP standards.

[0205] Exemplary hES cell markers include, but are not limited to, alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), Tra-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth and differentiation factor 3 (GDF3), expression reduction protein 1 (REX1), fibroblast growth factor 4 (FGF4), embryonic cell specific gene 1 (ESG1), developmental pluripotency-associated protein 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-cadherin, cluster tag 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor and stem cell factor (SCF or c-Kit ligand). In addition, embryonic stem cells can express Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, TRA1 60 and / or TRA1 81.

[0206] ESCs may initially be co-cultured with or without feeder cells (such as mouse embryonic feeder cells (MEFs) or human feeder cells, such as human dermal fibroblasts (HDFs)) in any culture medium known in the art for maintaining ESC pluripotency. Before ESCs are inoculated in co-culture, MEF cells or human feeder cells may be mitotically inactivated, for example, by exposure to mitomycin C, gamma radiation, or by any other known method, and thus MEFs are not propagated in culture. In addition, ESC cell cultures may be microscopically examined, and colonies containing non-ESC cell morphologies may be selected and discarded, for example, using a stem cell cutting tool, by laser ablation or otherwise. Typically, no additional MEF cells or human feeder cells are used after the point at which ESCs are harvested for inoculation to form embryoid bodies.

[0207] Alternatively, hES cells can be cultured on a solid surface, such as an extracellular matrix, under feeder-free conditions by any method known in the art, e.g., Klimanskaya et al., Lancet 365:1636-1641 (2005). Thus, the hES cells used in the methods described herein can be cultured in feeder-free cultures.

[0208] As used herein, "embryo-derived cells" (EDC) broadly represent cells derived from pluripotent morulae, cells derived from embryonic sacs, cells including inner cell mass, embryonic shield or ectoderm, or pluripotent stem cells of other early embryos, including primitive endoderm, ectoderm and mesoderm and derivatives thereof."EDC" also includes schizonts and cell masses from aggregated single schizonts or from embryos at different developmental stages, but does not include human embryonic stem cells that are passaged as cell lines.

[0209] As used herein, "efficacy" broadly represents the concentration, for example, number, of cells (such as hemangioblast-derived MSCs) that produce a determined effect. Efficacy can be defined according to effective concentration (EC50), which does not involve the measurement of the maximum effect, but instead measures the effect at different positions along the concentration axis of the dose-response curve. Efficacy can also be determined from a quantitative response dose-response curve (EC50) or a qualitative response dose-response curve (ED50, TD50, and LD50); however, efficacy is preferably measured by EC50. The term "EC50" refers to the concentration at which a drug, antibody, or poison causes a half-response between the baseline and the maximum effect after a certain specific exposure time. Therefore, the EC50 of the quantitative response dose-response curve represents the concentration of the compound at which 50% of its maximum effect is observed. The EC50 of the qualitative response dose-response curve represents the concentration of the compound at which 50% of the population exhibits a response after a specific exposure time. EC50 can be determined using animal studies, where a defined animal model shows measurable physiological changes in response to drug application; cell-based assays using specific cell systems that exhibit measurable biological responses when a drug is added; and / or enzymatic reactions, where the biological activity of a drug can be measured by the accumulation of products following a drug-assisted chemical reaction. Preferably, EC50 is determined using an immunomodulatory assay. Non-limiting examples of these immunomodulatory assays include intracellular cytokines, cytotoxicity, regulatory capacity, cell signaling capacity, proliferation capacity, apoptosis evaluation, and other assays.

[0210] As used herein, "mesenchymal stem cells" (MSC) refer to pluripotent stem cells with the ability to self-renew and differentiate into osteoblasts, chondrocytes and adipocytes and other mesenchymal cell lineages. Unless specifically specified otherwise, the MSC of the present invention is an MSC produced by in vitro differentiation of pluripotent stem cells, and it may be referred to as HMC herein. In one embodiment, HMC can be produced by in vitro differentiation of pluripotent stem cells, followed by differentiation into hemangioblasts, and then differentiation of hemangioblasts into HMCs. HMCs can be identified by the expression of one or more markers as further described herein. HMCs can also have any of the features described in WO 2013 / 082543, U.S. Patent No. 8,962,321 and U.S. Patent No. 8,961,956, the entire contents of which are incorporated herein by reference.

[0211] HMC cells may be genetically or otherwise modified to improve lifespan, potency, homing, or delivery of desired factors within the HMCs or from cells differentiated from these HMCs. As non-limiting examples thereof, HMCs can be genetically modified to express Sirt1 (thereby extending lifespan), express one or more telomerase subunit genes, optionally under the control of an inducible or repressible promoter, incorporate a fluorescent marker, incorporate iron oxide particles or other such agents (which can 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 can improve lifespan (see Go et al., J. Biochem. 142, 741-748 (2007)), express CXCR4 for homing (see Shi et al., Haematologica. 2007 Jul; 92(7):897-904), express recombinant TRAIL to induce caspase-mediated apoptosis in cancer cells, such as gliomas (see Sasportas et al., Proc Natl Acad Sci USA. 2009 Mar 24;106(12):4822-7).

[0212] As used herein, the term "extracellular vesicle" or "EV" refers to a lipid-bound vesicle secreted by a cell into the intercellular space. The three main subtypes of EV are microvesicles (MV), exosomes, and apoptotic bodies, which are distinguished according to their biogenesis, release pathways, size, content, and function (Zaborowski MP, et al. Bioscience. 2015; 65: 783-797). Typically, the diameter of an extracellular vesicle ranges from 20nm to 5000nm, and may be contained in an internal space (i.e., cavity), displayed on the outer surface of the extracellular vesicle, and / or across a membrane. The payload may include nucleic acids, for example, microRNA (miRNA), long non-coding RNA (lncRNA), mRNA, DNA fragments; proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example and without 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), vesicled organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived from / secreted from living or dead organisms, transplanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells.

[0213] As used herein, "optic neuropathy" includes any disease, disorder or condition involving damage to the optic nerve. Optic neuropathy includes hereditary (e.g., autosomal dominant optic atrophy (Kjer's disease) and maternally inherited Leber's hereditary optic neuropathy) and non-hereditary optic neuropathy (e.g., ischemic optic neuropathy). In one embodiment, the optic neuropathy is glaucoma / glaucomatous optic neuropathy.

[0214] As used herein, "therapy", "therapeutic", "treatment" broadly refers to treating a disease, preventing or alleviating the development of a disease or its clinical symptoms, and / or alleviating a disease, causing a disease or its clinical symptoms to regress. "Treatment", "therapeutic", "treatment" covers prevention, prevention, treatment, cure, remedy, reduction, alleviation and / or alleviation of a disease, a sign and / or symptom of a disease. "Therapy", "therapeutic", "treatment" covers the alleviation of a sign and / or symptom in a patient with ongoing signs and / or symptoms of a disease. "Therapy", "therapeutic", "treatment" also covers "prevention" and "prevention". Prevention includes preventing a disease from occurring after treating a disease in a patient or reducing the incidence or severity of a disease in a patient. For therapeutic purposes, the terms "alleviate", "therapeutic", "treatment" broadly refer to clinically significant alleviation of a sign and / or symptom. "Therapy", "therapeutic", "treatment" include treating recurring or recurrent conditions and / or symptoms. "Therapy", "therapeutic", "treatment" covers (but is not limited to) eliminating the appearance of signs and / or symptoms at any time, as well as reducing existing signs and / or symptoms and eliminating existing signs or symptoms. "Therapy," "therapeutic," and "treatment" include treatment of chronic conditions ("maintenance") and acute conditions. For example, treatment includes treating or preventing the recurrence or recurrence of signs and / or symptoms.

[0215] As used herein, the term "effective amount" is intended to include an amount of HMC and / or HMC-EVs sufficient to achieve disease treatment (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of a disease) when administered to a subject with brain damage. Ameliorating a disease includes slowing the progression of a disease or reducing the severity of a subsequent disease. An "effective amount" may vary depending on the nature of the HMC and / or HMC-EVs, the mode of administration of the HMC and / or HMC-EVs, the disease and its severity and history, age, weight, family history, genetic makeup, type of prior or concomitant therapy (if any), and other individual characteristics of the subject to be treated.

[0216] "Effective amount" also includes the amount of HMC and / or HMC-EV that produces a desired effect at a reasonable benefit / risk ratio applicable to any treatment. HMC and / or HMC-EV used in the methods described herein can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0217] As used herein, "normalizing a pathology" refers to restoring the abnormal structure and / or function caused by the disease to a more normal state. Normalization indicates that the development of the pathology can be controlled and improved by correcting the abnormalities in the structure and / or function of tissues, organs, cell types, etc. produced by the disease. For example, brain abnormalities caused by brain injury, such as traumatic brain injury, can be improved, corrected, and / or reversed after treatment with the HMC of the present invention.

[0218] As used herein, "induced pluripotent stem cells" or "iPSC" or "iPS cells" refer to pluripotent stem cells produced by somatic cell reprogramming. iPSC can be produced by expression or expression of a combination of inducing factors ("reprogramming factors"). Fetal, postnatal, neonatal, infantile or adult somatic cells can be used to produce iPS cells. iPS cells can be obtained from a cell bank. Alternatively, iPS cells can be newly produced (by methods known in the art) before starting to differentiate into MSC or another cell type. The preparation of iPS cells can be the initial step in producing differentiated cells. iPS cells can be specifically produced using materials from a specific patient or a matching donor for the purpose of producing tissue-matched MSC cells. iPS cells can be produced by cells that are not substantially immunogenic in the intended recipient, for example, by autologous homologous cells or cells that are compatible with the intended recipient tissue. As discussed further above (see "Pluripotent Cells"), pluripotent cells, including iPS cells, can be genetically or otherwise modified to improve lifespan, potency, homing, or delivery of desired factors in cells differentiated from these pluripotent cells (e.g., MSCs and hemangioblasts).

[0219] As a further example, induced pluripotent stem cells can be generated by reprogramming somatic cells or other cells by contacting the cells with one or more reprogramming factors. For example, the reprogramming factor can be expressed by the cell, for example, by an exogenous nucleic acid added to the cell, or by an endogenous gene in response to a factor that promotes or induces the expression of the gene, such as a small molecule, microRNA, etc. (see Suh and Blelloch, Development 138, 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) 1-3; Anokye-Danso et al., Cell Stem Cell 8, 376-388, April 8, 2011; Orkin and Hochedlinger, Cell 145, 835-850, June 10, 2011, each of which is incorporated herein by reference in its entirety). Reprogramming factors can be provided by exogenous sources, for example, by adding to culture medium, and can be introduced into cells by methods known in the art, such as by coupling with cell entry peptides, proteins or nucleic acid transfection agents, lipofection, electroporation, bioballistic 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 referred to as Oct 3 / 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 Oct-4, Sox2, Nanog and Lin28. In other embodiments, somatic cells are reprogrammed by expressing at least 2 reprogramming factors, at least 3 reprogramming factors or 4 reprogramming factors. In another embodiment, somatic cells are reprogrammed by expressing Oct4, Sox2, MYC, Klf4, Nanog and Lin28. In other embodiments, other 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 usually be identified by the expression of the same markers as embryonic stem cells, although specific iPS cell lines may differ in their expression profiles.

[0220] Induced pluripotent stem cells can be produced by expressing or inducing the expression of one or more reprogramming factors in somatic cells. In one embodiment, the somatic cell is a fibroblast, such as a skin fibroblast, a synovial fibroblast or a lung fibroblast, or a non-fibroblast somatic cell. In one embodiment, the somatic cell is reprogrammed by expressing at least 1, 2, 3, 4, 5 reprogramming factors as described above. In another embodiment, the expression of the reprogramming factor can be induced by contacting the somatic cell with at least one reagent that induces the expression of the reprogramming factor, such as an organic small molecule reagent.

[0221] Somatic cells can also be reprogrammed using a combination of methods, wherein reprogramming factors are expressed (e.g., using viral vectors, plasmids, etc.) and the expression of reprogramming factors is induced (e.g., using organic small molecules). For example, reprogramming factors can be expressed in somatic cells by infection using viral vectors, such as retroviral vectors or lentiviral vectors. In addition, reprogramming factors can be expressed in somatic cells using non-integrating vectors, such as episomal plasmids or mRNA. See, for example, Yu et al., Science. 2009 May 8; 324 (5928): 797-801, which is incorporated herein by reference in its entirety. When reprogramming factors are expressed using non-integrating vectors, electroporation, transfection, or transformation of somatic cells with vectors can be used to express the factors in cells.

[0222] In some embodiments, the reprogramming factor is expressed in the cell, and the cell can be cultured by any method known in the art. As time goes by, the cell with ES characteristics appears in the culture dish. Can be based on (for example) ES morphology or based on the expression of selectable or detectable markers to select and subculture cells. These cells can be cultivated to produce a cell culture similar to ES cells--these are assumed iPS cells. IPS cells can usually be identified by the expression of the same marker as other embryonic stem cells, although specific iPS cell lines can be different in its expression spectrum. Exemplary iPS cells can express Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, TRA160 and / or TRA181.

[0223] In order to confirm the pluripotency of iPS cells, cells can be tested in one or more pluripotency assays. For example, cells can be tested for expression of ES cell markers; when transplanted into SCID mice, the ability of cells to produce teratomas can be assessed; the ability of cells to differentiate and produce cell types with all three germ layers can be assessed. Once pluripotent iPS cells are obtained, the cells can be used to produce hemangioblasts and MSC cells.

[0224] As used herein, "angioblasts" or "HBs" refer to pluripotent cells and are used as common precursors of both hematopoietic and endothelial cell lineages. During embryonic development, they are considered to be produced as transitional cell types, which appear and colonize in primitive blood islands during early mesoderm development (Choi et al., Development 125 (4): 725-732 (1998)). Once colonized, angioblasts are able to produce primitive and determined hematopoietic cells, HSCs and endothelial cells (Mikkola et al., J. Hematother. Stem Cell Res 11 (1): 9-17 (2002)).

[0225] Hemangioblasts can be obtained in vitro from mouse PSCs (Kennedy et al., Nature (386): 488-493 (1997); Perlingeiro et al., Stem Cells (21): 272-280 (2003)) and human PSCs (Refs. 14, 15, Yu et al., Blood 2010 116: 4786-4794). Other studies claim to have isolated hemangioblasts from umbilical cord blood (Bordoni et al., Hepatology 45 (5) 1218-1228), from peripheral blood (Ciraci et al., Blood 118: 2105-2115) and from mouse uterus (Sun et al., Blood 116 (16): 2932-2941 (2010)) to isolate circulating CD34-lin-CD45-CD133- cells. By culturing and differentiating cell clusters grown in liquid culture medium, and then growing cells in semi-solid culture medium containing a variety of cytokines and growth factors, mouse and human PSC-derived angioblasts have been obtained (Kennedy, Perlingeiro, References 14, 15); see also, U.S. Patent No. 8,017,393, which is incorporated herein by reference in its entirety. In one embodiment, angioblasts can be produced 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 WO 2013 / 082543, which are incorporated herein by reference in their entirety. The term angioblast also includes vascular colony-forming cells described in U.S. Patent No. 8,017,393 (incorporated herein by reference in its entirety), which, in addition to being able to differentiate into hematopoietic and endothelial cell lineages, are also able to become smooth muscle cells and are not positive for CD34, CD31, KDR, and CD133. In another embodiment, the hemangioblasts are positive for the blood markers CD43 and CD45 and express low levels of or are negative for the pericyte markers CD146, PDGRb and / or NG2.

[0226] Useful hemangioblasts in the methods described herein can be produced or obtained by any of these known methods or any method described herein. For example, embryoid bodies can be formed by culturing pluripotent cells under non-adherent conditions, for example, on low-adherent substrates, in "hanging drops", or by Able Biott rotary bioreactors. In these cultures, PSCs can form cell clusters or cell clusters named as embryoid bodies. See Itskovitz-Eldor et al., Mol Med. 2000 Feb; 6 (2): 88-95, which is incorporated herein by reference in its entirety. Typically, embryoid bodies are initially formed as solid clusters or clusters of pluripotent cells, and over time, some embryoid bodies begin to include a cavity filled with liquid, the former being referred to as "simple" EB in the literature, and the latter being referred to as "cystic" embryoid bodies. The same. The cells (solid and cystic forms) in these EBs can differentiate and produce cells whose number gradually increases over time. Alternatively, EBs can then be cultured as adherent cultures and allowed to form growths. Likewise, pluripotent cells that are allowed to outgrow and form multilayered cell populations can differentiate over time.

[0227] In one embodiment, hemangioblasts are produced by the following steps, the steps comprising (a) culturing PSC lines for 2, 3, 4, 5, 6 or 7 days to form cell masses (embryoid bodies; EB) and (b) inducing the cell masses or EBs to differentiate into hemangioblasts. In other embodiments, the cell masses or EBs in step (b) are cultured in a culture medium based on serum-free methylcellulose rich in cytokines. In one embodiment, hemangioblasts are produced by inducing the differentiation of any pluripotent cells as described herein.

[0228] In one embodiment, the cell mass or EB is cultured for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days in a serum-free methylcellulose medium containing one or more selected from penicillin / streptomycin (pen / strp), Growth supplement (containing 9.0-11.0 g / L cholesterol and 13.0-18.0 g / L lipoprotein and fatty acid water-soluble concentrate at 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) and produce hemangioblasts. In a preferred embodiment of the present invention, the hemangioblasts are harvested between 6-14 days 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 culture medium at the following concentrations: 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), 50 ng / ml stem cell-derived factor (SCF), 20 ng / ml granulocyte macrophage colony-stimulating factor (GM-CSF), 20 ng / ml interleukin 3 (IL3) and 20 ng / ml interleukin 6 (IL6).

[0229] In vitro generation of mesenchymal stem cells

[0230] One embodiment of the present invention includes a method for producing mesenchymal stem cells (hereinafter referred to as "HMC") by in vitro differentiation of hemangioblasts. Hemangioblasts can be obtained by any of the methods described herein. In one embodiment, hemangioblasts are obtained by in vitro differentiation of pluripotent stem cells. Pluripotent stem cells can be cultured on feeder cells (e.g., human skin fibroblasts or mouse embryonic fibroblasts) or under conditions without feeder cells. In some embodiments, hemangioblasts are cultured under conditions without feeder cells, and then they are plated on an extracellular matrix. In another embodiment, the extracellular matrix is ​​selected from laminin, fibronectin, vitronectin, proteoglycan, nestin, collagen, collagen I, collagen IV, heparan sulfate, soluble preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, Matrigel and human basement membrane extracts. In a further embodiment, the extracellular matrix can be derived from any mammalian source, including human sources.

[0231] 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 an HMC preparation. In one embodiment, the hemangioblasts can be plated at about 50,000 to about 100,000 cells / cm 2 Initial plating of angioblasts on substrate-coated tissue culture dishes is performed at a concentration of about 100 μg / cm. During the culture period of the angioblasts, a portion of the angioblasts adhere to the culture plates and begin to differentiate into HMCs. Based on their growth rate, plating density, and perceived confluence, adherent cells are passaged every 3-6 days or more than 6 days, for example, about 6-10 days or about 10-15 days. For passage, the harvest density can be about 5,000 to about 20,000 cells / cm 2 , or about 20,000 to about 40,000 cells / cm 2 After harvesting the cells, the cells are counted and can be counted at about 2500 to about 6000 cells / cm 2 In one embodiment, HMCs are produced by the steps of (a) culturing ESCs for 8-12 days and producing hemangioblasts, (b) harvesting the hemangioblasts, (c) re-plating the hemangioblasts of step (b), and (d) culturing the hemangioblasts of step (c) for 14-30 days.

[0232] In one embodiment, hemangioblasts are harvested, re-plated and cultured in a liquid medium without feeder cells, wherein the culture does not contain a cell feeder layer, such as mouse embryonic fibroblasts, OP9 cells, or other cell types known to those of ordinary skill in the art. In a preferred embodiment, hemangioblasts are cultured on an extracellular matrix. In other preferred embodiments, hemangioblasts are cultured on an extracellular matrix, wherein the matrix comprises a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, which gels at room temperature to form a restored basement membrane (Matrigel). In a further preferred embodiment, hemangioblasts are formed according to the steps comprising the following: (a) the hemangioblasts are cultured on an extracellular matrix for at least 7 days, (b) the hemangioblasts of step (a) are transferred to an uncoated tissue culture plate, and the hemangioblasts of step (b) are further cultured for about 7 to 14 days. The hemangioblasts can be cultured in the presence of one or more factors selected from the following: transforming growth factor β (TGF-β), 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 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 main component can be of mammalian origin, or more specifically of human origin. In one embodiment, hemangioblasts are cultured in a serum-containing liquid culture medium on an extracellular matrix protein-coated plate, wherein the culture medium may contain a component selected from the group consisting of αMEM (Sigma-Aldrich) supplemented with 10-20% fetal calf serum (αMEM+20% FCS), αMEM supplemented with 10-20% heat-inactivated human AB serum, and IMDM supplemented with 10-20% heat-inactivated AB human serum.

[0233] In another embodiment, the hemangioblasts are cultured in a medium containing serum or a serum replacement, such as αMEM supplemented with 20% fetal bovine serum. In another embodiment, the hemangioblasts are cultured in a serum-free medium.

[0234] In other 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 yet a further embodiment of the invention, HMCs are produced by the steps comprising: (a) culturing the hemangioblasts on the extracellular matrix for about 7 days, (b) transferring the hemangioblasts of step (a) to an uncoated tissue culture dish and culturing the hemangioblasts for an additional 9-100 days, 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 produced by the steps comprising: (a) culturing hemangioblasts on an extracellular matrix for about 7 days, (b) transferring the hemangioblasts of step (a) to a coated tissue culture dish and culturing the hemangioblasts for an additional 9-100 days, 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.

[0235] In one embodiment of the invention, angioblasts are differentiated from PSCs by the following steps, comprising: (a) culturing PSCs to form cell clusters or EBs in the presence of vascular endothelial growth factor (VEGF) and / or bone morphogenetic protein 4 (BMP-4) (by way of non-limiting example); (b) culturing PSCs to form cell clusters 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), Flt; (c) culturing the hemangioblasts in a medium comprising at least one other 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 other growth factor is provided in an amount sufficient to expand the cell cluster in the culture, and wherein copper is optionally added to any one of steps (a)-(c).

[0236] In one embodiment of the present invention, HMCs are produced by culturing hemangioblasts, wherein the hemangioblasts are differentiated from PSCs according to the following steps, the steps comprising: (a) culturing PSCs to form cell clusters or EBs in the presence of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 4 (BMP-4) within 0-48 hours of the start of the culture; (b) culturing the cell clusters or EBs in the presence of at least one growth factor sufficient to induce the cell clusters or EBs to differentiate into hemangioblasts, the growth factor being selected from basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), Flt; 3L (FL), thrombopoietin (TPO) and tPTD-HOXB4; and (c) culturing the hemangioblasts in a culture medium containing at least one other growth factor, wherein the growth factor is selected from 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 tPTD-HOXB4, wherein the at least one other growth factor is provided in an amount sufficient to expand the hemangioblasts in the culture.

[0237] In another embodiment, HMCs are produced by the steps comprising: (a) harvesting hemangioblasts after at least 6, 7, 8, 9, 10, 11, 12, 13 or 14 days of inducing differentiation of PSCs into 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 of inducing differentiation of the hemangioblasts from step (a) into said mesenchymal cells.

[0238] In yet another embodiment, a preparation of at least 80, 85, 90, 95, 100, 125, or 125 million HMCs is produced from about 200,000 hemangioblasts within about 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days of culturing the hemangioblasts, wherein the preparation of HMCs comprises less than 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%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8 ... 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.

[0239] Extracellular vesicles secreted by mesenchymal stem cells

[0240] The present invention also provides extracellular vesicles isolated, derived, secreted or released from cells, e.g., HMCs of the present invention.

[0241] As used herein, the term "extracellular vesicle" or "EV" refers to a lipid-bound vesicle secreted by a cell into the intercellular space. The three main subtypes of EV are microvesicles (MV), exosomes, and apoptotic bodies, which are distinguished according to their biogenesis, release pathways, size, content, and function (Zaborowski MP, et al. Bioscience. 2015; 65: 783-797). Typically, the diameter of an extracellular vesicle ranges from 20nm to 5000nm, and may be contained in an internal space (i.e., cavity), displayed on the outer surface of the extracellular vesicle, and / or across a membrane. The payload may include nucleic acids, for example, microRNA (miRNA), long non-coding RNA (lncRNA), mRNA, DNA fragments; proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example and without limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived / secreted from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solutions), vesicled organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived / secreted from living or dead organisms, transplanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells.

[0242] As used herein, the term "exosome" refers to a small vesicle derived from a cell that contains a membrane (i.e., a lumen) that encloses an internal space (i.e., a cavity) and is formed by the cell either by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. ., et al. J. Extracell. Vesicles. 2015; 4: 27066). Specifically, exosomes are involved in the sorting, recycling, storage, transport, and release of proteins. Exosomes are typically between 20-300 nm in diameter. 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.

[0243] Exosomes have been found to participate in cell-cell communication, cell maintenance and tumor development. In addition, it has been found that exosomes stimulate immune responses by acting as antigen presenting vesicles (Bobrie A. et al., Traffic. 2011; 12: 1659-1668). In the nervous system, it has been found that exosomes help promote myelin formation, axon growth and neuronal survival, and therefore play a role in tissue repair and regeneration (Faure J., et al. Mol. Cell. Neurosci. 2006; 31: 642-648). At the same time, it has been found that exosomes in the central nervous system (CNS) contain pathogenic proteins, such as beta amyloid peptide, superoxide dismutase and alpha synuclein that can help disease development (Fevrier B., et al., Proc. Natl. Acad. Sci. USA. 2004; 101: 9683-9688). Exosomes have also been shown to be carriers of disease markers. The use of exosomes as carriers of biomarkers is ideal because these vesicles are present in bodily fluids, such as blood and urine, which allows for minimally to noninvasive "liquid biopsy"-type approaches to diagnose and even monitor a patient's response to treatment.

[0244] In addition to their natural role in cell-cell interactions, exosomes can be loaded with different cargoes, such as drugs and exogenous nucleic acids or proteins, and deliver the cargoes to different cells. The cargoes can be coupled to extracellular vesicles, embedded in extracellular vesicles, encapsulated in extracellular vesicles, or otherwise carried by extracellular vesicles or any combination thereof. Therefore, as used herein, reference to cargoes "present in" extracellular vesicles or their cavities should be understood to include any of the above-mentioned ways of carrying cargoes.

[0245] The cargo can be an endogenous cargo, an exogenous cargo, or a combination thereof. Examples of cargo that can be coupled, embedded, encapsulated within, or carried by the extracellular vesicles described herein include, without limitation: nucleic acid molecules (e.g., DNA, cDNA, antisense oligonucleotides, mRNA, inhibitory RNA (e.g., antisense RNA, miRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA) and 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 drugs, or any combination thereof. Importantly, exosomes are natural carriers of miRNA and other non-coding RNAs, and direct membrane fusion with target cells allows the contents to be delivered directly into the cytosol. This makes exosomes an excellent delivery system for small molecules (Lai RC, et al. Biotechnol. Adv. 2013; 31: 543-551).

[0246] Microvesicles are EVs formed by direct outward budding or contraction of the plasma membrane. The size of microvesicles is generally in the range of 100nm to 1000nm in diameter. The pathway of microvesicle formation is not very clear, however, it is believed to require cytoskeletal components such as actin and microtubules, as well as molecular motors (kinesin and myosin) and fusion mechanisms (SNARE 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 donor cells (Zaborowski MP, et al. Bioscience. 2015; 65: 783-797). Similarly, it has been previously confirmed that the number of microvesicles consumed depends on the physiological state and microenvironment of recipient cells. Like exosomes, microvesicles are also involved in cell-cell communication between local cells and distant cells (distant cells). The ability of these 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). EVs are unique in their ability to package active cargo (proteins, nucleic acids, and lipids) and deliver these cargoes to another cell, either adjacent or distant, and alter the function of the recipient cell through EV delivery.

[0247] Apoptotic bodies are released into the intercellular space by dying cells. It is reported that their size ranges from 50nm to 5000nm in diameter, and the size of most apoptotic bodies tends to be on the larger side (Borges F. et al., Braz. J. Med. Biol. Res. 2013; 46: 824-830). Due to the increase in hydrostatic pressure after cell contraction, the plasma membrane of the cell is separated from the cytoskeleton, thereby forming these bodies (Wickman G., et al. Cell Death Differ. 2012; 19: 735-742). The composition of apoptotic bodies is exactly the opposite of exosomes and microvesicles. Unlike exosomes and microvesicles, apoptotic bodies contain complete organelles, chromatin and a small amount 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).

[0248] Methods for isolating extracellular vesicles

[0249] The EVs of the present invention can be isolated, secreted, obtained or separated from culture medium or other source materials (e.g., HMCs of the present invention) using conventional methods known in the art and as described in the following examples (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) 293-304 and the techniques described in the references cited therein). The most commonly used methods include multiple centrifugation and ultracentrifugation steps.

[0250] The physical properties of EVs (e.g., HMC-EVs) can be used for EV separation, purification, or enrichment, including separation based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieve separation, etc.), density (e.g., conventional or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external forces, etc.). Alternatively or in addition, separation can be based on one or more biological properties, and include methods that can use surface markers (e.g., for precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, non-specific ligand binding, immunomagnetic capture of EVs using magnetic beads coated with antibodies to proteins exposed on the EV membrane, etc.).

[0251] Methods based on the use of size exclusion polymers such as PEG have recently been described by several different groups (US Patent Application 20130273544, US Patent Application 20130337440). Two such products are ExoQuick (System Biosciences, Mountain View, USA) and Total Exosome Isolation Reagent (Total Exosome Isolation Reagent, Life Technologies, Carlsbad, USA). These polymers work by binding water molecules and forcing less soluble components such as extracellular vesicles and proteins to precipitate out of solution, allowing them to be collected by short, low-speed centrifugation.

[0252] In some embodiments, separation, purification and enrichment can be carried out in a conventional and non-selective manner (generally including continuous centrifugation). Alternatively, separation, purification and enrichment can be carried out in a more specific and selective manner (e.g., using production cell-specific surface markers). For example, specific surface markers can be used for immunoprecipitation, FACS sorting, affinity purification or for the part of the bead binding of magnetic separation.

[0253] In some embodiments, tangential flow filtration can be used to isolate or purify EVs (e.g., HMC-EVs).

[0254] In some embodiments, size exclusion chromatography can be used to separate 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, separation of EVs (e.g., HMC-EVs) can include ion chromatography, such as anion exchange, cation exchange, or mixed mode chromatography. In some embodiments, separation of EVs (e.g., HMC-EVs) can include desalting, dialysis, tangential flow filtration, ultrafiltration or diafiltration, or any combination thereof. In some embodiments, separation of EVs (e.g., HMC-EVs) can include a combination of methods, including (but not limited to) differential centrifugation, size-based membrane filtration, concentration, and / or differential zonal centrifugation. In some embodiments, separation of EVs (e.g., HMC-EVs) can include one or more centrifugation steps. Centrifugation can be performed at about 50,000 to 150,000 × g. Centrifugation can be performed at about 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-membrane particles using 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 separation are commercially available from (e.g.) Qiagen, InVitrogen and SBI. Methods for loading therapeutic agents to EVs are known in the art and include lipid transfection, electroporation, and any standard transfection method.

[0255] In some embodiments, the present invention provides a method for isolating HMC-EVs secreted from HMC obtained by in vitro differentiation of pluripotent stem cells. The method includes providing HMC 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 separated by tangential flow filtration. In some embodiments, HMC-EVs are separated by ultracentrifugation. In some embodiments, HMC-EVs are separated by cation exchange chromatography. In some embodiments, HMC-EVs are separated by anion exchange chromatography.

[0256] Characteristics and composition of HMC and / or HMC-EV

[0257] The present invention further provides a composition comprising HMC obtained by in vitro differentiation of pluripotent stem cells and / or extracellular vesicles (HMC-EV) secreted from HMC of the present invention. In one embodiment, HMC is obtained by in vitro differentiation of hemangioblasts. The expression level of certain phenotypic markers can be determined by any method known in the art, such as immunohistochemistry. The expression of certain genes can be determined by any method known in the art, such as RT-PCR and RNA-Seq.

[0258] In one embodiment, the HMC of the present invention expresses at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 markers selected from the group consisting of CD9, CD13, CD29, CD44, CD73, CD90, CD105, CD166, and HLA-ABC. In yet further embodiments, the HMC of the present invention expresses at least 2, at least 3, at least 4, at least 5, or at least 6 markers selected from the group consisting of CD9, CD13, CD29, CD44, CD73, CD90, and CD105, and wherein the HMC does 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 HMC of the present invention expresses at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 markers selected from the group consisting of AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1.

[0259] In one embodiment, the composition comprises HMCs, wherein after about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days in culture, 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. In one embodiment of the invention, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the HMC in the composition of the invention express at least 2, at least 3, at least 4, at least 5, at least 6, At least 7 or at least 8 markers: CD9, CD13, CD29, CD44, CD73, CD90, CD105, CD166 and HLA-ABC and lacking expression of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD61, CD62E, CD133 and Stro-1. The HMCs in the composition of the invention may also express at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 markers selected from the following: AIRE-1, IL-11, CD10, CD24, ANG-1 and CXCL1.

[0260] In one embodiment, the composition comprises HMCs, wherein at least 30% of the HMCs are positive for CD10. In addition, at least 60% of the HMCs may be positive for 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 markers CD31, CD34, CD45, CD133, FGFR2, CD271, Stro-1, CXCR4, and TLR3.

[0261] In another embodiment, the composition comprises HMCs, wherein at least 50% of the HMCs are positive for CD105 or CD73 within about 7-20 (e.g., 15) days of culture. In a preferred embodiment of the invention, at least 50% of the HMCs are positive for CD105 or CD73 after about 7-15 days of culture. In other embodiments of the invention, at least 80% of the HMCs are positive for CD105 and CD73 within about 20 days of culture. In other embodiments of the invention, at least 80% of the HMC compositions are positive for CD105 and CD73 within about 20 days of culture.

[0262] In one embodiment, the composition comprises HMCs, wherein 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, CD 44, 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 (i) may be positive for CD105, CD73 and CD90; (ii) may be positive for CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD 44, CD166, CD274 and HLA-ABC and / or (ii) may be negative for CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4 and / or TLR3 or less than 5% or less than 10% of the cells express CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4 and / or TLR3. At least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the HMCs may be positive for: (i) one or more of 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, CD 44, CD166, CD274 and HLA-ABC.

[0263] In another embodiment, the composition comprises HMCs, wherein at least 20%, 30%, 40% or 50% of the HMCs (i) may be positive for all CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD 44, CD166, CD274 and HLA-ABC and (ii) may be negative for CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4 and / or TLR3 or less than 5% or less than 10% of the cells express CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4 and / or TLR3.

[0264] In other embodiments, the composition comprises HMCs, wherein 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.

[0265] In another embodiment, the composition comprises HMCs, wherein at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% 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; or (ii) at least one of CD73, CD90, CD105, CD13, CD29, CD 44, CD166, CD274 and HLA-ABC.

[0266] In another embodiment, the HMCs may not express or less than 5% or less than 10% of the HMCs may express at least one of CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, or TLR3.

[0267] In addition to the features described above, compared to adult-derived MSCs, the HMCs of the present invention may have the phenotype of younger cells. In one embodiment, the HMCs are able to undergo at least or 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 2-3 doublings in culture. In another embodiment, compared to adult-derived MSCs, the HMCs of the present invention have longer telomere lengths, greater immunosuppression, fewer vacuoles, faster divisions, easier divisions in culture, higher CD90 expression, less lineage orientation, or a combination thereof. In another embodiment, compared to adult-derived MSCs, the HMCs of the present invention have increased expression of transcripts that promote cell proliferation (i.e., have a higher proliferation capacity) and reduced expression of transcripts involved in terminal cell differentiation.

[0268] In one embodiment, the HMC is an "early passage" HMC and may be passaged no more than 1, 2, 3, 4, 5, 6, 7 or 8 times. In one embodiment, the early passage HMC is passaged no more than 4 times. In another embodiment, the early passage HMC is passaged no more than 5 times. In another embodiment, the early passage HMC is passaged no more than 6 times. In addition to the HMC characteristics described above, the early passage HMC may express mRNA encoding interleukin-6 (IL-6) in a resting or basal state, and the level of IL-6 expressed by the early passage HMC may be less than 10% of the IL-6 mRNA level expressed by BM-MSC or AD-MSC in a resting or basal state. The VEGF mRNA level in the early passage HMC in a resting or basal state may also be downregulated compared to BM-MSC in a resting or basal state. In another embodiment, the HMC in a resting or basal state may express mRNA encoding CD24, and the level of HMC expressing mRNA encoding CD24 is higher than the level of CD24 mRNA expressed by BM-MSC or AD-MSC preparations in a resting or basal state. Other mRNA levels that may be upregulated in early passage HMCs in a resting or basal state compared to BM-MSCs in a resting or basal state include AIRE, ANGPT1 (ANG-1), CXCL1, CD10, and IL-11. In addition, early passage HMCs in a resting or basal state may be negative for one or more mRNAs encoding ANGPT2, CD31, CD34, CD45, HLA-G, IL2RA, IL3, IL12B.

[0269] In other embodiments, 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, 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.

[0270] In one embodiment, the expression level of CD10 in early passage HMCs is upregulated as 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 may be about the same as the expression level of CD10 in BM-MSCs. In another embodiment, the expression level of Stro-1 in early passage HMCs is downregulated as 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, wherein about 5-10% of the early passage HMCs express Stro-1.

[0271] In other embodiments, the HMCs of the present invention express higher levels of certain genes compared to BM-MSCs, UCB-MSCs or AD-MSCs. For example, the HMCs of the present invention may express higher levels of any gene listed in Table 3 compared to BM-MSCs, and / or the HMCs of the present invention may express higher levels of any gene listed in Table 5 compared to UCB-MSCs, and / or the HMCs of the present invention may express higher levels of any gene listed in Table 7 compared to AD-MSCs. In another embodiment, the HMCs of the present invention may express lower levels of any gene listed in Table 4 compared to BM-MSCs, and / or the HMCs of the present invention may express lower levels of any gene listed in Table 6 compared to UCB-MSCs, and / or the HMCs of the present invention may express lower levels of any gene listed in Table 8 compared to AD-MSCs.

[0272] In one embodiment, genes associated with increased migration and chemotaxis, such as MMP9, are expressed at higher levels in the HMCs of the present invention compared to BM-MSCs or UCB-MSCs. In another embodiment, Lgr5 (a marker of pluripotent stem cells) is expressed at higher levels in the HMCs of the present invention compared to BM-MSCs or UCB-MSCs. In other embodiments, CD24 is expressed at higher levels in the HMCs of the present invention compared to BM-MSCs, while IL-6 is expressed at lower levels in the 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 (axon guidance factor -1 (Netrin-1)) are expressed at higher levels in the HMCs of the present invention compared to BM-MSCs or UCB-MSCs. The MSCs of the present invention can be selected or purified based on any differentially expressed genes.

[0273] In some embodiments, HMCs of the present invention may express lower levels of any miRNA listed in Table 21 compared to HMC-EVs. In some embodiments, HMCs of the present invention may express higher levels of any miRNA listed in Table 22 compared to HMC-EVs.

[0274] In other embodiments, the HMC-EVs of the present invention express higher levels of certain miRNAs, genes or proteins compared to BM-MSC-EVs, UCB-MSC-EVs or AD-MSC-EVs.

[0275] In some embodiments, the HMC-EVs of the present invention may express higher levels of any miRNA listed in Table 9 compared to UCB-MSC-EVs, and / or the HMC-EVs of the present invention may express higher levels of any miRNA listed in Table 11 compared to BM-MSC-EVs, and / or the HMC-EVs of the present invention may express higher levels of any miRNA listed in Table 13 compared to AD-MSC-EVs. In another embodiment, the HMC-EVs of the present invention may express lower levels of any miRNA listed in Table 10 compared to UCB-MSC-EVs, and / or the HMC-EVs of the present invention may express lower levels of any miRNA listed in Table 12 compared to BM-MSC-EVs, and / or the HMC-EVs of the present invention may express lower levels of any miRNA listed in Table 13 compared to AD-MSC-EVs. In some embodiments, the HMC-EVs of the present invention may express higher levels of any protein listed in Table 15 compared to UCB-MSC-EVs, and / or the HMC-EVs of the present invention may express higher levels of any protein listed in Table 17 compared to BM-MSC-EVs, and / or the HMC-EVs of the present invention may express higher levels of any miRNA listed in Table 19 compared to AD-MSC-EVs. In another embodiment, the HMC-EVs of the present invention may express lower levels of any protein listed in Table 16 compared to UCB-MSC-EVs, and / or the HMC-EVs of the present invention may express lower levels of any protein listed in Table 18 compared to BM-MSC-EVs, and / or the HMC-EVs of the present invention may express lower levels of any protein listed in Table 20 compared to AD-MSC-EVs.

[0276] 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 than BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs.

[0277] In some embodiments, HMC-EVs express at least one protein 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, K PNA2, 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.

[0278] In some embodiments, HMC-EVs express at least one protein 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, LGA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3, GA3 LS1, 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.

[0279] 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 than BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0280] 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 than BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

[0281] In some embodiments, the HMC-EV of the present invention may express higher levels of any miRNA listed in Table 21 compared to the HMC of the present invention. In some embodiments, the HMC-EV of the present invention may express lower levels of any miRNA listed in Table 22 compared to the HMC of the present invention.

[0282] In one embodiment, genes associated with or involved in neuronal lineage development, 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.

[0283] In another embodiment, the HMCs of the present invention have different expression profiles compared to mature MSCs, e.g., AD-MSCs or BM-MSCs or UCB-MSCs. Specifically, the HMCs of the present invention are able to confer neuroprotection 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 similar profiles with the HMCs from which they are derived. Similar signal transduction pathways enriched in HMCs are also enriched in HMC-EVs when compared to other tissue-derived MSCs and EVs.

[0284] In one embodiment, with respect to pluripotent stem cells, the composition comprising HMC of the present invention is substantially purified. In other embodiments, the HMC composition of the present invention is substantially purified relative to pluripotent stem cells so that the composition comprises at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% HMC. The pluripotent stem cells can be any pluripotent stem cells described herein.

[0285] The composition may contain less than 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. 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.

[0286] In some embodiments, the compositions comprising HMC-EVs of the invention are substantially purified relative to HMC. In other embodiments, the HMC-EV compositions of the invention are substantially purified relative to HMC such that the composition comprises 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.

[0287] The composition may contain less than 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% HMC.

[0288] In another embodiment of the present invention, the composition of HMC and / or HMC-EV produced by any one or more of the methods of the present invention does not form a teratoma when introduced into a host.

[0289] In an exemplary aspect, the present disclosure provides a composition comprising at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 In a specific embodiment, the composition comprises 10 6 The invention relates to a method for producing a mesenchymal stem cell comprising: a) a plurality of HMCs and less than 1% of any other cell type, wherein the mesenchymal stem cells have the capacity to replicate to undergo at least 10 population doublings in cell culture, wherein less than 25% of the cells undergo cell death, senescence, or differentiation into non-HMC cells by the 10th population doubling.

[0290] HMC can have a replication rate that experiences at least 10 population doublings in cell culture in less than 25 days. HMC can have an average terminal restriction fragment length (TRF) that can be longer than 8kb. Relative to the mesenchymal stem cell preparation derived from the bone marrow that has experienced 5 population doublings, HMC can have a statistically significantly reduced participation in (i) cell cycle regulation and cell aging, (ii) cell energy and / or lipid metabolism, and (iii) apoptosis in one or more of the protein content and / or enzyme activity. Relative to the mesenchymal stem cell preparation derived from the bone marrow, HMC can have a statistically significantly increased participation in cytoskeletal structure and the content and / or enzyme activity of the protein associated with it. By flow cytometry measurement, in 10 population doublings in culture, in cells with forward scattered light values ​​greater than 5,000,000, HMC may not experience an increase of more than 75%.

[0291] In one embodiment of the invention, a subject HMC preparation (e.g., produced by culturing hemangioblasts) is provided, wherein the preparation comprises substantially similar levels of p53 and p21 proteins, or wherein the level of p53 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than p21. In one embodiment of the invention, a pharmaceutical preparation of the subject HMC (e.g., produced by culturing hemangioblasts) is provided, wherein the pharmaceutical preparation comprises substantially similar levels of p53 and p21 proteins, or wherein the level of p53 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than p21.

[0292] In one embodiment, the invention provides a composition comprising HMCs, wherein the composition comprises substantially similar percentages of HMCs that are positive for p53 and p21 proteins, or wherein the percentage of p53-positive HMCs is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than that of p21-positive HMCs.

[0293] In one embodiment, the present invention provides a method comprising at least about 10 3 to about 10 13 In another embodiment, the present invention provides a composition comprising at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 1012 or 10 13 HMC-EV.

[0294] Methods for determining neurite outgrowth of HMC and / or HMC-EV populations.

[0295] The present invention also provides methods for determining the effects of HMC and / or HMC-EV on neurons, such as neurite growth. In one aspect, the present invention provides methods for determining neurite growth of HMC and / or HMC-EV populations. In one embodiment, the method includes (a) preparing a mixed neuronal culture from isolated cerebral cortex, (b) plating HMC and / or HMC-EV populations on a permeable membrane, (c) applying tension to the mixed neuronal culture, (d) covering the tensioned mixed neuronal culture with the permeable membrane of step (b), and (e) measuring neurite growth of the mixed neuronal culture. In one embodiment, the method also includes determining the gene expression of the mixed neuronal culture in the presence and absence of HMC and / or HMC-EV populations. In another embodiment, the tension is a physical scratch produced in the mixed neuronal culture. In another embodiment, the tension is vacuum pressure and positive air pressure applied to the mixed neuronal culture. In another embodiment, tension can be applied under tensile oscillations of 15% to 0%. In one embodiment, the stretch oscillations may be applied at 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 0% of the cycles.

[0296] Pharmaceutical preparations containing HMC and HMC-EV

[0297] The pharmaceutical preparations of the present invention may comprise any HMC or HMC composition described herein, and / or HMC-EV. Pharmaceutical preparations comprising HMC and / or HMC-EV of the present invention may be formulated with a pharmaceutically acceptable carrier. For example, HMC and / or HMC-EV of the present invention may be administered alone or as a component of a pharmaceutical preparation, wherein HMC and / or HMC-EV may be formulated for administration in any convenient manner for medical use. One embodiment provides a pharmaceutical preparation of HMC and / or HMC-EV, comprising HMC and / or HMC-EV in combination with one or more pharmaceutically available sterile isotonic water or non-aqueous solutions, the sterile isotonic water or non-aqueous solutions being selected from: dispersions, suspensions, emulsions, sterile powders (optionally reconstituted into sterile injectable solutions or dispersions before use), antioxidants, buffers, bactericides, solutes or suspending agents, and thickeners.

[0298] Exemplary pharmaceutical formulations of the present disclosure can be any formulation suitable for treating a human patient, such as a pyrogen-free or substantially pyrogen-free and pathogen-free formulation.

[0299] The preparations containing HMC and / or HMC-EV used in the methods described herein can be transplanted in suspensions, gels, colloids, slurries, or mixtures. In addition, at the time of injection, cryopreserved HMC and / or HMC-EV can be resuspended with a commercially available balanced salt solution to achieve the desired osmotic pressure and concentration for injection administration (i.e., bolus injection or intravenous injection).

[0300] One aspect of the present invention relates to a pharmaceutical preparation suitable for use in a mammalian patient, the pharmaceutical preparation comprising at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 Another aspect of the present invention provides a frozen cell bank, wherein the cell bank comprises at least 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or even 10 13 Another aspect of the present invention provides a pharmaceutical preparation that is free of or substantially free of non-human cells and / or non-human animal products, the pharmaceutical preparation comprising at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 HMCs and / or HMC-EVs, and less than 1% of any other cell type, more preferably less than 0.1%, 0.01% or even 0.001% of any other cell type.

[0301] The concentration of the pharmaceutical preparation for administration of HMC and / or HMC-EV can be in any amount that is effective and (e.g.) substantially free of PSC. For example, the pharmaceutical preparation can include the number and type of HMC and / or HMC-EV described herein. In a specific embodiment, the pharmaceutical preparation of HMC and / or HMC-EV includes about 1×10 6 About 1×10 7 , about 1×10 7 About 1×10 8 , about 1×108 About 1×10 9 , about 1×10 9 About 1×10 10 , about 1×10 10 About 1×10 11 , about 1×10 11 About 1×10 12 or about 1×10 12 About 1×10 13 HMC and / or HMC-EV (for systemic administration to a host in need thereof) or about 1×10 4 About 1×10 5 , about 1×10 5 About 1×10 6 , 1×10 6 About 1×10 7 , about 1×10 7 About 1×10 8 , about 1×10 8 About 1×10 9 , about 1×10 9 About 1×10 10 , about 1×10 10 About 1×10 11 , about 1×10 11 About 1×10 12 or about 1×10 12 About 1×10 13 HMC and / or HMC-EV (for local administration to a host in need thereof).

[0302] Treatments for brain damage

[0303] The HMC and / or HMC-EV described herein and the pharmaceutical preparations containing HMC and / or HMC-EV can be used to treat brain injury, for example, stroke or optic neuropathy. Specifically, the present invention provides a method for treating or preventing brain injury described herein, the method comprising administering an effective amount of HMC and / or HMC-EV, wherein HMC is obtained by in vitro differentiation of pluripotent stem cells. In another embodiment, HMC is obtained by in vitro differentiation of hemangioblasts.

[0304] 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.

[0305] The HMC and / or HMC-EV of the present invention may be administered systemically or locally. HMC and / or HMC-EV may be administered using methods known in the art, including, but not limited to, injection by intravenous, intracranial, intrathecal, intracerebral, intracisternal, intramuscular, intraperitoneal, intravitreal, or other routes of administration, or local implantation, depending on the specific pathology to be treated.

[0306] 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 produced using a material selected from the following: biocompatible fibers, biocompatible yarns, biocompatible foams, aliphatic polyesters, poly(amino acids), copoly(ether-esters), oxalic acid alkylene glycol esters, polyamides, tyrosine-derived polycarbonates, poly(imino carbonates), polyorthoesters, polyoxyesters, polyamide esters, polyoxyesters containing amine groups, poly(anhydrides), polyphosphazenes, biopolymers; homopolymers and copolymers of lactide, glycolide, ε-caprolactone, p-dioxanone, trimethylene carbonate; homopolymers and copolymers of lactide, glycolide, ε-caprolactone, p-dioxanone, trimethylene carbonate; Oxanone, homopolymers and copolymers of trimethylene carbonate, fibrillar collagen, non-fibrillar collagen, collagen not treated with pepsin, 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, cleavage protein-C, laminin, anti-rejection agents, analgesics, antioxidants, anti-apoptotic agents, anti-inflammatory agents and cell growth inhibitors. In some embodiments, HMC and / or HMC-EV are delivered by a sustained release device, for example, a transdermal microneedle patch.

[0307] The specific treatment regimen, route of administration, and adjunctive therapy can be tailored to the specific pathology, severity of the pathology, and the overall health of the patient. Administration of HMC and / or HMC-EV can effectively reduce the severity of pathological manifestations and / or prevent further degradation of pathological manifestations.

[0308] In some embodiments, the administration of HMC leads to the maintenance of myelin. In some embodiments, the administration of HMC leads to the inhibition of neuroinflammatory reactions in subjects. In some embodiments, the administration of HMC leads to the reduction of microglia and astrocyte activation in the brain. In some embodiments, the administration of HMC leads to the stimulation and / or activation of pathways involved in cell survival. In some embodiments, the administration of HMC leads to the 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 secretory phosphoprotein 1 (SPP1). In some embodiments, the administration of HMC leads to the stimulation and / or activation of pathways involved in synaptic transmission in the brain. In some embodiments, the administration of HMC leads to a decrease in apoptosis. In some embodiments, the administration of HMC leads to the stimulation and / or activation of pathways involving neuronal lineage development, such as axon guidance, BREB signal transduction in neurons, or synaptic signal transduction.

[0309] In some embodiments, the administration of HMC-EVs results in an increase in oligodendrocytes and precursor cells in the brain. In some embodiments, the administration of HMC-EVs results in the maintenance of myelin in the brain. In some embodiments, the administration of HMC-EVs results in the inhibition of neuroinflammatory responses in subjects. In some embodiments, the administration of HMC-EVs results in a decrease in the activation of microglia and astrocytes in the brain. In some embodiments, the administration of HMC-EVs results in the prevention or reduction of oxidative damage in neurons. In some embodiments, the administration of extracellular HMC-EVs results in the prevention or reduction of neuronal death caused by glutamate excitotoxic damage.

[0310] The treatment methods of the present invention may include the administration of a single dose of HMC and / or HMC-EV. Alternatively, the treatment methods described herein may include a course of treatment in which HMC and / or HMC-EV are administered multiple times over a period of time. An exemplary course of treatment may include treatment once a week, every two weeks, every month, every quarter, every six months, or every year. Alternatively, treatment may be performed in stages, with multiple doses (e.g., daily doses for the first week) being required initially, and fewer and less frequent doses being required subsequently.

[0311] HMC and / or HMC-EV can be administered alone or in combination. In some embodiments, the method comprises administering an effective amount of HMC to the subject. In other embodiments, the method comprises administering an effective amount of HMC-EV to the subject. In another embodiment, the method comprises administering an effective amount of HMC and an effective amount of HMC-EV to the subject.

[0312] HMC and HMC-EV can be administered simultaneously or sequentially. In one embodiment, HMC and HMC-EV can be mixed together before administration to the subject. In another embodiment, the subject receives an effective amount of HMC and then an effective amount of HMC-EV. Alternatively, the subject receives an effective amount of HMC-EV and then an effective amount of HMC.

[0313] In one embodiment, HMC and / or HMC-EV are regularly administered to the patient once or more throughout the patient's life span. In other embodiments of the invention, HMC and / or HMC-EV are administered once a year, once every 6-12 months, once every 3-6 months, once every 1-3 months, or once every 1-4 weeks. Alternatively, more frequent administration may be desirable for certain conditions or disorders. In one embodiment of the invention, HMC and / or HMC-EV are administered once, more than once, regularly throughout the patient's life span, or as required for the specific patient to be treated and the patient's pathology. Similarly, treatment regimens that vary over time are contemplated. For example, more frequent treatments (e.g., daily or weekly treatments) may be required at the beginning. Over time, as the patient's condition improves, a lower frequency of treatment may be required, or even no treatment may be required.

[0314] 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 600 million, about 700 million, about 800 million, about 900 million, about 100 million, about 150 million, about 160 million, about 180 million, about 190 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 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 to a subject. In some embodiments, about 1 billion, about 2 billion, about 3 billion, about 4 billion, or about 5 billion HMCs and / or HMC-EVs or more are administered. In some embodiments, the number of HMCs and / or HMC-EVs is between about 20 million and about 4 billion, between about 40 million and about 1 billion, between about 60 million and about 750 million, between about 80 million and about 400 million, between about 100 million and about 350 million, and between about 175 million and about 250 million.

[0315] The methods described herein may also include the step of monitoring the effectiveness of the treatment or prevention using methods known in the art.

[0316] Example

[0317] The following examples are not intended to limit the invention in any way.

[0318] Example 1 - Generation of HMCs from hemangioblasts

[0319] Angioblasts were generated from the human ESC line MA09 (Klimanskaya et al., Nature 444 (2006) 481-485) derived from a single schizont. First, 10 cm plates were coated with 0.1% gelatin and plated one day before ESCs were added to the plates at approximately 25,000 cells / cm in MEF medium (high glucose DMEM + 10% FCS). 2 Add irradiated MEFs at a concentration of . Then aspirate the MEF culture medium, rinse with PBS, and replace with Reprocell primate culture medium (Reprocell) plus 10ng / mL bFGF. Add the split MA09 cells to a culture dish and feed with fresh culture medium every day. Cultivate MA09 in Reprocell primate culture medium plus 10ng / mL bFGF until about 90% confluence. Then harvest MA09 with 0.05% trypsin / EDTA or Reprocell dissociation buffer (Reprocell). After separating the cells, rinse and collect the cells. Spin the cells at 300×g for 10min. Aspirate the supernatant and resuspend the cell pellet in Stemline II (Sigma) (with penicillin / streptomycin and L-glutamine) plus 50ng / mL VEGF and 50ng / mL BMP4. MA09 ESCs were plated in 2×10 cm ultra-low attachment plates (Corning) in 15 ml 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, resulting in a final bFGF concentration of 20-22.5 ng / ml bFGF, and incubation was continued for another 40-48 hours (i.e., 3.5-4 days in total).

[0320] Cell clumps (embryoid bodies; EBs) were dissociated and plated as single cells in serum-free semisolid embryonic cell-colony growth medium (BGM). Specifically, cell clumps were dissociated with trypsin for 2-5 minutes, or until the clumps began to break up. The cell suspension was pipetted up and down, and then DMEM + 10% FCS was added to inactivate trypsin. Then, the cells were passed through a 40 μm or 70 μm filter to obtain a single cell suspension. Then, the cells were counted and counted at 1-1.5×10 6 The cells / ml were then suspended in Stemline II medium.

[0321] The single cell suspension was mixed with hemangioblast (HB) growth medium (H4536-based medium formulation: basal medium methylcellulose product H4536 (StemCell Technologies) plus penicillin / streptomycin (pen / strp), Excyte growth supplement (Millipore), and cytokines, 50 ng / ml of Flt3-ligand (FL), 50 ng / ml of vascular endothelial growth factor (VEGF), 50 ng / ml of thrombopoietin (TPO), and 20-30 ng / ml of basic fibroblast growth factor (bFGF)) by brief vortexing to obtain approximately 1 × 10 5 The final concentration of cells / ml was obtained and the bubbles were allowed to settle. The cell mixture was then transferred to a 4×10 cm ultra-low adhesion plate using a syringe (30 ml) attached to an 18G needle and incubated at 37°C, 5% CO2 for 8-12 days. HBS will begin to appear within 3 or 4 days and continue to multiply on the plate and can be harvested between days 7-12 of culture. HBs were harvested on day 9 of culture and frozen.

[0322] Frozen HBs were thawed and re-plated onto Matrigel-coated tissue culture plates in MSC medium [α-MEM without nucleosides (Hyclone), 20% defined FBS—heat-inactivated (Hyclone), 1×Glutamax (Gibco), 1×MEM non-essential amino acids (Gibco), and 1×penicillin / streptomycin]. Cells were cultured for approximately 4-5 days before passage, and were repeated up to 3 generations (P3) to generate HMCs. P3 HMCs (“MARP12” cells) were frozen for further use.

[0323] Example 2 - In vivo studies of traumatic brain injury (TBI)

[0324] The HMC obtained according to Example 1 was thawed and cultured in a T225 culture flask at 37°C, 5% CO2 at a density of about 4500 cells / cm 2Culture in the MSC medium described above for about 4 days. To harvest cells for administration, wash the cells with PBS, dissociate from the flask with trypsin, and inactivate the trypsin by adding MSC medium. Collect the cells in a 50 ml conical tube and centrifuge at 300×g for 10 min. Aspirate the supernatant and add 1 ml of GS2 buffer to each tube [for 552.2 ml GS2: 0.9% sodium chloride rinse USP (408.6 mL); 5% glucose / 0.9% sodium chloride, injection USP (33.2 mL) and BSS rinse solution (110.4 mL)], which is described in WO 2017 / 031312 and is incorporated herein by reference in its entirety. Filter the cells through a 100 μm cell strainer and centrifuge at 300×G for 5 min. Aspirate the supernatant and resuspend in GS2. The cells obtained are passage 4 (P4) HMCs.

[0325] 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)). The cells were injected locally or systemically (iv) into the rats by intracerebral (IC) transplantation and sacrificed at early or late time points according to Table 1.

[0326] Table 1.

[0327]

[0328]

[0329] The rats were studied according to the following schedule:

[0330] Early

[0331] Day -1: Pendulum test and Bederson test at baseline

[0332] Day 0: Controlled cortical impact for all groups

[0333] Day 7: All groups were treated topically or intravenously with cells or vehicle; pendulum test and Bederson test were performed on all groups after treatment

[0334] Day 14: Swing test and Bederson test for all groups; sacrifice all groups; H&E staining, CA3 neuron counting, DCX, OX6, IBA-1 staining, IHC for human cells for all groups

[0335] Late stage

[0336] Day -1: Pendulum test and Bederson test at baseline for all groups

[0337] Day 0: Controlled cortical impact for all groups

[0338] Day 7: All groups were treated topically or intravenously with cells or vehicle; pendulum test and Bederson test were performed on all groups after treatment

[0339] Day 14: Pendulum test and Bederson test for all groups

[0340] Day 28: Pendulum test and Bederson test for all groups

[0341] Day 35: Pendulum test and Bederson test for all groups

[0342] Day 42: Pendulum test and Bederson test for all groups

[0343] Day 49: Pendulum test and Bederson test for all groups

[0344] Day 56: Swing test and Bederson test for all groups; sacrifice all groups; H&E staining, CA3 neuron counting, DCX, OX6, IBA-1 staining, IHC of human cells for all groups.

[0345] Behavioral test results

[0346] The CCI in vivo model of TBI resulted in significant behavioral deficits in rats up to 56 days post-injury. Intracerebral (IC) transplantation of HMCs significantly rescued behavioral deficits compared with their respective vehicles, including elevated body swing test (EBST) ( Figure 1 ), forelimb akinesia from day 28 to day 56 after transplantation ( Figure 2 ), and paw grasping from day 14 to day 56 after transplantation ( Figure 3 Compared with their respective vehicles, intravenous (IV) transplantation of HMCs also significantly rescued behavioral deficits, including EBST from day 14 to day 56 after transplantation ( Figure 1 ), forelimb akinesia from day 42 to day 56 after transplantation ( Figure 2 ), and claw grasping at day 28 after transplantation ( Figure 3 ). These findings support the use of HMC in the treatment of TBI.

[0347] Histological results

[0348] The CCI in vivo model caused significant histopathological effects after injury in rats. Compared with their respective vehicles, IV and IC transplantation of HMCs showed neuroprotective effects. For example, H&E staining showed reduced tissue loss compared with vehicle ( Figure 4A-B), Nissl staining demonstrated that HMC administration had a neuroprotective effect by reducing cell death ( Figure 5A -F), and doublecortin (DCX) staining showed a slight increase in neurogenesis after HMC administration after injury ( Fig. 6A -F).

[0349] IV and IC transplantation of HMCs also significantly reduced microglia and macrophage activation compared to their respective vehicles. Fig. 7A -D) and OX6( Fig. 8A -D) Staining confirmed that HMC reduced the presence of microglia and macrophages in the cortex and striatum after injury, respectively.

[0350] Furthermore, IV and IC transplantation of HMCs significantly reduced inflammatory markers in the spleen compared with their respective vehicles. Fig. 9A -B) and TNF-α( Fig. 10A -B) Decreased staining indicates that HMC reduces inflammation following injury.

[0351] IV and IC transplantation of HMCs also resulted in the migration of HMCs across the blood-brain barrier (BBB) ​​to the cortex, striatum, and hippocampus, as shown by HuNu staining ( Fig.11A -F).

[0352] These findings support the use of HMCs in the treatment of TBI.

[0353] Example 3 - In vitro migration assay of HMC

[0354] HMCs were produced from the same frozen hemangioblast bank described in Example 1. Three separate batches of HMCs were produced, frozen at P4, thawed and cultured for 4 days, and the 5th generation (P5) cells were harvested according to the method described in Example 1. MSCs isolated from bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs) were used as controls. Each of HMCs, BM-MSCs and UCB-MSCs was inoculated into two wells of the ibidi plug-in with a defined gap between the two wells and allowed to adhere overnight. The plug-in was removed, leaving a gap of 500 μm. The cells were washed and MSC culture medium (as described in Example 1) was added to the chamber and stimulated with or without 25 ng / mL TNF-α+50 ng / mL IFN-γ. The cells were incubated at 37°C for 6 hours. Then, photos of non-stimulated cells were taken ( Fig. 12A ), and cells that migrated to the center of the gap (middle ~250 μm) were visually counted using the open source image processing program ImageJ (Schneider et al., Nature Methods 9:671-675 (2012)) Fig. 12B ). Fig. 12A As can be seen in Figure 2A, HMC (hESC-MSC) has greater cell migration ability than BM-MSC or UCB-MSC.

[0355] Example 4 - In vitro neurite outgrowth / neuronal migration in the presence of HMC

[0356] Rat primary mixed neuronal cultures were prepared from 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 isolated from the tissue and cultured for 14 days to mature. Although the tissue was from embryonic rat pups, after 14 days of culture, the neurons had shown mature receptors and electrophysiological profiles. Mixed neuronal cultures were used in adaptive migration assays to study neural 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. 2013 Oct 15. 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)).

[0357] On day 0, the mixed neuronal cultures were plated. On day 9, frozen and thawed MARP12 cells were plated in flasks for expansion as described in Example 1. On day 13, MARP12 cells were harvested and plated on transwell inserts in MSC culture medium at a ratio of 10:1 neurons to MARP12 cells. On day 14, two scratches were made per well in the mixed neuronal cultures prepared as described above (Liang et al., Nat. Protoc. 2:329-333 (2007)). The MSC culture medium in the transwell was changed to neuronal culture medium (Neurobasal TM Plus (ThermoFisher); 1× gentamicin; 1× GlutaMAX TM (ThermoFisher); 1×B27 TMPlus (Thermo Fisher)) to remove all traces of serum, and the transfer plate insert containing MARP12 cells was added to the wells containing the mixed neuronal culture. Fig.13 As shown, co-culture with MARP12 (hESC-MSC or HMC) promoted neurite outgrowth and increased migration.

[0358] RNA-Seq data also showed that the presence of co-cultured HMCs and / or HMC-EVs can affect gene expression in neurons. Neurons were isolated from the cerebral cortex of E18 Sprague-Dawley rats and cultured at 1.2 × 10 6 The cells were plated at a density of 10 cells / mL in 6-well BioFlex culture plates (FlexCell Int.) coated with poly-D-lysine (Sigma). Neurons were supplemented with Neurobasal Plus / B27 Plus medium (Gibco) and maintained for 14 days in vitro (DIV) at 37°C in a humidified CO2 incubator. Half of the medium was replaced every 3 days. For HMC treatment, HMCs were cultured in α-MEM medium (α-MEM (Hyclone) with 1× GlutaMAX (Gibco), 1× MEM-NEAA (Gibco), and Pen-Strep (Gibco)) for 4 days and then harvested and plated at 1.2×10 per insert. 5The cells were plated on a transfer plate insert (Corning) at a density of 10 cells / mL. After one day of culture, the α-MEM medium was replaced with Neurobasal Plus / B27 Plus medium for 1 hour, and then the insert was added to a 6-well plate containing DIV14 neurons. For EV treatment, EVs were purified from HMC (HMC-EV) by tangential flow filtration. HMC-EVs were added to the plate containing neurons. Then, TNF-α was added at a concentration of 100 ng / mL where appropriate, and the plate was placed on the FlexCell FX-6000. The culture was subjected to 15%-0% stretch oscillation (15%, 12.5%, 10%, 7.5%, 5%, 2.5% and 0% cycles) overnight. The neurons were then removed from the BioFlex plate, centrifuged, washed with PBS, and RNA was isolated using the RNeasy Mini kit (Qiagen). RNA (300 ng) was then submitted to BGIAmericas for RNAseq analysis, and the data were analyzed by Rosalind software (https: / / rosalind.onramp.bio / ). Cutadapt was used to trim the reads, and FastQC was used to assess the quality scores. The reads were aligned to the Rattus norvegicus genome build rn5 using STAR. HTseq was used to quantify individual sample reads, and they were normalized by relative log expression (RLE) using the DESeq2 R library.

[0359] Example 5 - In vivo neonatal hypoxia-ischemia model of cerebral palsy

[0360] The HMC of the present invention was tested in an in vivo neonatal hypoxia-ischemia (HI) model of cerebral palsy. The HMC used was the MARP12 cells described in Example 1 as 5th generation (P5) cells melted and passaged for 4 days, at which time the cells were harvested, rinsed and prepared for injection. To establish an in vivo model of cerebral palsy, the common carotid artery of male Sprague Dawley rat pups on postnatal day 7 (PND) was ligated to induce ischemia. After recovery, the pups were subjected to a hypoxic episode and then to another 25 minutes of normoxia. The pups in the sham control group received the same exposure, except that normoxia was present instead of hypoxia. On the 7th day after surgery and hypoxic exposure (i.e., PND14), the pups were humanely euthanized, and blood, cerebrospinal fluid (CSF) and brain tissue were harvested for further testing. The pups were treated according to Table 2.

[0361] Table 2. Treatment groups

[0362]

[0363] End points evaluated

[0364] For the inflammation group, CSF and blood were used for ELISA, while for the other groups it depended on the sample amount.

[0365] Brain tissue for the following analyses:

[0366] Cell death-TUNEL;

[0367] Infarct volume-H&E;

[0368] Iba-1-activation of microglia in peri-infarct tissue;

[0369] GFAP-Astrocyte activation in peri-infarct tissue;

[0370] Olig2-oligodendrocyte precursor cells in the hippocampus

[0371] MBP - myelin basic protein in mature oligodendrocytes in the corpus callosum and hippocampus.

[0372] result

[0373] like Fig.14A -TUNEL staining shown in B demonstrates the neuroprotective effect of MARPS12 (Batch B) with reduced cell death. Fig.15 H&E staining shown demonstrates the neuroprotective effect of MARPS12 (Batch B) with reduced lesion size. Fig.16A As shown in Figure 3-C, the anti-inflammatory effect of MARPS12 (Batch B) was demonstrated by the reduction of microglial activation stained by IBA-1. Fig.17A The anti-inflammatory effect of MARPS12 (Batch B) was also demonstrated by a modest reduction in astrocyte activation as measured by GFAP staining (Fig. Fig.18A -C shows the maintenance of myelin in the corpus callosum by MBP staining, indicating the beneficial effects of MARPS12 on oligodendrocytes. Fig.19A -C shows that Olig2 expression was partially rescued by administration of MARPS12.

[0374] These results support the use of HMC in the treatment of cerebral palsy.

[0375] Example 6 - RNAseq analysis of HMC vs BM-MSC vs UCB-MSC

[0376] HMCs were generated from the same frozen hemangioblast bank described in Example 1. Three independent batches of HMCs were generated according to the method described in Example 1 and passaged to passage 5 (P5). RNA seq analysis was performed on 3 batches of HMCs under basal conditions. MSCs isolated from bone marrow (BM-MSCs) (9 batches) and umbilical cord blood (UCB-MSCs) (9 batches) under basal conditions were used as controls.

[0377] 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. HMCs of the present invention can be selected or purified based on any differentially expressed genes.

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[0467] Example 7 - In vivo middle cerebral artery occlusion (MCAO) stroke model

[0468] The HMCs and HMC-EVs of the present invention were tested in an in vivo model of middle cerebral artery occlusion (MCAO) stroke.

[0469] HMCs were generated from the same frozen hemangioblast bank described in Example 1.

[0470] For HMC-EVs, early passage (passage 4) HMCs were thawed, washed, counted, and plated at 5000 cells / cm in RoosterBioRoosterNourish-MSC-XF medium. 2 Cells were plated at a density of 100 μg / mL in Corning CellBIND flasks. Cells were grown for 96 h to approximately 70-90% confluence to accommodate the medium and cell expansion. After 96 h, cells were removed from the flasks by TripLE dissociation, viable cells were counted, and counted at 5,000 cells / cm 2 Re-plating at passage 5 in new culture flasks and fresh medium. At this passage, the medium can be collected after 96 hours for EV isolation. The cells can be passaged again to passage 7 to collect a larger volume of medium. After harvesting the medium for EV isolation, the medium is clarified by differential low-speed centrifugation at 300×g for 10 minutes and 2000×g for 20 minutes, and then by 0.2μm vacuum filtration to remove cells and debris. EVs are separated from clarified medium using tangential flow filtration (TFF) on a Repligen KR2i system equipped with a hollow fiber, 300kDa pore, mPES membrane filter. The filter's pore size of approximately 100nm removes small impurities and retains EVs. The combined clarification and TFF parameters allow the separation of particles between 100nm and 200nm in size. The medium is first concentrated approximately 10-fold and then diafiltered with DPBS to improve sample purity and remove non-EV-associated proteins during the TFF process. The diafiltered medium is further concentrated, resulting in a final product that is concentrated approximately 100-fold. The resulting isolated and concentrated EVs in DPBS are then ready for downstream analysis and can also be further purified using chromatographic techniques.

[0471] In vivo effects of HMC and HMC-EV on motor skills

[0472] As described herein, MCAO animal model is produced. Briefly, male Sprague-Dawley rats (300-400g) were used to perform body swing test to establish baseline performance one day before surgical injury. For each rat, the rat was kept about one inch from the base of its tail. Then, the rat was lifted one inch above the table. The rat was kept on a vertical axis, which was defined as not deviating to the left or right side by more than 10 °. Whenever the rat moved its head from the vertical axis to either side, the swing was recorded. The rat must return to the vertical position to count the next swing. A total of 30 swings were counted. Normal rats usually swing equally to both sides. After focal ischemia, rats tend to swing to the contralateral (left side). After testing one day, a modification of the method of Tamura et al. was used to produce focal cerebral infarction by permanent occlusion of the proximal right middle cerebral artery (MCA). Rats were anesthetized with 1-3% isoflurane in a mixture of N2O:O2 (2:1) and maintained with 1.5-2% isoflurane in a mixture of N2O:O2 (2:1). The temporalis muscle was bisected and reflected by making an incision midway between the eye and the tympanic tube. The proximal MCA was exposed by a subtemporal craniectomy without resecting the zygomatic arch and without transecting the facial nerve. The artery was then occluded by microbipolar coagulation only from the proximal olfactory tract to the inferior cerebral vein. Body temperature was maintained at 37.0±1°C throughout the surgery. Cefazolin (40 mg / kg) was injected intraperitoneally (ip) before MCAO to prevent infection. Buprenorphine (~0.1 mg / kg Simbadol) was administered (sc) as analgesia before MCAO surgery. For sham conditions, animals underwent the same procedure as above, but the middle cerebral artery was not coagulated.

[0473] Treatments were administered on day 1 and day 7 after MCAO surgery (24 hours and 7 days + / - 10%). For HMC treatment, 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-LyteA. For HMC-EV treatment, 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 directly as prepared.

[0474] On days 1 and 7 after MCAO (24 hours and 7 days + / - 10%), animals were anesthetized with 1-3% isoflurane in O2 and anesthesia was maintained with 1.5-2% isoflurane in O2. 0.5 ml of vehicle or cells were injected into the jugular vein by intrajugular injection using a 1 ml syringe connected to a 25G (3 / 4") needle. The injection site was pressed for approximately 1 minute to ensure the absence of bleeding. Local injections were performed using a 50 μL Hamilton syringe connected to a 26G needle, with 10 μL of vehicle, cells, or EVs injected into the peri-infarct area at 3 locations, 3 to 4 μL per site. 40 μL of vehicle, cells, or EVs were injected intrathecally between the last lumbar vertebra and the first sacral vertebra (L6-S1) using a 25G subcutaneous injection needle and an insulin syringe (0.5 mL).

[0475] Body swing test was performed on days 1, 7, 14, 21 and 28 after injury, and animals were sacrificed after testing on day 28 after injury. On day 28 after MCAO (day 28), rats were deeply anesthetized with ketamine / xylazine (91 mg / kg ketamine, 9 mg / kg xylazine, respectively). After the rats were in the deep anesthesia stage, the rats were perfused with saline (containing 2 units / ml of heparin) and then perfused with 4% paraformaldehyde. The brain was removed and stored in 4% paraformaldehyde for 24 hours, then replaced with 1× PBS and stored at 0-4°C. All data are expressed as mean ± SEM. Body swing test data were analyzed by two-way analysis of variance and Tukey's multiple comparison test. Significance is expressed as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0476] The effects of HMC and HMC-EV of the present invention on locomotion were evaluated in the MCAO model.

[0477] HMC cells were injected by 3 routes of administration, including intravenous (IV), intracerebral (IC), and intrathecal (IT). Cells were administered at a dose of 4 million per IV injection in 0.5 mL; 400,000 per IC injection in 10 microliters; and 500,000 or 1 million per IT injection in 40 microliters. Fig. 20 As shown, all treatment groups showed improved recovery from deficits in the body swing test, with IV and IC treatments being the most significant.

[0478] In another study, animals were subjected to MCAO injury as described above. On days 1 and 7 after MCAO surgery (24 hours and 7 days + / - 10%), HMC, specifically HMC derived from C-GS1 cells (C-GS1-HMC) and N-batch QR57 cells (N-HMC), were used for cell treatment. The cell dose was 4 million per IV injection in 0.5 mL. On days 1 and 7 after MCAO surgery (24 hours and 7 days + / - 10%), extracellular vesicle (EV) treatment was administered using EVs derived from N-HMC (N-HMC-EV). For intracerebral and intracisternal administration, the EV dose was 10×10 10 All treatment groups showed significant improvements in the limb placement test ( Fig.21 ). In the body swing test, all treatment groups recovered, with C-GS1-HMC, N-HMC, and N-HMC-EV injected via intracerebral injection showing significant improvements.

[0479] In a separate study, treatment with N-HMC-EVs (N-batches P6 and P7 treated with 50 ng / ml IFNγ for 96 hours) was administered on days 1 and 7 after MCAO surgery (24 hours and 7 days + / - 10%). For N-HMC-EVs (N-batches stimulated) injected via intracisternal injection, the EV dose was a total of 10 × 10 10 or 30×10 10 All groups showed significant improvements in all three behavioral tests, with the most significant improvements being shown in the forelimb placing test and the body swing test ( Fig. 22 ).

[0480] In another study, treatment was administered with HMC-EVs (N-batches) or HE-VPC-EVs on days 1 and 7 after MCAO surgery (24 hours and 7 days + / - 10%). The exosome dose of HMC-EVs administered via intrathecal injection was 10×10 10 ,30×10 10 and 10×10 11 The exosome dose for VPC-EV was 10 × 10 10 HMC-EV depletion injections were performed as negative controls. All groups showed significant improvements in all three behavioral tests, with the most significant improvements being shown in the forelimb placing test and the body swing test ( Fig.23 ).

[0481] Therefore, HMC and HMC-EV 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 provide improved motor recovery in behavioral tests.

[0482] In vivo effects of HMC on histopathological outcomes

[0483] The effect of HMC of the present invention on histopathological outcomes was evaluated. Specifically, animals were subjected to MCAO injury as described above. On the 1st and 7th day after MCAO surgery (24 hours and 7 days + / - 10%), HMC, specifically HMC derived from C-GS1 cells (C-GS1-HMC) and N- batches of QR57 cells (N-HMC) were used for cell treatment. The dosage of cells was 4 million per IV injection in 0.5 mL.

[0484] Sham-treated, vehicle-treated, and cell-treated groups were prepared for histopathological analysis of white matter loss (MBP) and neuroinflammatory markers, such as microglial activation (Iba-1) and astrocyte activation (GFAP).

[0485] Figure 24 shows the maintenance of myelin treated with HMC cells in the striatum. Specifically, for MBP, there are statistically significant differences between the sham treated group and the vehicle group, but in the ipsi portion of the cortex, there are no statistically significant differences between the vehicle group and the treated group. In the contralateral cortex, there are statistically significant differences between the vehicle group and the N-line cell treated group, however, there are no statistically significant differences between the groups in the ipsi, and between the sham treated group and the vehicle group in the contralateral portion of the cortex. For the ipsi and contralateral in the striatum, there are statistically significant differences between the sham treated group and the vehicle group, and only in the ipsi portion of the striatum, there are statistically significant differences between the vehicle group and the two cell treated groups. There are no statistically significant differences between the groups in the contralateral portion of the striatum.

[0486] Figure 25 is presented at HMC and uses after microglia activation reduces.Specifically, for Iba-1, between sham treatment group and vehicle group (corresponding cortical ipsi and contralateral part) and between sham treatment group and cell treatment group (only in cortical ipsi part) there is statistically significant difference.In cortical contralateral part, there is no statistically significant difference between vehicle group and treatment group.For striatal ipsi and contralateral part, there is statistically significant difference between sham treatment group and vehicle group, and only in striatal ipsi part, there is statistically significant difference between vehicle group and C-GS1 cell treatment group.In striatal contralateral part, there is no statistically significant difference between vehicle group and treatment group.

[0487] Figure 26 shows the reduction of astrocyte reactivity after HMC treatment. Specifically, for GFAP, for both ipsi and contralateral parts of the cortex, there were statistically significant differences between the sham and vehicle groups and between the vehicle and cell-treated groups. For both ipsi and contralateral parts of the striatum, there were statistically significant differences between the sham and vehicle groups and between the vehicle and cell-treated groups.

[0488] Therefore, these results demonstrate that the MSCs of the present invention not only improve the maintenance of myelin, and therefore white matter, but also lead to a robust reduction in neuroinflammatory markers by reducing the number of reactive astrocytes and microglia.

[0489] In vivo effects of HMC-EVs on histopathological outcomes

[0490] The effect of HMC-EV on histopathological outcomes was also evaluated. Specifically, animals were subjected to MCAO injury as described above. Treatment was administered with HMC-EV (N-batches p6 and p7 treated with 50 ng / ml IFNγ for 96 hours) on day 1 and day 7 after MCAO surgery (24 hours and 7 days + / - 10%). For HMC-EV (N-batches stimulated), a total of 10×10 EVs were administered via intracisternal injection. 10 or 30×10 10 .

[0491] Sham, vehicle and cell treated groups were prepared for histopathological analysis of MBP, Iba-1, GFAP, Olig-2 and NG2. Figure 27 shows the maintenance of myelin delivered in the cisterna magna of EV. Specifically, MBP IF staining showed stable staining areas in the range of 0.81-0.88 in all treatment groups. The average ratio of the vehicle group was the lowest (0.64). The difference between the vehicle group and all treatment groups was significant.

[0492] Figure 28 shows the effect of HMC-EV treatment on microglial activation. Specifically, Iba-1 IF staining showed that the activation of microglial cells was significantly increased in the vehicle group and in the HMC-EV group. 10 and HMC-EV 30 10 The average ratio (R / L) of the number of positive cells was the same in the treatment groups (~2.5).

[0493] Figure 29 shows the effect of intracisternal HMC-EV delivery on astrocyte reactivity. Specifically, GFAP IF staining did not show any differences between the control group and all treatment groups, and showed a stable average ratio of the number of positively stained cells (R / L).

[0494] Figure 30 shows that intracisternal delivery of HMC-EVs increased oligodendrocytes. Specifically, Olig-2 IF staining showed the highest average ratio of positively stained cells (R / L) in all exosome-treated groups (compared to the vehicle group). Vehicle group vs. HMC-EV 10 10 and HMC-EV 30 10 The difference between them is significant.

[0495] Figure 31 shows that intracisternal delivery of HMC-EVs increased oligodendrocyte precursor cells. Specifically, NG2 IF staining showed that HMC-EVs 10 10 and HMC-EV 30 10 The average ratio of positive staining areas (R / L) was statistically significantly increased.

[0496] Thus, these results demonstrate that HMC-EVs enhance myelin maintenance. Additionally, EV treatment also enhances oligodendrocytes and oligodendrocyte precursors.

[0497] Example 8 - In vitro glucose-oxygen deprivation stroke model

[0498] The neuroprotective effect of the MSCs of the present invention was tested in vitro. The oxygen-glucose deprivation (OGD) assay combines hypoxic conditions and glucose-deprived medium, and is used to model stroke in vitro.

[0499] Determination profile Fig.32 As shown. For primary neuronal culture, embryonic day 18 (E18) rat cortex samples (#SDECX) from Sprague Dawley rats were ordered from Brain Bits, LLC (Springfield, IL). The cortex was washed three times in dissection medium (DM). DM consisted of 50 mL 10× HBSS (w / o Ca and Mg; Gibco 14185-052), 500 μL gentamicin, 5 mL pyruvate (Gibco: 11360070), 5 mL Hepes (Gibco 15630080, 10 mM final concentration), 15 mL glucose (30 mM final concentration, 1 M stock solution), and 425 mL water. After washing, the DM was aspirated and the tissue was cut into pieces of equal size using a scalpel. Prepare DM, papain, and DNase I solutions while washing tissues, measuring 1 mL DM, 40 uL papain (Worthington LS003126), and 2.5 uL DNase I (DNAse (Sigma #DN-25) per brain; activate papain by incubating in a 37°C water bath for 30 minutes; and sterile filter using a 0.22 micron filter. Add DM, activated papain, and DNase I solutions to cortical samples and incubate at 37°C for 30 minutes to dissociate tissues.

[0500] During this period, neuronal culture medium (NM0) was also prepared and incubated at 37°C. NM0 consists of Neurobasal plus culture medium (Neurobasal Plus and B27; Life Tech CorpA3653401) with freshly added 1×B27 Plus, 1×Glutamax (Gibco#35050-061) and gentamicin sulfate (MP Biomedical#0916760-CF). Dissociation pipettes were prepared by fire polishing Pasteur pipettes with successively smaller tip diameters (1=fire polishing only, 2=3 / 4 of the original diameter, 3=1 / 2 of the original diameter). After incubation for 30 minutes, the tissue was removed from the water bath. Gently aspirate DM / papain / DNase I solution and add 5mL preheated NM0 with freshly added B27. The tissue was allowed to settle and NM0 was gently aspirated. The tissue was cleaned again with 5mL fresh NMO (with B27), and the step was repeated for a total of 3 times. After the last wash, NMO was removed. Starting from the largest pipette, the brain tissue was gently crushed by a fire-polished Pasteur pipette to dissociate the tissue. This was carried out by the following steps: 3mL of NMO was added, gently crushed 4-5 times, and the tissue was distributed on the tube wall to prevent bubble formation, because the neurons trapped in the bubbles will die. After the remaining tissue precipitation, the supernatant was removed and added to a fresh 50mL falcon tube. This operation was repeated for all pipette sizes, and then the cell mixture was passed through a 70 micron cell strainer. Cells were counted and diluted to 600,000 cells per milliliter. Cells were plated on tissue culture plates pre-coated with poly-D-lysine (PDL). For 6-well plates, 2mL was added, and 1.2 million cells were totaled per well. For 24-well plates, 0.5mL was added, and 300,000 cells were totaled per well. The cultures were then fed by replacing 1 / 2 of the medium every 3 days to prevent accumulation of metabolic byproducts. After one week, the cells were subjected to the OGD assay.

[0501] 5 days before the end point treatment of neurons, N-batch HMCs were thawed in a 37°C water bath and gently swirled. Once thawed, cells were moved dropwise into pre-warmed MSC culture medium (αMEM without nucleosides (Hyclone, #SH30568.01), 20% defined heat-inactivated FBS (Hyclone, #SH30070.03HI), 1×Glutamax (Gibco#35050-061), 1×MEM NEAA (Gibco#11140-050), 1×Pen / Strep (Gibco#15140-120)). The cells were then centrifuged at 300×g for 5 min, resuspended and counted. 1 million MSCs were plated in a T225 culture flask using 50 mL of MSC culture medium and allowed to continue culturing for 4 days. HMCs were then harvested by first aspirating the culture medium. The culture flask was washed with 10 mL PBS, the PBS was aspirated, 3 mL TripLE 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 culture medium, collected in a 50 mL conical tube, the plate was washed with MSC culture medium to remove the remaining cells, and the cells were centrifuged at 300 × g for 5 minutes. Then, the cells were resuspended in MSC culture medium and counted. Then, HMCs were plated in MSC culture medium in the transfer plate insert to achieve a 1:10 ratio of HMCs to neurons (for 6-well transfer plate inserts, 120,000 HMCs were plated per well, and for 24-well transfer plate inserts, 30,000 HMCs were plated per well). HMCs were allowed to recover for 24 hours, and the MSC culture medium was replaced with NM0 to remove traces of FBS. HMCs were incubated in NM0 culture medium for 24 hours until they were used for recovery in the glucose-oxygen deprivation (OGD) assay.

[0502] For OGD determination, OGD culture medium is used to deprive neurons of glucose.OGD culture medium is by 1mM CaCl2, 5mM KCl, 137mM NaCl, 0.4mM KH2PO4, 0.3mM Na3HPO4, 0.5mM MgCl2, 0.4mM MgSO4, 25mM HEPES, 4mM NaHCO3, 1 × Pen / Strep diluted in 450mL DI water.pH is adjusted to 7.3, and water is added to make final volume 500mL.Then 0.2 μm filter is used to sterile filter the culture medium.One day before starting OGD experiment, OGD culture medium is placed in T75 ventilation culture bottle, and incubated overnight in hypoxia chamber (C-Chamber, with ProOx C21 oxygen CO2 single chamber controller, BioSpherix, Parish, NY), so that oxygen diffuses out of culture medium. On the second day, OGD culture medium was removed from the hypoxic chamber, and neurons were cleaned once with OGD culture medium to remove traces of NMO. OGD culture medium was removed, and the entire culture medium was replaced with OGD culture medium immediately before cells were added to the chamber, i.e., the culture medium of 3hr OGD duration was changed, but the culture medium of 2hr time point was not changed until cells were added to the chamber, etc. This ensured that the recovery time of all conditions was the same. Neurons were incubated in the hypoxic chamber with OGD culture medium for 1, 2 or 3 hours. Once completed, neurons were removed and the entire culture medium was replaced with NMO culture medium (+B27). For undamaged controls, NMO was replaced with OGD culture medium, but neurons were not incubated in the hypoxic chamber. The OGD culture medium in the non-damaged control was replaced with NMO simultaneously with the damaged cells. HMC co-culture conditions were implemented to both undamaged controls and damaged cells. Immediately after replacing OGD culture medium with NMO, a transfer plate plug-in with HMC was added under co-culture conditions. Under normal cell culture conditions, the recovery of OGD damage was continued in an incubator for 24 hours. Neurons were collected for RNA isolation or fixed and subjected to TUNEL staining.

[0503] In vitro OGD assay TUNEL analysis

[0504] Primary neuronal cultures were generated from embryonic day 18 (E18) rat cortical samples derived from Sprague Dawley rats ordered from Brain Bits, LLC (Springfield, IL) as described above. N-batches of cells were used at a 1:10 ratio of HMC to neurons, HMC co-culture conditions were performed using a transplate insert (no direct contact), and were initiated immediately after OGD injury for a total duration of 24 hours.

[0505] In order to evaluate the effect of HMC co-culture to prevent neuronal cell death caused by OGD determination, TUNEL staining, imaging and quantification were implemented. After OGD determination, the transfer plate was removed in the co-culture condition and the neurons were first fixed with 4% paraformaldehyde. In order to fix the cells, NM0 was removed, 4% paraformaldehyde was applied to each well, and incubated at room temperature for 10 minutes. After fixation, the cells were washed 3 times with PBS and permeabilized with 0.02% Triton-X in PBS for 10 minutes at room temperature. Then, the cells were washed 3 times with PBS. A positive control was designated and treated with DNase I (Sigma #4536282001) in DNase I reaction buffer (20mM Tris-HCl, pH 8.4, 2mM MgCl2, 50mM KCl) at room temperature at 37°C for 30 minutes. Then, the positive control was washed 3 times in PBS.

[0506] To achieve TUNEL staining, TUNEL labeling mixture (Sigma # 11767291910) and TUNEL enzyme kit (Sigma # 11767305001) were used according to the manufacturer's protocol with minor changes. Typically, two kits were used per experiment and diluted in PBS to accommodate the larger volume of a 24-well plate. The instructions indicate that the kit was used directly with a volume of 50uL per well, but to ensure coverage of the 24-well plate, the sample was diluted to 150uL per well using PBS. For negative controls, TUNEL labeling reagents without TUNEL enzyme diluted in PBS were used. For all samples, 200uL DAPI staining solution (VWR # 10791-650) was added to the mixed solution. TUNEL labeling reagents with TUNEL enzyme diluent were added to the desired wells and the samples were incubated at 37°C for 1hr. The samples were washed 3 times with PBS. Imaging was performed on a Leica DMi8 microscope and quantified using LeicaLAS X navigation software. For each condition, 3 wells were stained and 9 images were collected and quantified per well, resulting in 27 images analyzed per condition. TUNEL staining and analysis showed a significant increase in cell death with increasing duration of OGD injury.

[0507] 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 rather exerts its effects on target neurons through paracrine effects.

[0508] Thus, in vitro analysis demonstrates that HMCs of the invention can protect against ischemic injury (ie, glucose-oxygen deprivation) in isolated neuronal culture preparations, demonstrating the benefit of direct access to the central nervous system in stroke.

[0509] RNAseq analysis of glucose-oxygen deprived rat neurons

[0510] Primary rat neuronal cultures were subjected to oxygen-glucose deprivation (OGD) for different durations (e.g., 0, 1, 2, and 3 hours of injury duration). After OGD treatment, neurons were then co-cultured with HMCs for 24 hours. RNA samples were collected 24 hours after OGD treatment. RNA-seq analysis was performed to detect transcriptome and pathway enrichment after OGD in vivo injury with or without subsequent HMC co-culture.

[0511] For RNA isolation, neurons were collected by washing with PBS, scraping, and centrifuging at 500g in a microcentrifuge tube for 5 minutes. The PBS was aspirated, and the cell pellet was quickly frozen and placed at -80°C, or immediately processed by 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 / uL, and the total amount of 1ug was submitted to GeneWiz for RNAseq analysis, with the aim of analyzing changes in gene expression in response to OGD injury and HMC co-culture. The conditions were control, control plus HMC, 1hr OGD, 1hr OGD plus HMC, 2hr OGD, 2hr OGD plus HMC, 3hr OGD, and 3hr OGD plus HMC. For each condition, 3 biological replicates were provided.

[0512] Library preparation was performed using the NEB Ultra II RNA library preparation kit followed by Illumina sequencing. For each sample, 20–30 million reads were achieved. Bioinformatics analysis was performed and RNAseq data were analyzed. Reads were trimmed using cutadapt1. Quality scores were evaluated using FastQC2. Reads were aligned to the Rattus norvegicus genome build rn6 using STAR3. Individual sample reads were quantified using HTseq4 and normalized by relative log expression (RLE) using the DESeq2R library5. As part of the QC step, read distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated using RSeQC6. DEseq2 was also used to calculate fold change and p-values, and to perform optional covariate correction. The final heatmap of differentially expressed genes was gene clustered using the PAM (partitioning around the center) method using the fpc R library7. Hypergeometric distribution was used to analyze pathway enrichment, gene ontology, domain structure, and other ontologies. The topGO R library8 was used to determine local similarities and dependencies between GO terms in order to perform Elim pruning correction. Enrichment analysis referenced several database sources, including Interpro, NCBI, MSigDB REACTOME, WikiPathways. Enrichment was calculated relative to a set of background genes related to the experiment. Although multiple gene expression changes were observed, genes involved in neuroprotection were highlighted.

[0513] A therapeutic effect of HMC-enriched culture on OGD neuronal growth was observed for neurons subjected to 3-hour OGD insult. Using the Qiagen Ingenuity pathway analysis framework, differentially expressed pathway enrichment analysis was performed between neurons subjected to 3-hour OGD insult and grown in HMC-enriched medium and control medium. Fig.34A -C, the pathways enriched by this differential expression include (a) STAT3 pathway (p value: 4×10 -11 ), inactivated in HMC-cultured OGD neurons; (b) CREB signaling in neurons (p value: 4.4×10 -8 ) and (c) multiple inflammatory activity pathways (e.g., IL-6 signaling, IL-10 signaling, Th1 / 2 activation pathways) that were downregulated in HMC-cultured OGD neurons.

[0514] For gene ontology terms enriched for differential expression between OGD neurons grown on HMC-enriched medium and control medium ( Fig.34C -F) conversely shows the increase in cell viability of OGD neurons grown on HMC-enriched culture ( Fig.34C ), direct neuroprotective effect ( Fig.34C ,exist Fig.34D Genes involved in neuroprotection up-regulated) and up-regulated pathways involved in synaptic transmission ( Fig.34C ). At the same time, the pathway involved in cell apoptosis ( Fig.34E ,exist Fig.34F The top shows genes that are downregulated by the effect of HMC-enriched growth culture) and are strongly downregulated in general response to cell death. This reflects a relationship between complete differential expression and displacement of molecular markers of OGD damage induced by the presence of HMC-enriched growth medium.

[0515] To verify the increase in expression of these genes, the same RNA samples used for RNAseq analysis were used for qPCR analysis. For qPCR analysis, Taqman probes (ThermoFisher Scientific) were designed and used with Taqman FastAdvanced Master Mix (ThermoFisher Scientific #4444556), and samples were analyzed on a QuantStudioFlex 7RT-PCR system (Applied Biosystems #4485698). Three biological replicates were repeated for each sample, and the analysis demonstrated a similar increase in gene expression in the presence of HMC. Statistical significance was achieved by two-way ANOVA and Sidak multiple comparison test (*p<0.05, **p<0.01, ****p<0.0001).

[0516] As shown in Figure 35, qPCR analysis validated the RNAseq results for genes involved in cell viability and neuroprotection. 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.

[0517] Example 9 - In vitro oxidative damage model

[0518] The HMC-EVs of the present invention were tested in an in vitro oxidative injury model. Briefly, neurons were subjected to H2O2 oxidative injury and treated with HMC-EVs at a dose of about 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 out of the total number of cells.

[0519] As shown in Figure 36, HMC-EV treatment resulted in a dose-dependent attenuation of cell death. Significant rescue of cell death by HMC-EV was observed at 30K and 100K doses. The overall cell death rate was approximately 44% lower than that of the control group without EV treatment.

[0520] Therefore, these results suggest that HMC-EVs can protect against oxidative damage in neurons.

[0521] Example 10 - In vitro glutamate excitotoxicity model

[0522] 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 different concentrations of L-glutamate (about 0, 30, 300, and 3000 μM) and treated with HMC-EVs at a dose of about 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.

[0523] like Fig.37 As shown, HMC-EV treatment maintained cells in the nuclear swelling phase and preserved viability after glutamate-induced injury. Staining with TMRM, a cell-permeable dye that accumulates in active mitochondria with intact membrane potential, showed that HMC-EV treatment also maintained mitochondrial activity in damaged cells.

[0524] Thus, these results suggest that HMC-EVs protect against neuronal death caused by glutamate excitotoxic insults.

[0525] Example 11 - RNAseq analysis of HMC vs bone marrow-MSC vs adipose tissue-MSC

[0526] RNAseq analysis was performed on HMCs of the present invention under basal and stimulated conditions. HMCs were generated from N-line (N-HMC) and GMP-1 (GMP-HMC) cell lines, and 3 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 3 different adult donors, and 2 technical replicates were prepared for each biological replicate. BM-MSCs were also collected from 3 different adult donors.

[0527] HMC vs. Adipose Tissue-Derived MSC

[0528] Principal component analysis of the transcriptomes of HMC (derived from N-cell line) and AD-MSCs showed that HMCs were different from the latter in both basal and interferon-γ stimulated states ( Fig.38 ). The first principal component mainly describes the effect of stimulation with interferon-γ, while the second principal component describes the difference between HMCs and AD-MSCs.

[0529] The weights of the different genes contributing to the second principal component determined the differences between HMC and AD-MSC. It is particularly noteworthy that collagen genes (COL1A1, COL3A1, etc.), mitochondrial function genes, and TGFβ1 (one of the main factors promoting angiogenesis) were downregulated in HMC compared with AD-MSC, indicating a certain degree of immaturity of HMC ( Fig.39 ).

[0530] Hierarchical clustering demonstrated similarities between biological / technical replicates of the same biological type, as well as clear differences between HMCs and AD-MSCs in basal cell states and in cell states stimulated with IFN-γ ( Fig.40 ).

[0531] like Fig.41 As shown, the genes in this cluster were upregulated in HMCs (basal and INFNγ stimulated) compared to AD-MSCs. The 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. According to the hierarchical clustering plot, the differential expression of these genes between HMCs and AD-MSCs was consistent in biological and technical replicates.

[0532] Reactome (https: / / reactome.org / ) was used to Fig.41 Functional annotation of biological pathways enriched in the clusters on . The top ranked pathways enriched by the corresponding genes are related to axon guidance. Other significantly enriched pathways include cellular stress response and developmental biology (related to the relative immaturity of HMCs).

[0533] like Fig.42As shown, genes in this cluster were downregulated in HMCs (basal and INFNγ stimulated) compared to AD-MSCs. The 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. According to the hierarchical clustering plot, the differential expression of these genes between HMCs and AD-MSCs was consistent in biological and technical replicates.

[0534] Reactome (https: / / reactome.org / ) was used to Fig.42 Functional annotation of biological pathways enriched in the clusters above was performed. The top ranked pathways enriched by the corresponding genes were related to respiratory electron transport and mitochondrial function as well as collagen biosynthesis.

[0535] Typical 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, among others. Fig.43 ). These results suggest that HMCs have a different expression profile compared to AD-MSCs and that HMCs can confer neuroprotection and provide neurotrophic factors, factors involved in supporting neuronal health and recovery.

[0536] The list of gene contributors to this differential activation pathway is as follows Figures 44-47 shown.

[0537] Fig.44The top 15 most strongly differentially expressed genes contributing to activation of neuronal CREB signaling in HMCs are shown. The experimental logarithm ratio (Expr log ratio) represents the base 10 logarithm of the fold change between the mean TPM expression of a gene in HMCs and its mean TPM expression in adipose tissue-derived MSCs, i.e., an Expr log ratio above 2 means that the gene expression has increased by more than 100-fold.

[0538] Fig.45 The top 15 most strongly upregulated genes contributing to the enrichment of the axon guidance pathway in HMCs are shown. Although the activation pattern of the axon guidance signaling pathway has not been determined by Qiagen Ingenuity pathway analysis, this pathway is enriched in HMCs compared to AD-MSCs with a p-value of approximately 1.38e-4.

[0539] Fig.46 The top 15 most strongly expressed genes contributing to activation of synaptogenic signaling pathways in HMCs are shown. Enrichment p-value 1.14e-3, activation pattern z-score 3.578, highest among all pathways differentially upregulated in HMCs.

[0540] Fig.47 The top 15 most strongly upregulated genes contributing to activation of neuroinflammatory signaling pathways in HMC are shown. Pathway enrichment p-value 4.97e-3, activation z-score 1.508.

[0541] HMCs were also generated from a different pluripotent stem cell, namely 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 basal and stimulated conditions ( Fig.48 ).

[0542] Hierarchical cluster analysis showed that GMP1-HMC and N-HMC had similar spectra ( Fig.49 ).like Fig.50 As shown, compared with AD-MSC, the genes in this cluster are upregulated in N-HMC and GMP1-HMC (basal and INFNγ stimulated). The genes include: TMSB4X, ACTG1, GSTP1, KRT18, IGFBP5, NPY, KRT8, PRDX6, MDK, DKK3, UCHL1, TUBB3, HN1, PTMA, HSP90AB1, HMGA1, HSPA8, TAGLN2, ANKRD1, PFN1, CYBA and UBB. According to the hierarchical clustering diagram, the differential expression of these genes between N-HMC, GMP1-HMC and adipose tissue-derived MSC lines is consistent in biological and technical replicates.

[0543] Reactome (https: / / reactome.org / ) was used to Fig.50 Functional annotation of biological pathways enriched in the clusters on . The top ranked pathways enriched by the corresponding genes are related to axon guidance. Other significantly enriched pathways include cellular stress response and developmental biology.

[0544] like Fig.51 As shown, the genes in this cluster were downregulated in N-HMC and GMP1-HMC under basal conditions compared to AD-MSC. The genes included: 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. According to the hierarchical clustering diagram, the differential expression of these genes between N-HMC, GMP1-HMC and AD-MSC lines was consistent in biological and technical replicates.

[0545] like Fig.52 As shown, compared with AD-MSC, the genes in this cluster were downregulated in N-HMC and GMP1-HMC under INFNγ-stimulation conditions. 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. According to the hierarchical clustering diagram, the differential expression of these genes between N-HMC, GMP1-HMC and AD-MSC lines was consistent in biological and technical replicates.

[0546] Reactome (https: / / reactome.org / ) was used to Fig.51 and 52 Functional annotation of biological pathways enriched in the clusters above was performed. The top ranked pathways enriched by the corresponding genes were related to general extracellular matrix organization and collagen biosynthesis.

[0547] Similarly, the canonical pathway enrichment of the differential gene expression signatures between N-HMC, GMP1-HMC and AD-MSCs showed 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. ( Fig.53A -C and 54A-C). Thus, N-HMC and GMP1-HMC have similar profiles and both show enrichment in axon guidance.

[0548] Therefore, it is concluded that the HMCs of the present invention are different from AD-MSCs. Specifically, when compared to AD-MSCs, the MSCs of the present invention have a different expression profile and can confer neuroprotection, providing neurotrophic factors, i.e., factors involved in supporting neuronal survival, growth, health and recovery.

[0549] HMC vs. Bone Marrow-Derived MSC

[0550] Principal component analysis of the transcriptome of HMC (derived from N-lineage cells) and BM-MSCs showed that HMCs differed from the latter in the basal state and in the INFN-γ stimulated state. The first principal component mainly described the effect of stimulation with interferon-γ, while the second principal component described the difference between HMCs and BM-MSCs ( Fig.55 ).

[0551] The weights of the different genes contributing to the second principal component determined the differences between HMCs and BM-MSCs. Of particular note, collagen genes (COL1A1, COL1A2, COL3A1, COL6A2, etc.), mitochondrial function genes, and TGFβ1 (one of the main factors promoting angiogenesis) were downregulated in HMCs compared to BM-MSCs, indicating a certain degree of immaturity of HMCs compared to the latter ( Fig.56 ).

[0552] Hierarchical clustering demonstrated similarities between biological replicates of the same type, as well as clear differences between HMCs and BM-MSCs in the basal cell state and in the cell state stimulated with interferon-γ ( Fig.57 ).

[0553] Genes in this cluster were upregulated in HMCs (basal and INFNγ stimulated) compared with BM-MSCs ( Fig.58). The 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. According to the hierarchical clustering diagram, the differential expression of these genes between HMCs and BM-MSCs was consistent in biological and technical replicates.

[0554] Reactome (https: / / reactome.org / ) was used to Fig.58 Functional annotation of biological pathways enriched in the clusters on . Among the top ranked pathways enriched by the corresponding genes, there is axon guidance. Other significantly enriched pathways include cellular stress response and developmental biology (related to the relative immaturity of HMCs).

[0555] Genes in this cluster were downregulated in HMCs (basal and INFNγ stimulated) compared to BM-MSCs ( Fig.59 ). The 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, POSTN. According to the hierarchical clustering diagram, the differential expression of these genes between HMCs and BM-MSCs was consistent in biological and technical replicates.

[0556] Reactome (https: / / reactome.org / ) was used to Fig.59 Functional annotation of biological pathways enriched in the clusters above was performed. The top ranked pathways enriched by the corresponding genes were related to collagen biosynthesis / assembly (demonstrating the similarity between BM-MSCs and AD-MSCs).

[0557] Canonical pathway enrichment of the differential gene expression signature between HMCs and BM-MSCs again showed HMC-specific upregulation of pathways involved in neuronal lineage development, such as CREB signaling in neurons.

[0558] Fig.61 The top 15 most strongly differentially expressed genes contributing to the activation of neuronal CREB signaling in HMCs compared to BM-MSCs are shown. Fig.62Shown are the top 15 most strongly upregulated genes contributing to activation of synaptogenic signaling in HMCs compared to BM-MSCs.

[0559] Therefore, the following conclusions were obtained: the HMCs of the present invention are different from BM-MSCs. Specifically, when compared with BM-MSCs, the HMCs of the present invention have different expression profiles and provide neuroprotective effects.

[0560]

[0561]

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[0595] Example 12. miRNAs of HMC-EV vs BM-MSC-EV vs UCB-MSC-EV vs AD-MSC-EV NanostringnCounter analysis

[0596] HMCs were generated from the same frozen hemangioblast bank described in Example 1. HMCs were generated and passaged up to 6 generations (P6) according to the method described in Example 1. Extracellular vesicles (EVs) were purified from HMCs (HMC-EVs) by tangential flow filtration (TFF). Three batches of HMC-EVs were analyzed for miRNA profiles using the Nanostring nCounter analysis system under basal conditions. EVs isolated from bone marrow (BM-MSC-EVs) (3 batches), umbilical cord blood (UCB-MSC-EVs) (3 batches) and adipose tissue (AD-MSC-EVs) under basal conditions were used as controls.

[0597] Table 9 shows the miRNAs that are more highly expressed in HMC-EVs compared to UCB-MSC-EVs. Table 10 shows the miRNAs that are more highly expressed in UCB-MSC-EVs compared to HMC-EVs. Table 11 shows the miRNAs that are more highly expressed in HMC-EVs compared to BM-MSC-EVs. Table 12 shows the miRNAs that are more highly expressed in BM-MSC-EVs compared to HMC-EVs. Table 13 shows the miRNAs that are more highly expressed in HMC-EVs compared to AD-MSC-EVs. Table 14 shows the miRNAs that are more highly expressed in AD-MSC-EVs compared to HMC-EVs.

[0598] HMC-EVs of the present invention can be selected or purified based on any differentially expressed miRNA.

[0599]

[0600]

[0601]

[0602]

[0603]

[0604] Example 13. Proteome mapping of HMC-EV vs BM-MSC-EV vs UCB-MSC-EV vs AD-MSC-EV system

[0605] HMCs were generated from the same frozen hemangioblast bank described in Example 1. HMCs were generated and passaged up to 6 generations (P6) according to the method described in Example 1. Extracellular vesicles (EVs) were purified from HMCs (HMC-EVs) by tangential flow filtration (TFF). Proteomic profiles of three batches of HMC-EVs were mapped by standard mass spectrometry under basal conditions. EVs isolated from bone marrow (BM-MSC-EVs) (3 batches), umbilical cord blood (UCB-MSC-EVs) (3 batches) and adipose tissue (AD-MSC-EVs) under basal conditions were used as controls.

[0606] The t-test statistical analysis was used to identify proteins with significant abundance differences between EV types. Table 15 shows proteins with higher abundance in HMC-EVs compared to UCB-MSC-EVs. Table 16 shows proteins with higher abundance in UCB-MSC-EVs compared to HMC-EVs. Table 17 shows proteins with higher abundance in HMC-EVs compared to BM-MSC-EVs. Table 18 shows proteins with higher abundance in BM-MSC-EVs compared to HMC-EVs. Table 19 shows proteins with higher abundance in HMC-EVs compared to AD-MSC-EVs. Table 20 shows proteins with higher abundance in AD-MSC-EVs compared to HMC-EVs. The HMC-EVs of the present invention can be selected or purified based on any protein with a difference in abundance.

[0607] The proteomic data were then analyzed to determine how the global protein expression profile could affect different signaling pathways. Fig.63A Pathway enrichment showing differential expression patterns between HMC-EVs and BM-MSC-EVs. Fig.64A Pathway enrichment of differential expression patterns between HMC-EVs and AD-MSC-EVs is shown. Fig.65A Pathway enrichment showing differential expression patterns between HMC-EVs and EVs secreted from umbilical cord blood-derived MSCs (UCB-MSC-EVs). Fig.63A , 64A As shown in Figures 65A and 65A, some pathways were upregulated in HMC-EVs compared with EVs secreted from MSCs of other tissue sources (see orange columns), such as pathways involved in LXR / RXR activation, acute phase response signaling, B cell receptor signaling, and systemic lupus erythematosus in B cell signaling pathways. In addition, proteins contributing to certain pathways were enriched, such as IL-15 signaling, Claritin-mediated endocytosis signaling, and FXR / RXR activation (see white and gray columns).

[0608] Proteins differentially expressed in HMC-EVs and EVs secreted from tissue-derived MSCs were also analyzed for disease or functional annotations. Fig.63B Functional annotations of proteins upregulated in HMC-EVs when compared to BM-MSC-EVs are shown. Fig.63C Functional annotations of proteins downregulated in HMC-EVs when compared to BM-MSC-EVs are shown. Fig.64B Functional annotations of proteins upregulated in HMC-EVs when compared to AD-MSC-EVs are shown. Fig.64C Functional annotations of proteins downregulated in HMC-EVs when compared to AD-MSC-EVs are shown. Fig.65B Functional annotations of proteins upregulated in HMC-EVs when compared to UCB-MSC-EVs are shown. Fig.65C Functional annotations of proteins downregulated in HMC-EVs when compared to UCB-MSC-EVs are shown. Activation z scores greater than 2 or less than -2 were considered as thresholds. The analysis showed that proteins involved in cell viability / survival, cell motility, cell-to-cell signaling and interaction pathways were upregulated in HMC-EVs, while proteins involved in cell death or apoptosis were downregulated in HMC-EVs.

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[0645] Example 14. smRNAseq profiling of HMC cells vs HMC-EVs

[0646] HMCs were generated from the same frozen hemangioblast bank described in Example 1. HMCs were generated and passaged up to 6 passages (P6) according to the method described in Example 1. Extracellular vesicles (EVs) were purified from HMCs (HMC-EVs) by tangential flow filtration (TFF). smRNAseq profiling was performed for HMC-EVs (n=3) and HMCs (n=3).

[0647] Table 21 shows smRNAs with higher abundance in HMC-EVs compared to HMC. Table 22 shows smRNAs with higher abundance in HMCs compared to HMC-EVs.

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[0659] Although the above description and the accompanying drawings show exemplary embodiments of the present disclosure, it will be understood that various additions, changes and substitutions can be made therein without departing from the equivalent spirit and scope of the attached claims. Specifically, it will be clear 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, various changes can be made in the method / process described herein within the scope of the present invention without departing from the principles described herein. Therefore, in all aspects, the embodiments disclosed by the present invention should be considered to be illustrative and not restrictive. The attached claims should be broadly understood to include other variants and embodiments of the present disclosure, which can be made by those skilled in the art without departing from the scope of equivalents.

Claims

1. A method for treating brain damage in a subject suffering from or suspected of suffering from brain damage, the method 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 damage in the subject.

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

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

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

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

7. The method of any one of claims 1 to 3, wherein the method comprises preventing neuronal death due to glutamate excitotoxic injury after administering the HMC-EV to the subject.

8. The method of any one of claims 1 to 3, wherein the method comprises 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, wherein the method comprises 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, wherein the method comprises stimulating a pathway involved in neuronal lineage development 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 according to 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-EV is administered intracisternal.

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

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

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

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

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

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

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

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 than EVs secreted from umbilical cord blood-derived mesenchymal stem cells (UCB-MSC-EVs).

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 than UCB-MSC-EVs.

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 than EVs secreted from bone marrow-derived mesenchymal stem cells (BM-MSC-EVs).

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 than BM-MSC-EVs.

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 than EVs secreted from adipose tissue-derived mesenchymal stem cells (AD-MSC-EVs).

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

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

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

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

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

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

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

34. The method of any one of claims 1 to 33, wherein the HMC-EV expresses at least one miRNA in Table 21 at a higher level than the HMC.

35. The method of any one of claims 1 to 34, wherein the HMC-EV expresses at least one miRNA in Table 22 at a lower level than the HMC.

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 than BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs.

37. The method of any one of claims 1 to 36, wherein the HMC-EVs express at least one protein 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.

38. The method of any one of claims 1 to 37, wherein the HMC-EVs express at least one protein 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.

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 than BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

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 than BM-MSC-EVs, UCB-MSC-EVs, and / or AD-MSC-EVs.

41. The method of any one of claims 1 to 40, wherein about 1×10 6 About 1×10 13 HMC-EV.

42. The method of any one of claims 1 to 41, wherein about 10×10 10 or about 30×10 10 HMC-EV.

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

44. The method of claim 43, wherein 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, which maintains the solution at a physiological osmotic pressure.

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

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

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

48. The method according to 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. A method for treating brain damage in a subject suffering from or suspected of suffering from brain damage, the method 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 damage in the subject.

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

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

53. The method of any one of claims 49 to 51, wherein the method comprises inhibiting a neuroinflammatory response after administering the HMC to the subject.

54. The method of any one of claims 49 to 51, wherein the method comprises reducing microglial and astrocyte activation in the brain after administering the HMC to the subject.

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

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

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 secretory phosphoprotein 1 (SPP1).

58. The method of any one of claims 49 to 51, wherein the method comprises stimulating a pathway involved in synaptic transmission in the brain after administering the HMC to the subject.

59. The method of any one of claims 49 to 51, wherein the method comprises stimulating a pathway involved in neuronal lineage development after administering the HMC to the subject.

60. The method of any one of claims 49 to 51, wherein the method comprises reducing apoptosis after administering the HMC to the subject.

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

62. The method of claim 61, wherein the method comprises reducing tissue loss in the brain after administering the HMC to the subject.

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

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

65. The method of any one of claims 61 to 64, wherein the method comprises reducing the presence of microglia and macrophages in the cortex and striatum after administering the HMC to the subject.

66. The method of any one of claims 61 to 65, wherein the method comprises reducing spleen inflammation after administering the HMC to the subject.

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

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

69. The method of claim 68, wherein the method comprises reducing apoptosis in the brain after administering the HMC to the subject.

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

71. The method of any one of claims 68 to 70, wherein the method comprises reducing microglial and astrocyte activation in the brain after administering the HMC to the subject.

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

73. The method of any one of claims 68 to 72, wherein the method comprises at least partially rescuing Olig2 in the brain after administering the HMC to the subject.

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

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

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

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

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

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

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

81. according to each described method in claim 49 to 80, wherein said pluripotent stem cell is further differentiated into hemangioblast.

82. according to any one of claims 49 to 81 methods, wherein the pluripotent stem cell is an embryonic stem cell.

83. according to the method described in any one in claim 49 to 82, wherein said pluripotent stem cell is an induced pluripotent stem cell.

84. according to the method described in any one in claim 49 to 83, wherein said pluripotent stem cell is a human pluripotent stem cell.

85. The method of any one of claims 49 to 84, wherein the HMCs have been passaged no more than 5 times in vitro prior to administration to the subject.

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

87. The method of any one of claims 49 to 85, wherein HMC express at least one gene in Table 4 at a lower level than BM-MSC.

88. The method of any one of claims 49 to 85, wherein HMC express at least one gene in Table 5 at a higher level than umbilical cord blood-derived MSCs (UCB-MSCs).

89. The method of any one of claims 49 to 85, wherein HMC express at least one gene in Table 6 at a lower level than UCB-MSC.

90. The method of any one of claims 49 to 85, wherein HMC express at least one gene in Table 7 at a higher level than adipose tissue-derived MSCs (AD-MSCs).

91. according to the method described in any one of claims 49 to 85, wherein HMC expresses at least one gene in Table 8 at a lower level than AD-MSC.

92. The method of any one of claims 49 to 85, wherein HMCs express mRNA encoding interleukin-6 (IL-6) at a level lower than 10% of the level of IL-6 mRNA expressed by BM-MSCs at a basal state, and wherein HMCs express mRNA encoding CD24 at a level higher than the level of CD24 mRNA expressed by BM-MSCs at a basal state.

93. The method of any one of claims 49 to 85, wherein HMCs express at a higher level than AD-MSCs 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.

94. The method of any one of claims 49 to 85, wherein HMCs express at a higher level than AD-MSCs 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.

95. The method of any one of claims 49 to 85, wherein HMCs express at a lower level than AD-MSCs at least one gene selected from the group consisting of SERPINE1, ACTA2, TPM2, CTGF, SERPINE2, CRYAB, ELN, MFGE8, ANXA2, POSTN, VIM, MFAP5, ISLR, THBS1, TIMP3, DKK1, COL6A3, COL6A1, TPT1, BCYRN1, COL1A1, SPARC, TPM1, B GN, 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.

96. The method of any one of claims 49 to 85, wherein 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 than AD-MSCs.

97. The method of any one of claims 49 to 85, wherein 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 than AD-MSCs.

98. The method of any one of claims 49 to 85, wherein HMCs express at a higher level than BM-MSCs 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.

99. The method of any one of claims 49 to 85, wherein HMCs express at a lower level than BM-MSCs 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.

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

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

102. The method of any one of claims 49 to 101, wherein about 1×10 6 About 1×10 13 HMCs.

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

104. The method of claim 103, wherein 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, which maintains the solution at a physiological osmotic pressure.

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

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

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

108. 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 than AD-MSCs.

109. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at a higher level than AD-MSCs 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.

110. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at a higher level than BM-MSCs 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.

111. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at a lower level than AD-MSCs at least one gene selected from the group consisting of SERPINE1, ACTA2, TPM2, CTGF, SERPINE2, CRYAB, ELN, MFGE8, ANXA2, POSTN, VIM, MFAP5, ISLR, THBS1, TIMP3, DKK1, COL6A3, COL6A1, TPT1, BCYRN1, COL1A1, SPARC, TP M1, 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.

112. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at a lower level than AD-MSCs 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.

113. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at a lower level than AD-MSCs 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.

114. A composition comprising HMCs obtained by in vitro differentiation of pluripotent stem cells, wherein the HMCs express at a lower level than BM-MSCs 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.

115. The composition of any one of claims 108 to 114, wherein HMC also expresses at least one gene in Table 3 at a higher level than BM-MSC.

116. The composition of any one of claims 108 to 114, wherein HMC also expresses at least one gene in Table 4 at a lower level than BM-MSC.

117. The composition of any one of claims 108 to 114, wherein HMC further expresses at least one gene in Table 5 at a higher level than UCB-MSC.

118. The composition of any one of claims 108 to 114, wherein HMC further expresses at least one gene in Table 6 at a lower level than UCB-MSC.

119. The composition of any one of claims 108 to 114, wherein HMC also expresses at least one gene in Table 7 at a higher level than AD-MSC.

120. The composition of any one of claims 108 to 114, wherein HMC also expresses at least one gene in Table 8 at a level lower than AD-MSC.

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

122. A HMC-EV population according to any one of claims 108 to 114.

123. An EV population according to claim 122, wherein the HMC-EVs express at least one miRNA in Table 9 at a higher level than UCB-MSC-EVs.

124. An EV population according to claim 122 or 123, wherein the HMC-EVs express at least one miRNA in Table 10 at a lower level than UCB-MSC-EVs.

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

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

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

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

129. An EV population according to any one of claims 122 to 128, wherein the HMC-EVs express at least one protein in Table 15 at a higher level than UCB-MSC-EVs.

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

131. An EV population according to any one of claims 122 to 130, wherein the HMC-EVs express at least one protein in Table 17 at a higher level than BM-MSC-EVs.

132. An EV population according to any one of claims 122 to 131, wherein the HMC-EVs express at least one protein in Table 18 at a lower level than BM-MSC-EVs.

133. An EV population according to any one of claims 122 to 132, wherein the HMC-EVs express at least one protein in Table 19 at a higher level than AD-MSC-EVs.

134. An EV population according to any one of claims 122 to 133, wherein the HMC-EVs express at least one protein in Table 20 at a lower level than AD-MSC-EVs.

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

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

137. An EV population according to 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 a higher level than BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs.

138. The EV population of any one of claims 122 to 137, wherein the HMC-EVs express at least one protein 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, H5-17, H6-18, H7-20, H7-34, H8-19, H9-10, H10-11, H10-12, H10-13, H10-14, H10-15, H10-16, H10-17 LA-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.

139. The EV population of any one of claims 122 to 138, wherein the HMC-EVs express at least one protein 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, LA MP1, LAP3, LGALS1, LTBP3, MAPK3, MARCKS, MBTD1, MDH1, MOB1B, MYL12B, MYO1F, MYO3A, NIBAN2, PEBP1, PF4, PGAP1, PLOD1, PPP2R1A, PRSS23, PXDN, RALA, RAP2A, RPS1 3. RPS3, RPSA, S100A11, SLC44A1, SLC44A2, SLTM, SMG1, SPARC, SRSF8, STRADB, STX11, STXBP2, TGM2, TPP1, TPTE2, TRIM5, TRPM2, TUBA8, TUBB3, VCAN, YWHAE and ZFN607.

140. A HMC-EV population according to 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 than BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs.

141. A HMC-EV population according to 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 than BM-MSC-EVs, UCB-MSC-EVs and / or AD-MSC-EVs.

142. A pharmaceutical composition comprising the HMC-EV according to 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 miRNA in Table 9 at a higher level than 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 miRNA in Table 10 at a lower level than 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 miRNA in Table 11 at a higher level than 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 miRNA in Table 12 at a lower level than 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 miRNA in Table 13 at a higher level than 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 miRNA in Table 14 at a lower level than 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 protein in Table 15 at a higher level than 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 protein in Table 16 at a lower level than UCB-MSC-EVs.

151. A HMC-EV population obtained by in vitro differentiation of pluripotent stem cells, wherein the HMC-EVs express at least one protein in Table 17 at a higher level than (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 protein in Table 18 at a lower level than 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 protein in Table 19 at a higher level than 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 protein in Table 20 at a lower level than 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 than 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 at least one protein 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, H5-17, H6-18, H7-20, H8-19, H9-30, H10-11, H11-31, H12-32, H13-33, H14-34, H15-35, H16-37, H17-38, H18-39, H19-40, H20-41, H20-42, H20-43, H20-44, H30-45, H30-47, H18-39, H19-48, H20-49, H30-50, H30-51, H30-52, H30-53, H30-54, H30-55, H30-56, H30-57 LA-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.

157. 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 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, LA MP1, LAP3, LGALS1, LTBP3, MAPK3, MARCKS, MBTD1, MDH1, MOB1B, MYL12B, MYO1F, MYO3A, NIBAN2, PEBP1, PF4, PGAP1, PLOD1, PPP2R1A, PRSS23, PXDN, RALA, RAP2A, RPS1 3. RPS3, RPSA, S100A11, SLC44A1, SLC44A2, 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 than 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 than 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 the HMC-EVs express at least one miRNA in Table 21 at a higher level than the HMCs.

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

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

163. A method of determining neurite outgrowth of a HMC population, the method comprising: (a) Preparation of mixed neuronal cultures from isolated cerebral cortex, (b) Plating the HMC population on a permeable membrane, (c) applying tension to mixed neuronal cultures, (d) covering the strained mixed neuronal culture with the permeable membrane of step (b); and (e) The neurite outgrowth of the mixed neuronal cultures was measured.

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

165. The method of claim 163, wherein the tension is a physical scratch created in the mixed neuronal culture.

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

167. The method of claim 163, wherein the tension is applied at a stretching oscillation of 15% to 0%.

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