# US 2023/0056533 A1 · Radial Glia and Astrocyte Differentiation From Human Pluripotent Stem Cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2023/0056533 A1 - Document ID: US20230056533A1 - Status: Published application - Kind: A1 - Application number: 17/796,044 - Filed: 2021-02-19 - Published: 2023-02-23 - Earliest claimed: 2020-02-21 - Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services - Applicants: The United States o fAmerica, as represented by the Secretary, Dept. of Health and Human Services - Inventors: Ilyas Singec; Vukasin Jovanovic; Anton Simeonov - Web page: https://patentgrep.com/patent/US20230056533A1 ## CPC - C12N 5/0622: {Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells} - C12N 2500/36: Lipids - C12N 2500/38: Vitamins - C12N 2501/13: Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins - C12N 2501/135: Platelet-derived growth factor [PDGF] - C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor - C12N 2501/2306: Interleukin-6 (IL-6) - C12N 2501/235: Leukemia inhibitory factor [LIF] - C12N 2501/237: Oncostatin M [OSM] - C12N 2501/42: Notch; Delta; Jagged; Serrate - C12N 2501/999: Small molecules not provided for elsewhere - C12N 2506/02: from embryonic cells - C12N 2506/45: from artificially induced pluripotent stem cells - C12N 2533/52: Fibronectin; Laminin - C12N 2533/54: Collagen; Gelatin - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Methods for generating multipotent radial glia-like cells and astrocyte-like cells from human pluripotent stem cells are provided along with the related compositions. ## Claims ### Claim 1 A method of producing, in culture, radial glia-like cells, the method comprising: (a) plating vertebrate pluripotent stem cells on a substrate-coated surface of a culture vessel at a density of 1,000-100,000 cells/cm2; (b) incubating the plated vertebrate pluripotent stem cells in a first culture medium; (c) replacing the first culture medium with a second culture medium comprising: (i) an effective amount or concentration of one or more inhibitors of BM P pathway, (ii) an effective amount or concentration of one or more activators of Notch pathway, (iii) one or more cytokines of interleukin-6 family; and (d) culturing the plated vertebrate pluripotent stem cells in the second culture medium; thereby producing radial glia-like cells. ### Claim 2 (depends on 1) The method of claim 1, wherein the substrate comprises vitronectin, laminin 521, Matrigel, and/or Geltrex. ### Claim 3 (depends on 1) The method of claim 1, wherein plating vertebrate pluripotent stem cells, comprises plating at the cell density of 2,000-90,000 cells/cm2; 3,000-80,000 cells/cm2; 4,000-70,000 cells/cm2; 5,000-50,000 cells/cm2, and/or 10,000-30,000 cells/cm2. ### Claim 4 (depends on 1) The method of claim 1, wherein incubating the plated vertebrate pluripotent stem cells in the first culture medium comprises incubating for 12-48 hours. ### Claim 5 (depends on 1) The method of claim 1, wherein culturing the plated vertebrate pluripotent stem cells in the second culture medium comprises culturing for at least 5-20 days. ### Claim 6 (depends on 1) The method of claim 1, wherein the first culture medium is a first defined culture medium, wherein the first defined culture medium is E8, E8 Flex, StemFlex, mTeSR, StemFit, or mouse embryonic fibroblast (MEF)-conditioned medium. ### Claim 7 (depends on 1, 2, 3, 4, 5, 6) The method of any one of claims 1-6, wherein the first culture medium comprises an effective concentration of Chroman 1 or a derivative thereof, an effective concentration of Emricasan or a derivative thereof, an effective concentration of trans-ISRIB, and an effective concentration of polyamines comprising putrescine, spermine, and spermidine. ### Claim 8 (depends on 7) The method of claim 7, wherein the effective concentration of Chroman 1 or the derivative thereof is about 4 nM to about 80 μM, the effective concentration of Emricasan or the derivative thereof is about 100 nM to about 80 μM, the effective concentration of trans-ISRIB is about 50 nM to about 80 μM, and wherein putrescine, spermine, and spermidine is each at a concentration of about 0.5 nM to 1 mM. ### Claim 9 (depends on 1) The method of claim 1, wherein the first culture medium further comprises at least one inhibitor of Rho-associated protein kinase (ROCK). ### Claim 10 (depends on 9) The method of claim 9, wherein the one or more ROCK inhibitors comprise one or more of Chroman 1 or a derivative thereof, Y27632, blebbistatin, or thiazovivin. ### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) The method of any one of claims 1-10, wherein, during the culturing in the second culture medium, the cells being cultured detectably express one or more radial glia cell markers at approximately 4-10 days after start of the culturing in the second culture medium. ### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) The method of any one of claims 1-11, wherein the radial glia-like cells detectably express one or more of Brain Lipid Binding Protein (BLBP), CD133 (Prominin 1), abnormal spindle-like microcephaly-associated protein (ASPM), baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5 or Survivin), FAT Atypical Cadherin 1 (FAT1), Hes family bHLH transcription factor 5 (HES5), SRY-Box Transcription Factor 21 (SOX21), and PAX6 protein. ### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) The method of any one of claims 1-12, wherein, during the culturing in the second culture medium, the cells being cultured detectably express one or more astrocyte markers at approximately 5-20 days after start of the culturing. ### Claim 14 (depends on 13) The method of claim 13, wherein the one or more astrocyte markers comprise S100 Calcium-Binding Protein B (S100B). ### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) The method of any one of claims 1-14, wherein, during the culturing in the second culture medium, cells being cultured detectably express one or more neural stem cell markers at approximately 2-10 days after start of the culturing. ### Claim 16 (depends on 15) The method of claim 15, wherein the one or more neural stem cell markers comprise PAX6. ### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) The method of any one of claims 1-16, wherein the radial glia-like cells are multipotent stem cells capable of differentiating into neuron-like cells, oligodendrocyte-like cells, and/or astrocyte-like cells. ### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) The method of any one of claims 1-17, wherein the vertebrate pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells. ### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) The method of any one of claims 1-18, wherein the vertebrate pluripotent stem cells are human pluripotent stem cells. ### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) The method of any one of claims 1-19, wherein the second culture medium is a second defined culture medium, wherein the second defined culture medium is DMEM-F12, E6, Neurobasal medium, or minimal essential medium (MEM). ### Claim 21 (depends on 20) The method of claim 20, wherein the second defined culture medium comprises N2 supplement and/or B27 supplement without vitamin A. ### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) The method of any one of claims 1-21, wherein the one or more inhibitors of the BMP pathway comprise one or more of LDN-193189, LDN-214117, LDN-212854, DMH2, ML 347, UK 383367, K 02288, Dorsomorphin, Noggin, Chordin, Follistatin, or Gremlin. ### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) The method of any one of claims 1-22, wherein the effective amount or concentration of the one or more inhibitors of the BMP pathway comprise 2 nM-40 μM LDN-193189. ### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) The method of any one of claims 1-23, wherein the second culture medium further comprises an effective amount or concentration of one or more Platelet-Derived Growth Factor protein. ### Claim 25 (depends on 24) The method of claim 24, wherein the one or more Platelet-Derived Growth Factor protein is Platelet-Derived Growth Factor-AA (PDGF-AA), Platelet-Derived Growth Factor-BB (PDGF-BB), or Platelet-Derived Growth Factor-AB (PDGF-AB). ### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) The method of any one of claims 1-25, wherein the effective amount or concentration of the one or more Platelet-Derived Growth Factor protein is about 1 ng/mL-800 ng/mL. ### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) The method of any one of claims 1-26, wherein the effective amount or concentration of the one or more activators of Notch pathway in the second culture medium comprise one or more of Jagged 1 protein, Jagged 2 protein, and Delta-Like protein 1 (DLL1), Delta-Like protein 2 (DLL2), or Delta-Like protein 3 (DLL3). ### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) The method of any one of claims 1-27, wherein the one or more activators of Notch pathway in the second culture medium comprise one or both of 1 ng/mL-800 ng/mL Jagged 1 protein and 1 ng/mL-800 ng/mL Delta-Like protein 1 (DLL1). ### Claim 29 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) The method of any one of claims 1-28, wherein the one or more cytokines of interleukin-6 family in the second culture medium comprise one or more of Oncostatin M protein, Ciliary-Derived Neurotrophic Factor protein (CNTF) and Leukemia-Inhibitory Factor protein (LIF). ### Claim 30 (depends on 29) The method of claim 29, wherein each of the one or more Oncostatin M protein, Ciliary-Derived Neurotrophic Factor protein (CNTF) and Leukemia-Inhibitory Factor protein (LIF) is present in the second culture medium in a concentration of 1 ng/mL-800 ng/mL. ### Claim 31 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) The method of any one of claims 1-30, wherein the culturing in the second culture medium comprises changing the second culture medium approximately every 20-28 hours. ### Claim 32 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) The method of any one of claims 1-31, wherein the culturing in the second culture medium comprises one or more steps of passaging cells being cultured when they become confluent. ### Claim 33 (depends on 32) The method of claim 32, wherein the one or more steps of passaging the cells are performed at 1:3 to 1:5 ratio of confluent cell culture to fresh medium. ### Claim 34 (depends on 32, 33) The method of claim 32 or 33, wherein the culturing in the second culture medium comprises 3-7 of the passaging steps. ### Claim 35 (depends on 1, 2, 3, 4, 5, 6, 7, 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) A method of producing a culture of astrocyte-like cells, comprising performing the method of any one of claims 1-34 and, after the step of generating the radial glia-like cells, culturing the radial glia-like cells for approximately 5-30 days in a third culture medium, an effective amount or concentration of one or more activators of Notch pathway, and an effective amount or concentration of one or more cytokines of Interleukin-6 (IL-6) family, thereby generating the culture of the astrocyte-like cells. ### Claim 36 (depends on 35) The method of claim 35, wherein the third culture medium is a third defined culture medium. ### Claim 37 (depends on 36) The method of claim 36, wherein the third defined culture medium is DMEM-F12, Neurobasal medium, minimal essential medium (MEM), or BrainPhys neuronal medium. ### Claim 38 (depends on 36, 37) The method of claim 36 or 37, wherein the third defined culture medium comprises N2 supplement and/or complete B27 supplement. ### Claim 39 (depends on 37, 38) The method of any one of claims 37-38, wherein the one or more activators of Notch pathway in the third culture medium comprise one or more of Jagged 1 protein, Jagged 2 protein, and Delta-Like protein 1 (DLL1), Delta-Like protein 2 (DLL2), or Delta-Like protein31 (DLL3). ### Claim 40 (depends on 35, 36, 37, 38, 39) The method of any one of claims 35-39, wherein the effective amount or concentration of the one or more activators of Notch pathway in the third culture medium comprise one or both of 1 ng/mL-800 ng/mL Jagged 1 protein and 1 ng/mL-800 ng/mL Delta-Like protein 1 (DLL1). ### Claim 41 (depends on 35, 36, 37, 38, 39, 40) The method of any one of claims 35-40, wherein the one or more cytokines of interleukin-6 family in the third culture medium comprise one or more of Oncostatin M protein, Ciliary-Derived Neurotrophic Factor protein (CNTF) and Leukemia-Inhibitory Factor protein (LIF). ### Claim 42 (depends on 41) The method of claim 41, wherein the effective amount or concentration of each of the one or more Oncostatin M protein, Ciliary-Derived Neurotrophic Factor protein (CNTF) and Leukemia-Inhibitory Factor protein (LIF) is present in the third culture medium in a concentration of 1-800 ng/mL. ### Claim 43 (depends on 35, 36, 37, 38, 39, 40, 41, 42) The method of any one of claims 35-42, wherein the culturing in the third culture medium comprises changing the third culture medium approximately every 24-72 hours. ### Claim 44 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43) The method of any one of claims 35-43, wherein the culturing in the third culture medium comprises one or more steps of passaging cells being cultured when they become confluent. ### Claim 45 (depends on 44) The method of claim 44, wherein the one or more passaging steps are performed at 1:2 ratio of confluent cell culture to fresh medium. ### Claim 46 (depends on 44, 45) The method of claim 44 or 45, wherein the culturing in the third culture medium comprises 1-3 passaging steps. ### Claim 47 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46) The method of any one of claims 35-46, wherein the astrocyte-like cells detectably express one or more of astrocyte markers. ### Claim 48 (depends on 47) The method of claim 47, wherein the one or more astrocyte markers comprise S100 Calcium-Binding Protein B (S100B), Nuclear Factor 1 A-Type Protein (NFIA), Glial Fibrillary Acidic Protein (GFAP) and vimentin (VIM). ### Claim 49 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48) The method of any one of claims 35-48, wherein the astrocyte-like cells exhibit flat and/or star-shaped morphology. ### Claim 50 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49) The method of any one of claims 35-49, wherein during the culturing in a third culture medium detectable neuron-like cells are present at 10% or less of total cells in culture. ### Claim 51 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) The method of any one of claims 35-50, wherein the third culture medium further comprises a chemically defined lipid concentrate at a concentration of approximately 2%, comprising one or more of arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid or fetal bovine serum at a concentration of approximately 2%. ### Claim 52 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51) A method of culturing the astrocyte-like cells, comprising performing the method of any one of claims 35-51, and further culturing the astrocyte-like cells in a fourth culture medium and an effective amount or concentration of one or more cytokines of interleukin-6 family, thereby enhancing maturation of astrocyte-like cells. ### Claim 53 (depends on 52) The method of claim 52, wherein the fourth culture medium is a fourth defined culture medium. ### Claim 54 (depends on 53) The method of claim 53, wherein the fourth defined culture medium is DMEM-F12, E6, Neurobasal medium, or minimal essential medium (MEM). ### Claim 55 (depends on 53, 54) The method of claim 53 or 54, wherein the fourth defined culture medium comprises N2 supplement and/or B27 supplement. ### Claim 56 (depends on 52, 53, 54, 55) The method of any one of claims 52-55, wherein the one or more cytokines of interleukin-6 family comprise one or both of Ciliary-Derived Neurotrophic Factor protein (CNTF) and Leukemia-Inhibitory Factor protein (LIF). ### Claim 57 (depends on 56) The method of claim 56, wherein the effective amount of concentration of each of the one or both of Ciliary-Derived Neurotrophic Factor protein (CNTF) and Leukemia-Inhibitory Factor protein (LIF) is present in a concentration of 1-800 ng/mL. ### Claim 58 (depends on 52) The method of claim 52, wherein the fourth medium optionally is an enriched fourth defined culture medium, comprising an effective amount or concentration of one or more activators of Notch pathway and/or one or more thyroid hormone, phorbol ester, forskolin, neuregulin, and ascorbic acid. ### Claim 59 (depends on 58) The method of claim 58, wherein the thyroid hormone is triiodothyronine and the one or more activators of Notch pathway in the fourth culture medium comprise one or more of Jagged 1 protein and Delta-Like protein 1 (DLL1). ### Claim 60 (depends on 58) The method of claim 58, wherein the one or more activators of Notch pathway is about 1 ng/mL to about 800 ng/mL Jagged 1 protein and 1 ng/mL to about 800 ng/mL Delta-Like protein 1 (DLL1), and the concentration of thyroid hormone is about 1 ng/Ml to about 1000 ng/mL, the concentration of phorbol ester is about 1 nM to about 1000 nM, the concentration of forskoline is about 1 μM to about 200 μM, the concentration of neuregulin is about 1 ng/mL to about 1000 ng/mL, and the concentration of ascorbic acid is about 1 μM to about 1000 μM. ### Claim 61 (depends on 52, 53, 54, 55, 56, 57, 58, 59, 60) The method of any one of claims 52-60, wherein the culturing in the fourth culture medium is performed for at least approximately 40-60 hours. ### Claim 62 (depends on 52, 53, 54, 55, 56, 57, 58, 59, 60, 61) The method of any one of claims 52-61, wherein the culturing in the fourth culture medium comprises changing the fourth culture medium approximately every 24-96 hours. ### Claim 63 (depends on 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62) The method of any one of claims 52-62, wherein during the culturing in the fourth culture medium the astrocyte-like cells detectably express one or more of Hepatic and Glial Cell Adhesion Molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, and vimentin (VIM). ### Claim 64 (depends on 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63) The method of any one of claims 52-63, wherein during the culturing in the fourth culture medium the astrocyte-like cells exhibit star-shaped morphology and/or sphere morphology. ### Claim 65 (depends on 1, 2, 3, 4, 5, 6, 7, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64) The method of any one of claims 1-64, wherein one or more steps of the method are performed by an automated system. ### Claim 66 (depends on 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65) The method of any one of claims 52-65, wherein the fourth culture medium further comprises a chemically defined lipid concentrate at a concentration of approximately 2%, comprising one or more of arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid or fetal bovine serum at a concentration of approximately 2%. ### Claim 67 A composition, comprising at least one cultured radial glia-like cell detectably expressing at least one marker, wherein the at least one marker is Brain Lipid Binding Protein (BLBP), CD133 (Prominin 1), abnormal spindle-like microcephaly-associated protein (ASPM), baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5 or Survivin), FAT Atypical Cadherin 1 (FAT1), Hes family bHLH transcription factor 5 (HES5), SRY-Box Transcription Factor 21 (SOX21), or PAX6 protein. ### Claim 68 (depends on 67) The composition of claim 67, wherein the at least one cultured radial glia-like cell is or was cryopreserved. ### Claim 69 (depends on 68) The composition of claim 68, wherein the at least one cultured radial glia-like cell is or was cryopreserved in a cryopreservation medium comprising Chroman 1 and/or a derivative thereof, Emricasan and/or the derivative thereof, trans-ISRIB and polyamines comprising putrescine, spermine and spermidine. ### Claim 70 (depends on 69) The composition of claim 69, wherein, in the cryopreservation medium, Chroman 1 and/or the derivative thereof is or was at a concentration of about 4 nM to about 80 μM, wherein Emricasan and/or the derivative thereof is or was at a concentration of about 100 nM to about 80 μM, wherein trans-ISRIB is or was at a concentration of about 50 nM to about 80 μM, and wherein each of putrescine, spermine and spermidine is or was at a concentration of about 0.5 μM to 1 mM. ### Claim 71 (depends on 1, 2, 3, 4, 5, 6, 7, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66) A composition, comprising at least one cultured radial glia-like cell produced by the method of any one of claims 1-66 and expressing at least one marker, wherein the at least one marker is Brain Lipid Binding Protein (BLBP), CD133 (Prominin 1), abnormal spindle-like microcephaly-associated protein (ASPM), baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5 or Survivin), FAT Atypical Cadherin 1 (FAT1), Hes family bHLH transcription factor 5 (HESS), SRY-Box Transcription Factor 21 (SOX21), or PAX6 protein. ### Claim 72 A cell culture, comprising at least one cultured radial glia-like cell detectably expressing at least one marker, wherein the at least one marker is Brain Lipid Binding Protein (BLBP), CD133 (Prominin 1), abnormal spindle-like microcephaly-associated protein (ASPM), baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5 or Survivin), FAT Atypical Cadherin 1 (FAT1), Hes family bHLH transcription factor 5 (HESS), SRY-Box Transcription Factor 21 (SOX21), or PAX6 protein. ### Claim 73 (depends on 72) The cell culture of claim 72, wherein the cell culture is grown from previously cryopreserved cells. ### Claim 74 (depends on 73) The cell culture of claim 73, wherein the previously cryopreserved cells were cryopreserved in a cryopreservation medium comprising Chroman 1 and/or a derivative thereof, Emricasan and/or the derivative thereof, trans-ISRIB and polyamines comprising putrescine, spermine and spermidine. ### Claim 75 (depends on 73, 74) The cell culture of claim 73 or 74, wherein the previously cryopreserved cells are vertebrate pluripotent stem cells. ### Claim 76 (depends on 75) The cell culture of claim 75, wherein the vertebrate pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells. ### Claim 77 (depends on 75, 76) The cell culture of claim 75 or 76, wherein the vertebrate pluripotent stem cells are human pluripotent stem cells. ### Claim 78 (depends on 73, 74) The cell culture of claim 73 or 74, wherein the previously cryopreserved cells are cultured radial glia-like cells detectably expressing Brain Lipid Binding Protein (BLBP), Brain Lipid Binding Protein (BLBP), CD133 (Prominin 1), abnormal spindle-like microcephaly-associated protein (ASPM), baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5 or Survivin), FAT Atypical Cadherin 1 (FAT1), Hes family bHLH transcription factor 5 (HESS), SRY-Box Transcription Factor 21 (SOX21), and PAX6 protein. ### Claim 79 (depends on 1, 2, 3, 4, 5, 6, 7, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66) A cell culture, comprising at least one cultured radial glia-like cell produced by the method of any one of claims 1-66 and expressing at least one marker, wherein the at least one marker is Brain Lipid Binding Protein (BLBP), CD133 (Prominin 1), abnormal spindle-like microcephaly-associated protein (ASPM), baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5 or Survivin), FAT Atypical Cadherin 1 (FAT1), Hes family bHLH transcription factor 5 (HES5), SRY-Box Transcription Factor 21 (SOX21), or PAX6 protein. ### Claim 80 A composition, comprising at least one cultured astrocyte-like cell exhibiting flat, star-shaped, and/or sphere morphology and detectably expressing at least one marker, wherein the at least one marker is S100 Calcium-Binding Protein B (S100B), Nuclear Factor 1 A-Type Protein (NFIA), Hepatic and Glial Cell Adhesion Molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM). ### Claim 81 (depends on 80) The composition of claim 80, wherein the at least one cultured astrocyte-like cell is or was cryopreserved in a cryopreservation medium comprising Chroman 1 and/or a derivative thereof, Emricasan and/or the derivative thereof, trans-ISRIB and polyamines comprising putrescine, spermine and spermidine. ### Claim 82 (depends on 81) The composition of claim 81, wherein, in the cryopreservation medium, Chroman 1 and/or the derivative thereof is or was at a concentration of about 4 nM to about 80 μM, wherein Emricasan and/or the derivative thereof is or was at a concentration of about 100 nM to about 80 μM, wherein trans-ISRIB is or was at a concentration of about 50 nM to about 80 μM, and wherein each of putrescine, spermine and spermidine is or was at a concentration of about 0.5 μM to 1 mM. ### Claim 83 (depends on 1, 2, 3, 4, 5, 6, 7, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66) A composition, comprising at least one cultured astrocyte-like cell exhibiting flat, star-shaped, and/or sphere morphology produced by the method of any one of claims 1-66 and expressing at least one marker, wherein the at least one marker is S100 Calcium-Binding Protein B (S100B), Nuclear Factor 1 A-Type Protein (NFIA), Hepatic and Glial Cell Adhesion Molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM). ### Claim 84 A cell culture, comprising at least one cultured astrocyte-like cell exhibiting flat, star-shaped, and/or sphere morphology and detectably expressing at least one marker, wherein the at least one marker is S100 Calcium-Binding Protein B (S100B), Nuclear Factor 1 A-Type Protein (NFIA), Hepatic and Glial Cell Adhesion Molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM). ### Claim 85 (depends on 84) The cell culture of claim 84, wherein detectable neuron-like cells are present at 10% or less of total cells in culture. ### Claim 86 (depends on 84, 85) The cell culture of claim 84 or 85, wherein the cell culture is grown from previously cryopreserved cells. ### Claim 87 (depends on 86) The cell culture of claim 86, wherein the previously cryopreserved cells were cryopreserved in a cryopreservation medium comprising Chroman 1 and/or a derivative thereof, Emricasan and/or the derivative thereof, trans-ISRIB and polyamines comprising putrescine, spermine and spermidine. ### Claim 88 (depends on 86, 87) The cell culture of claim 86 or 87, wherein the previously cryopreserved cells are vertebrate pluripotent stem cells. ### Claim 89 (depends on 88) The cell culture of claim 88, wherein the vertebrate pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells. ### Claim 90 (depends on 88, 89) The cell culture of claim 88 or 89, wherein the vertebrate pluripotent stem cells are human pluripotent stem cells. ### Claim 91 (depends on 86, 87) The cell culture of claim 86 or 87, wherein the previously cryopreserved cells are cultured radial glia-like cells detectably expressing Brain Lipid Binding Protein (BLBP), CD133 (Prominin 1), abnormal spindle-like microcephaly-associated protein (ASPM), baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5 or Survivin), FAT Atypical Cadherin 1 (FAT1), Hes family bHLH transcription factor 5 (HESS), SRY-Box Transcription Factor 21 (SOX21), and PAX6 protein. ### Claim 92 (depends on 86, 87) The cell culture of claim 86 or 87, wherein the previously cryopreserved cells are astrocyte-like cell exhibiting flat, star-shaped, and/or sphere morphology and detectably expressing one or more of S100 Calcium-Binding Protein B (S100B), Nuclear Factor 1 A-Type Protein (NFIA), CD44, HEPACAM, Glial Fibrillary Acidic Protein (GFAP), and vimentin (VIM). ### Claim 93 (depends on 1, 2, 3, 4, 5, 6, 7, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66) A cell culture, comprising at least one cultured astrocyte-like cell exhibiting flat, star-shaped, and/or sphere morphology produced by the method of any one of claims 1-66 and detectably expressing at least one marker, wherein the at least one marker is S100 Calcium-Binding Protein B (S100B), Nuclear Factor 1 A-Type Protein (NFIA), Hepatic and Glial Cell Adhesion Molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM).