Differentiation of radial glia and astrocytes from human pluripotent stem cells

Abstract

To provide a composition useful for culturing and differentiating pluripotent stem cells.SOLUTION: A composition comprising cultured radial glial-like cells expressing at least one marker of Brain Lipid-Binding Proteins (BLBP), CD133 (Prominin 1), Abnormal Spindle Microcephaly Associated Proteins (ASPM), Baculovirus Inhibitors of Apoptosis Repeat Containing 5 (BIRC5 or Survivin), FAT atypical cadherin 1 (FAT1), Hes family bHLH transcription factors 5 (HES5), SRY-Box transcription factors 21 (SOX21), or PAX6 proteins, A composition wherein the radial-glial-like cells are or have been cryopreserved in a cryopreservation medium comprising Chroman1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB and a polyamine comprising putrescine, spermine and spermidine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Government Licensing Rights This invention was made with government support awarded by the National Institutes of Health (NIH) Regenerative Medicine Program (NIH Common Fund) and the National Center for Advancing Translational Sciences (NCATS). The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 979,429, filed February 21, 2020, which is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION The present disclosure relates to the fields of biochemistry, cell biology, bioengineering, drug discovery, and stem cell biology, and related fields, and relates to compositions and methods useful for the culture and differentiation of pluripotent stem cells. [Background technology]

[0004] Pluripotency is a remarkable cellular state that allows stem cells to differentiate into any cell type in the human body. Vertebrate pluripotent stem cells (including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)) undergo extensive self-renewal and have the potential to differentiate into all somatic cell types. Generating desired cells from pluripotent stem cells holds enormous potential for drug discovery, disease modeling, and regenerative medicine. For example, the development of novel therapeutic agents for human use and neuroscience research would benefit greatly from the induction of differentiation of human pluripotent stem cells (hPSCs) into relevant cells of the nervous system (e.g., astrocytes). Unfortunately, existing procedures for producing astrocytes from vertebrate pluripotent stem cells can be inefficient, undefined, and time-consuming. Furthermore, they exhibit low reproducibility, require expensive supplements, and often generate a chaotic mixture of different cell lineages. Therefore, improved methods for generating cells exhibiting at least some characteristics of astrocytic cells from vertebrate pluripotent stem cells are needed. Summary of the Invention

[0005] Against the above background, the present invention offers certain advantages over the prior art.

[0006] Described and included in embodiments of the present invention are methods useful for producing and maintaining in culture differentiated vertebrate cells that exhibit at least some characteristics of radial glia-like cells of the vertebrate central nervous system. Among other characteristics, the radial glia-like cells produced by the methods described herein have the ability to differentiate into one or more cell types that exhibit characteristics of cells found in the vertebrate nervous system (e.g., neurons, oligodendrocytes, and / or astrocytes), as described and included in embodiments of the present invention. Also described and included in embodiments of the present invention are methods useful for producing and maintaining in culture vertebrate cells that exhibit at least some characteristics of astrocytes. Among other things, the methods described herein are highly efficient, cost-effective, reproducible, scalable, and amenable to automation. For example, some embodiments of the methods described herein can be performed using automated culture systems. The methods described herein are useful, for example, in drug discovery and development, and neuroscience research (including, but not limited to, high-throughput screening of compounds for various applications (including drug development and toxicity screening)), disease modeling and research, as well as regenerative medicine (e.g., cell replacement and repair of damaged central nervous systems), and cell and tissue engineering, among others. Advantages of the compositions, kits, and methods of the present invention are discussed throughout this document and illustrated in the accompanying figures.

[0007] The inventions disclosed herein may have particular advantages or functionality (e.g., methods for producing radial glia-like cells in culture, methods for producing cultures of astrocyte-like cells, methods for culturing astrocyte-like cells, compositions useful for the culture and differentiation of pluripotent stem cells, cell cultures useful for the culture and differentiation of pluripotent stem cells, compositions comprising at least one cultured radial glia-like cell that detectably expresses at least one marker disclosed herein, compositions comprising at least one cultured radial glia-like cell produced by a method disclosed herein, cell cultures comprising at least one cultured radial glia-like cell that detectably expresses at least one marker disclosed herein, cell cultures comprising at least one cultured radial glia-like cell produced by a method disclosed herein and that expresses at least one marker disclosed herein,

[0013] The present invention provides, but is not limited to, a composition comprising at least one cultured astrocyte-like cell exhibiting a flat, stellate, and / or spherical morphology that detectably expresses at least one disclosed marker; a composition comprising at least one cultured astrocyte-like cell exhibiting a flat, stellate, and / or spherical morphology produced by the methods disclosed herein; a cell culture comprising at least one cultured astrocyte-like cell exhibiting a flat, stellate, and / or spherical morphology and detectably expressing at least one disclosed marker; and a cell culture comprising at least one cultured astrocyte-like cell exhibiting a flat, stellate, and / or spherical morphology produced by the methods disclosed herein and expressing at least one marker disclosed herein.

[0014] The present invention provides methods for producing radial glia-like cells in culture, the methods comprising: (a) Vertebrate pluripotent stem cells are cultured on the substrate-coated surface of a culture vessel at a density of 1,000 to 100,000 cells / cm. 2 and plating at a density of (b) incubating the plated vertebrate pluripotent stem cells in a first culture medium; (c) replacing the first culture medium with a second culture medium, the second culture medium comprising: (i) an effective amount or concentration of one or more BMP pathway inhibitors; (ii) an effective amount or concentration of one or more Notch pathway activators; (iii) one or more cytokines of the interleukin-6 family; (d) culturing the plated vertebrate pluripotent stem cells in the second culture medium; This produces radial glia-like cells.

[0008] In one embodiment of the methods disclosed herein, the substrate comprises vitronectin, laminin 521, Matrigel, and / or Geltrex.

[0009] In one embodiment of the methods disclosed herein, plating the vertebrate pluripotent stem cells is performed at a density of 2,000 to 90,000 cells / cm. 2 , 3,000~80,000 cells / cm 2 , 4,000~70,000 cells / cm 2 , 5,000~50,000 cells / cm 2 , and / or 10,000 to 30,000 cells / cm 2 This involves plating at a cell density of

[0010] In one embodiment of the methods disclosed herein, incubating the plated vertebrate pluripotent stem cells in the first culture medium comprises incubating for 12 to 48 hours.

[0011] In one embodiment of the methods disclosed herein, culturing the plated vertebrate pluripotent stem cells in the second culture medium comprises culturing for at least 5 to 20 days.

[0012] In one embodiment of the methods disclosed herein, the first culture medium is a first defined culture medium, and the first defined culture medium is E8, E8 Flex, StemFlex, mTeSR, StemFit, or mouse embryonic fibroblast (MEF) conditioned medium.

[0013] In one embodiment of the methods disclosed herein, 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 effective concentrations of polyamines including putrescine, spermine, and spermidine.

[0014] In one embodiment of the methods disclosed herein, the effective concentration of Chroman 1 or a derivative thereof is about 4 nM to about 80 μM, the effective concentration of Emricasan or a 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 the effective concentrations of putrescine, spermine, and spermidine are each at a concentration of about 0.5 nM to 1 mM.

[0015] In one embodiment of the methods disclosed herein, the first culture medium further comprises at least one inhibitor of Rho-associated protein kinase (ROCK).

[0016] In one embodiment of the methods disclosed herein, the one or more ROCK inhibitors comprise one or more of Chroman 1 or a derivative thereof, Y27632, blebbistatin, or thiazovivin.

[0017] In one embodiment of the methods disclosed herein, during culture in the second culture medium, the cultured cells detectably express one or more radial glial cell markers approximately 4 to 10 days after initiation of culture in the second culture medium.

[0018] In one embodiment of the methods disclosed herein, 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 proteins.

[0019] In one embodiment of the methods disclosed herein, during culture in the second culture medium, the cultured cells detectably express one or more astrocyte markers approximately 5 to 20 days after initiation of culture.

[0020] In one embodiment of the methods disclosed herein, the one or more astrocyte markers comprises S100 calcium binding protein B (S100B).

[0021] In one embodiment of the methods disclosed herein, during culture in the second culture medium, the cultured cells detectably express one or more neural stem cell markers approximately 2 to 10 days after initiation of culture.

[0022] In one embodiment of the methods disclosed herein, the one or more neural stem cell markers include PAX6.

[0023] In one embodiment of the methods disclosed herein, the radial glia-like cells are multipotent stem cells that can differentiate into neuron-like cells, oligodendrocyte-like cells, and / or astrocyte-like cells.

[0024] In one embodiment of the methods disclosed herein, the vertebrate pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells.

[0025] In one embodiment of the methods disclosed herein, the vertebrate pluripotent stem cells are human pluripotent stem cells.

[0026] In one embodiment of the methods disclosed herein, the second culture medium is a second defined culture medium, and the second defined culture medium is DMEM-F12, E6, Neurobasal medium, or Minimum Essential Medium (MEM).

[0027] In one embodiment of the methods disclosed herein, the second defined culture medium comprises a vitamin A-free N2 supplement and / or a B27 supplement.

[0028] In one embodiment of the methods disclosed herein, the one or more bone morphogenetic protein (BMP) pathway inhibitors include one or more of LDN-193189, LDN-214117, LDN-212854, DMH2, ML347, UK383367, K02288, dorsomorphin, noggin, chordin, follistatin, or gremlin.

[0029] In one embodiment of the methods disclosed herein, the effective amount or concentration of one or more BMP pathway inhibitors comprises 2 nM to 40 μM LDN-193189.

[0030] In one embodiment of the methods disclosed herein, the second culture medium further comprises an effective amount or concentration of one or more platelet-derived growth factor proteins.

[0031] In one embodiment of the methods disclosed herein, the one or more platelet-derived growth factor proteins are platelet-derived growth factor-AA (PDGF-AA), platelet-derived growth factor-BB (PDGF-BB), or platelet-derived growth factor-AB (PDGF-AB).

[0032] In one embodiment of the methods disclosed herein, the effective amount or concentration of the one or more platelet-derived growth factor proteins is between about 1 ng / mL and 800 ng / mL.

[0033] In one embodiment of the methods disclosed herein, the effective amount or concentration of one or more Notch pathway activators in the second culture medium comprises 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).

[0034] In one embodiment of the methods disclosed herein, the one or more Notch pathway activators in the second culture medium comprise one or both of 1 ng / mL to 800 ng / mL of Jagged 1 protein and 1 ng / mL to 800 ng / mL of Delta-Like protein 1 (DLL1).

[0035] In one embodiment of the methods disclosed herein, the one or more interleukin-6 family cytokines 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).

[0036] In one embodiment of the methods disclosed herein, each of one or more of oncostatin M protein, ciliary-derived neurotrophic factor protein (CNTF), and leukemia inhibitory factor protein (LIF) is present in the second culture medium at a concentration of 1 ng / mL to 800 ng / mL.

[0037] In one embodiment of the methods disclosed herein, culturing in the second culture medium comprises changing the second culture medium approximately every 20 to 28 hours.

[0038] In one embodiment of the methods disclosed herein, culturing in the second culture medium includes one or more steps of passaging the cultured cells when they become confluent.

[0039] In one embodiment of the methods disclosed herein, one or more steps of passaging the cells are performed at a ratio of 1:3 to 1:5 of confluent cell culture:fresh medium.

[0040] In one embodiment of the method disclosed herein, the culturing in the second culture medium comprises 3 to 7 passage steps.

[0041] The present invention also provides a method for producing a culture of astrocyte-like cells, comprising performing at least one of the methods disclosed herein, and after the step of generating radial glia-like cells, culturing the radial glia-like cells for approximately 5 to 30 days in a third culture medium, an effective amount or concentration of one or more Notch pathway activators, and an effective amount or concentration of one or more interleukin-6 (IL-6) family cytokines, thereby generating a culture of astrocyte-like cells.

[0042] In one embodiment of the methods disclosed herein, the third culture medium is a third defined culture medium.

[0043] In one embodiment of the methods disclosed herein, the third defined culture medium is DMEM-F12, Neurobasal medium, Minimum Essential Medium (MEM), or BrainPhys Neuronal Medium.

[0044] In one embodiment of the methods disclosed herein, the third defined culture medium comprises N2 supplement and / or complete B27 supplement.

[0045] In one embodiment of the methods disclosed herein, the one or more Notch pathway activators in the third culture medium include 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).

[0046] In one embodiment of the methods disclosed herein, the effective amount or concentration of one or more Notch pathway activators in the third culture medium comprises one or both of 1 ng / mL to 800 ng / mL of Jagged 1 protein and 1 ng / mL to 800 ng / mL of Delta-Like protein 1 (DLL1).

[0047] In one embodiment of the methods disclosed herein, the one or more interleukin-6 family cytokines 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).

[0048] In one embodiment of the methods disclosed herein, an effective amount or concentration of each of one or more of oncostatin M protein, ciliary-derived neurotrophic factor protein (CNTF), and leukemia inhibitory factor protein (LIF) is present in the third culture medium at a concentration of 1 to 800 ng / mL.

[0049] In one embodiment of the methods disclosed herein, culturing in the third culture medium comprises changing the third culture medium approximately every 24 to 72 hours.

[0050] In one embodiment of the methods disclosed herein, culturing in the third culture medium comprises one or more steps of passaging the cultured cells when they become confluent.

[0051] In one embodiment of the methods disclosed herein, one or more passaging steps are performed at a 1:2 ratio of confluent cell culture:fresh medium.

[0052] In one embodiment of the methods disclosed herein, the culturing in the third culture medium comprises 1 to 3 passage steps.

[0053] In one embodiment of the methods disclosed herein, the astrocyte-like cells detectably express one or more astrocyte markers.

[0054] In one embodiment of the methods disclosed herein, the one or more astrocyte markers comprise S100 calcium binding protein B (S100B), nuclear factor 1 type A protein (NFIA), glial fibrillary acidic protein (GFAP), and vimentin (VIM).

[0055] In one embodiment of the methods disclosed herein, the astrocyte-like cells exhibit a flattened and / or stellate morphology.

[0056] In one embodiment of the methods disclosed herein, during culture in the third culture medium, detectable neuron-like cells are present at 10% or less of the total cells in culture.

[0057] In one embodiment of the methods disclosed herein, the third culture medium further comprises a chemically defined lipid concentrate 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 at a concentration of approximately 2%, or further comprises fetal bovine serum at a concentration of approximately 2%.

[0058] The present invention also provides a method for culturing astrocyte-like cells, comprising performing at least one of the methods disclosed herein and further culturing the astrocyte-like cells in a fourth culture medium and an effective amount or concentration of one or more cytokines of the interleukin-6 family, thereby enhancing maturation of the astrocyte-like cells.

[0059] In one embodiment of the methods disclosed herein, the fourth culture medium is a fourth defined culture medium.

[0060] In one embodiment of the methods disclosed herein, the fourth defined culture medium is DMEM-F12, E6, Neurobasal medium, or Minimum Essential Medium (MEM).

[0061] In one embodiment of the methods disclosed herein, the fourth defined culture medium comprises an N2 supplement and / or a B27 supplement.

[0062] In one embodiment of the methods disclosed herein, the one or more interleukin-6 family cytokines include one or both of ciliary derived neurotrophic factor protein (CNTF) and leukemia inhibitory factor protein (LIF).

[0063] In one embodiment of the methods disclosed herein, the effective amount or concentration of one or both of ciliary-derived neurotrophic factor protein (CNTF) and leukemia inhibitory factor protein (LIF) is present at a concentration of 1 to 800 ng / mL.

[0064] In one embodiment of the methods disclosed herein, the fourth culture medium is an enriched fourth defined culture medium optionally comprising an effective amount or concentration of one or more Notch pathway activators, and / or one or more thyroid hormones, phorbol esters, forskolin, neuregulin, and ascorbic acid.

[0065] In one embodiment of the methods disclosed herein, the thyroid hormone is triiodothyronine, and the one or more Notch pathway activators in the fourth culture medium include one or more of Jagged 1 protein and Delta-Like protein 1 (DLL1).

[0066] In one embodiment of the methods disclosed herein, the one or more Notch pathway activators are about 1 ng / mL to about 800 ng / mL of Jagged 1 protein and 1 ng / mL to about 800 ng / mL of Delta-Like protein 1 (DLL1), the thyroid hormone concentration is about 1 ng / mL to about 1000 ng / mL, the phorbol ester concentration is about 1 nM to about 1000 nM, the forskolin concentration is about 1 μM to about 200 μM, the neuregulin concentration is about 1 ng / mL to about 1000 ng / mL, and the ascorbic acid concentration is about 1 μM to about 1000 μM.

[0067] In one embodiment of the methods disclosed herein, the culturing in the fourth culture medium is carried out for at least approximately 40 to 60 hours.

[0068] In one embodiment of the methods disclosed herein, culturing in the fourth culture medium comprises changing the fourth culture medium approximately every 24 to 96 hours.

[0069] In one embodiment of the methods disclosed herein, during culture in the fourth culture medium, the astrocyte-like cells detectably express one or more of hepato-glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, and vimentin (VIM).

[0070] In one embodiment of the methods disclosed herein, the astrocyte-like cells exhibit a stellate and / or spherical morphology during culture in the fourth culture medium.

[0071] In one embodiment of the methods disclosed herein, one or more steps of the methods are performed by an automated system.

[0072] In one embodiment of the methods disclosed herein, the fourth culture medium further comprises a chemically defined lipid concentrate 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 at a concentration of approximately 2%, or further comprises fetal bovine serum at a concentration of approximately 2%.

[0073] The present invention also provides a composition comprising at least one cultured radial glia-like cell that detectably expresses 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.

[0074] In one embodiment of the compositions disclosed herein, the at least one cultured radial glia-like cell is or has been cryopreserved.

[0075] In one embodiment of the compositions disclosed herein, the composition comprises at least one cultured radial glia-like cell that detectably expresses 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.

[0076] In one embodiment of the compositions disclosed herein, the at least one cultured radial glia-like cell is or has been cryopreserved.

[0077] In one embodiment of the compositions disclosed herein, at least one cultured radial glia-like cell is or has been cryopreserved in a cryopreservation medium comprising Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine.

[0078] In one embodiment of the compositions disclosed herein, in the cryopreservation medium, the concentration of Chroman 1 and / or its derivatives is or was about 4 nM to about 80 μM, the concentration of Emricasan and / or its derivatives is or was about 100 nM to about 80 μM, the concentration of trans-ISRIB is or was about 50 nM to about 80 μM, and the concentrations of each of putrescine, spermine, and spermidine are or were about 0.5 μM to 1 mM.

[0079] The present invention also provides a composition comprising at least one cultured radial glia-like cell produced by the methods disclosed herein 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.

[0080] The present invention also provides a cell culture comprising at least one cultured radial glia-like cell that detectably expresses 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.

[0081] In one embodiment of the cell cultures disclosed herein, the cell cultures are grown from previously cryopreserved cells.

[0082] In one embodiment of the cell culture disclosed herein, the previously cryopreserved cells were cryopreserved in a cryopreservation medium comprising Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine.

[0083] In one embodiment of the cell cultures disclosed herein, the previously cryopreserved cells are vertebrate pluripotent stem cells.

[0084] In one embodiment of the cell cultures disclosed herein, the vertebrate pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells.

[0085] In one embodiment of the cell cultures disclosed herein, the vertebrate pluripotent stem cells are human pluripotent stem cells.

[0086] In one embodiment of the cell culture disclosed herein, the previously cryopreserved cells are cultured radial glia-like cells that detectably express 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 (HES5), SRY-Box transcription factor 21 (SOX21), and PAX6 proteins.

[0087] The present invention also provides a cell culture comprising at least one cultured radial glia-like cell produced by the methods disclosed herein 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.

[0088] The present invention also provides a composition comprising at least one cultured astrocyte-like cell that exhibits a flat, stellate, and / or spherical morphology and detectably expresses at least one marker, wherein the at least one marker is S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic-glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM).

[0089] In one embodiment of the compositions disclosed herein, at least one cultured astrocyte-like cell is or has been cryopreserved in a cryopreservation medium comprising Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine.

[0090] In one embodiment of the compositions disclosed herein, in the cryopreservation medium, the concentration of Chroman 1 and / or its derivatives is or was about 4 nM to about 80 μM, the concentration of Emricasan and / or its derivatives is or was about 100 nM to about 80 μM, the concentration of trans-ISRIB is or was about 50 nM to about 80 μM, and the concentrations of each of putrescine, spermine, and spermidine are or were about 0.5 μM to 1 mM.

[0091] The present invention also provides a composition comprising at least one cultured astrocyte-like cell produced by the methods disclosed herein, the cell expressing at least one marker and exhibiting a flat, stellate, and / or spherical morphology, wherein the at least one marker is S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic-glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM).

[0092] The present invention also provides a cell culture comprising at least one cultured astrocyte-like cell that exhibits a flat, stellate, and / or spherical morphology and detectably expresses at least one marker, wherein the at least one marker is S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic-glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM).

[0093] In one embodiment of the cell cultures disclosed herein, detectable neuron-like cells are present in 10% or less of the total cells in culture.

[0094] In one embodiment of the cell cultures disclosed herein, the cell cultures are grown from previously cryopreserved cells.

[0095] In one embodiment of the cell culture disclosed herein, the previously cryopreserved cells were cryopreserved in a cryopreservation medium comprising Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine.

[0096] In one embodiment of the cell cultures disclosed herein, the previously cryopreserved cells are vertebrate pluripotent stem cells.

[0097] In one embodiment of the cell cultures disclosed herein, the vertebrate pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells.

[0098] In one embodiment of the cell cultures disclosed herein, the vertebrate pluripotent stem cells are human pluripotent stem cells.

[0099] In one embodiment of the cell cultures disclosed herein, the previously cryopreserved cells are cultured radial glia-like cells that detectably express 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 proteins.

[0100] In one embodiment of the cell cultures disclosed herein, the previously cryopreserved cells are astrocyte-like cells that exhibit flat, stellate, and / or spherical morphology and detectably express one or more of S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), CD44, HEPACAM, glial fibrillary acidic protein (GFAP), and vimentin (VIM). The invention also provides a cell culture comprising at least one cultured astrocyte-like cell produced by the methods disclosed herein, detectably expressing at least one marker, and exhibiting flat, stellate, and / or spherical morphology, wherein the at least one marker is S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic-glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44, or vimentin (VIM).

[0101] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken in conjunction with the appended claims, which should be noted that the claims are defined by the description therein, and not by the specific discussion of the features and advantages set forth in the description herein.

[0102] The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which: [Brief explanation of the drawings]

[0103] [Figure 1] Figures A and B show schematic diagrams of the procedure for differentiating human pluripotent stem cells. Figure A shows the schematic pathway of human pluripotent stem cell differentiation. Figure B shows a schematic diagram of the procedure for differentiating human pluripotent stem cells into radial glia-like cells and astrocyte-like cells, as well as the medium used in this procedure. [Figure 2]Figures A-D show a schematic diagram of the procedure for differentiating human pluripotent stem cells into radial glia-like cells and astrocyte-like cells. Figure B shows the components of Astro 1 medium used from days 0 to 15. Figure C shows the components of Astro 2 medium used from days 15 to 30. Figure D shows the components of Astro 3 medium used from day 30 onwards. [Figure 3]Panels A–C are representative images of cells from different time points during the differentiation procedure (indicated on the left border of each panel). Images labeled "Phase" are phase-contrast microscopy images. Images labeled with specific protein names are micrographs of cells immunocytochemically stained with the indicated antibody combinations specific for the following proteins: TUJ1 (also known as β-III tubulin, a neuronal marker); PAX6: neural stem cell marker PAX6; BLBP: radial glial marker brain lipid-binding protein (BLBP); S100B: astrocyte marker S100β (S100B); NF-IA: astrocyte marker NFIA; VIM: astrocyte marker vimentin; GFAP: astrocyte marker glial fibrillary acidic protein; HEPACAM: astrocyte marker liver-glial cell adhesion molecule. Panel A shows that differentiated cells expressed the neural stem cell marker Paired Box Protein Pax-6 (PAX6) on "day 5" and the radial glial marker brain lipid-binding protein (BLBP) on "day 7." At day 15, the astrocyte marker S100 calcium-binding protein B (S100B) was widely expressed. (B) At day 30, the cultures consisted essentially of large cells with a flat morphology that expressed typical astrocyte markers S100B, nuclear factor type 1 A (NFIA), CD44, HEPACAM, glial fibrillary acidic protein (GFAP), and vimentin (VIM). (C) Astrocyte-like cells generated by the differentiation procedure were cryopreserved at day 30 or cultured for an additional 20 days and passaged twice, leading to further cell maturation, as indicated by stellate morphology and expression of hepatic glial cell adhesion molecule (HEPACAM), CD44, glial fibrillary acidic protein (GFAP), and NFIA. The designation "HOECHST" indicates Hoechst counterstaining, which marks cell nuclei. [Figure 4] 1 shows a bar graph illustrating the percentage of cells expressing the astrocyte markers NFIA and S100-beta and the neuronal marker TUJ1 (beta-III tubulin) at "day 30" of the differentiation procedure. [Figure 5]A-B show images of Western blots generated from differentiating radial glial cells at "day 7" of the differentiation procedure (A) and their demonstration of pluripotency (B). B shows exemplary images of cells at different time points of the differentiation procedure. [Figure 6] This shows the results of time-course gene expression profiling by RNA-seq of cells differentiated by the differentiation procedure. [Figure 7] Time course gene expression profiling by RNA-seq of cells generated by the differentiation procedure is shown compared with information available in the ARCHS4 human tissue RNA-seq database. [Figure 8] Single-cell RNA-seq results for cells generated by the differentiation procedure and comparison of these results with other indicated cell types (pluripotent stem cells, neuroectoderm, neurons, oligodendrocytes, microglia, and endothelial cells). [Figure 9] Figures A-B show functional analysis of astrocyte cells derived from iPSCs following a differentiation procedure. Figure A shows exemplary microscopic images illustrating the comparable glycogen accumulation capacity of iPSC-derived astrocyte-like cells produced by the differentiation procedure ("SCTL iPSC Astro") and commercially available iPSC-derived astrocyte-like cells ("Commercial iPSC Astro," sourced from Fujifilm Cellular Dynamics International). Figure B shows a bar graph illustrating the reduction in baseline glutamate levels in the culture medium after 3 hours of incubation with astrocytes. [Figure 10]Panels A-B show experimental results demonstrating that iPSC-derived astrocyte-like cells derived by the differentiation procedure promoted neuronal maturation and synaptic activity. The upper panel in A shows images of neuronal cells derived from a human ESC reporter cell line (SYN1:GFP; green fluorescent protein expressed under the control of the synapsin 1 promoter) cultured for 13 days with astrocyte-like cells generated by the differentiation procedure (+iPSC Astro) or without astrocyte-like cells (-iPSC Astro). The lower panel in A shows a line graph illustrating synapsin 1 expression in neurons. Panel B shows the results of a multielectrode array experiment (Axion Biosystems) performed using glutamatergic neurons (procured from Fujifilm Cellular Dynamics International) cocultured with astrocyte-like cells generated by the differentiation procedure described in Example 1. [Figure 11] 1 shows the cytoprotective effect of astrocyte-like cells on the activity of motor neurons upon exposure to glutamate. Motor neurons were purchased from Fujifilm Cellular Dynamics International and cultured with or without astrocyte-like cells produced by the differentiation procedure in Example 1. [Figure 12] Figures A-B show an automated procedure using the CompacT SelecT® system (Sartorius, Wilmington, USA) based on the procedure described in Example 1. Figure A shows a schematic diagram of the procedure for performing the automated differentiation procedure using CEPT at each passage (24-hour exposure to CEPT). Figure B shows a representative microscopic image of astrocyte-like cells produced by the exemplary automated procedure at "day 30" of the procedure. [Figure 13]Figures A-C show that 3D sphere formation and GFAP expression were enhanced by using enriched Astro-3 medium. A shows a schematic of the enhancement protocol for capturing the sphere formation stage (boxed area) from days 14 to 28 of differentiation. B shows a photomicrograph demonstrating increased GFAP expression and more mature morphology by astrocytes in cultures treated with enriched Astro-3 medium. C shows the components of enriched Astro-3 medium used to differentiate human pluripotent stem cells into radial glia-like and astrocyte-like cells from days 22 to 50. [Figure 14] Time-course gene expression profiling by RNA sequencing demonstrates the stepwise differentiation into radial glia and astrocytes. Heatmap (RNA-seq) showing genes expressed by pluripotent stem cells, radial glia, and astrocytes (days 0–50). [Figure 15] We demonstrate that iPSC-astrocytes display calcium transients in response to appropriate stimuli. iPSC-derived astrocytes exhibit typical physiological responses and elevated intracellular calcium levels in response to KCl, ATP, and L-glutamate. DMSO was used as a control treatment. DETAILED DESCRIPTION OF THE INVENTION

[0104] Those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated more than other elements to help to improve understanding of the embodiment(s) of the present invention.

[0105] All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0106] Before describing the present invention in detail, several terms are defined. These, together with the remainder of this disclosure and the accompanying figures, are intended to facilitate understanding of various embodiments of the present invention. These terms and concepts may be further clarified and understood based on accepted practice in the field of the present invention and the explanations provided throughout this document and / or the accompanying figures. Some other terms may be explicitly or implicitly defined in other sections of this disclosure and the accompanying figures, and may be used and understood based on accepted practice in the field of the present invention, the explanations provided throughout this document, and / or the accompanying figures. Terms not explicitly defined may also be defined and understood based on accepted practice in the field of the present invention and interpreted in the context of this document and / or the accompanying figures.

[0107] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0108] It should be noted that terms such as "preferably," "generally," and "typically" are not used herein to limit the scope of the claimed invention, or to suggest that a particular feature is essential, required, or even critical to the structure or function of the claimed invention. Instead, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in particular embodiments of the invention.

[0109] As used herein, the terms "invention," "the invention," "this invention," and "the present invention" are intended to broadly refer to all of the subject matter of this patent application and the claims that follow. It should be understood that statements containing these terms do not limit the subject matter described herein, nor do they limit the meaning or scope of the following claims. The embodiments of the invention that are encompassed are defined by the claims, not this summary or description. This description is a high-level overview of various aspects of the invention and introduces some of the concepts that are described and illustrated in this document and the accompanying figures. This description is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by reference to the entire specification, any figures, and appropriate portions of each claim. This document describes and refers to various embodiments of the invention. No particular embodiment is intended to define the scope of the invention. Instead, the embodiments merely provide non-limiting examples of various methods, compositions, kits, systems, and the like that are included, at least within the scope of the invention. Embodiments of the present invention are summarized below and are also described and illustrated elsewhere in this document.

[0110] For purposes of describing and defining the present invention, it should be noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree to which a quantitative representation may vary from the stated standard without resulting in a change in the basic functionality of the subject matter under consideration.

[0111] As used herein, "about" or "approximately" is used herein to indicate that a value includes the inherent variation for the device, the method being used to determine the value, or simply the tolerance for error in the value. For example, the term "about" or "approximately" can mean a ±1%, ±5%, ±10%, ±15%, or ±20% variation from a given value.

[0112] As used herein, "isolating," "separating," or "purifying," and related terms, are not necessarily used to refer to the removal of all material other than the component(s) of interest from a sample. Instead, in some embodiments, these terms are used to refer to procedures that enrich the amount of one or more components of interest relative to one or more other components present in a sample. In some embodiments, "isolating," "separating," or "purifying" can be used to remove or reduce the amount of one or more components from a sample. For example, the phrase "isolated cells" can refer to cells that have been substantially separated or purified from other cells in a cell culture or organism.

[0113] As used herein, the term "derived from" and related phrases in reference to cells or biological samples indicate that the cells or samples were obtained from the stated source at some point. For example, cells derived from an organism may represent primary cells (i.e., unmodified) obtained directly from an individual, or may be modified, for example, by introduction of a recombinant vector, exposure or culture under specific conditions, or immortalization. In some cases, cells derived from a given source may have undergone cell division and / or differentiation such that the original cells no longer exist, but surviving cells are understood to be derived from the same source. The terms "derived," "derived," and related terms and phrases may also be used in the present disclosure to refer to the creation of a cell population from a different starting or prior population or cells. For example, in each case of the populations of differentiated radial glia-like cells or astrocyte-like cells described herein, the starting population may be pluripotent stem cells (e.g., iPSCs). In the case of the populations of astrocyte-like cells described herein, the starting population may also be radial glia-like cells. Thus, astrocyte-like cells can be described as derived from radial glia-like cell(s) and / or pluripotent stem cell(s). Radial glia-like cells can be described as derived from pluripotent stem cell(s).

[0114] Throughout this specification, unless the context specifically dictates otherwise, when used in this disclosure to describe various embodiments of the invention, the term "comprising" and related terms (e.g., "comprise," "comprises," etc.) are open-ended, i.e., mean not excluding additional elements, and are synonymous with "including," "containing," or "having." When an embodiment of the invention is described using the term "comprising," it is intended to include embodiments in which the term "comprising" is replaced with the term "consisting of" or "consisting essentially of." That is, when an embodiment of the invention described in this disclosure is described using the term "comprising" and related terms, related embodiments are also described in which "consisting of" or "consisting essentially of" is used instead of "comprising." The term "consisting of" excludes any elements (steps, components, etc.) not expressly stated in the description. The term "consisting essentially of" is intended to exclude only those elements not specified in the description that do not materially affect the basic and novel characteristics of the embodiment.

[0115] As used in accordance with the present disclosure, unless otherwise defined, all technical and scientific terms should be understood to have the same meaning as commonly understood by one of ordinary skill in the art.

[0116] The percentages disclosed herein can vary from the disclosed values ​​by ±10, 20, or 30% amounts and remain within the contemplated disclosed range.

[0117] Unless otherwise indicated, or otherwise clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed herein as ranges can be taken to the tenth of the unit of the lower limit of the range, with any particular value or subrange within the stated range in different embodiments of this disclosure, unless the context dictates otherwise.

[0118] As used in this specification and drawings, ranges and amounts may be expressed by adding "about" to a particular value or range. "About" includes the exact amount. For example, "about 5%" means both "about 5%" and "5%." The term "about" may also refer to ±10% of a given value or range of values. Thus, about 5% also means, for example, 4.5% to 5.5%.

[0119] As used herein, the terms "or" and "and / or" are used to describe two or more elements in combination with each other or exclusively. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z."

[0120] As used herein, the terms "culture," "cell culture," and related terms can be used to refer to cells or cell populations present outside of an organism. These cells can be stem cells, primary cells isolated from an organism or obtained from a cell bank, animal, or blood bank, or secondary cells derived from such sources. Secondary cells can be immortalized for long-lived cell culture. Primary cells include egg cells, sperm cells, and any adult or fetal cell except stem cells. Examples of useful primary cells include, but are not limited to, skin cells, bone cells, blood cells, cells of internal organs, and cells of connective tissue. Secondary cells are derived from primary cells and can be immortalized for long-lived in vitro cell culture. A cell culture can be described as "pure" if it contains a sufficiently high proportion of cells of the desired type(s) and a sufficiently low proportion of cells of other types. It should be understood that "pure," as used in this disclosure in the context of cell culture and related processes, is a relative term, not an absolute term. For example, cell cultures and / or cell populations can be described as "pure" if they contain more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or approximately 100% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) of the desired cell type(s).

[0121] As used herein, the terms "cultivate," "culturing," "grow," "growing," "maintaining," "maintaining," "expanding," "expanding," and the like, when referring to the process of culturing or culturing cells, tissues, or organs, can be used interchangeably to mean that a cell or group of cells (the scope of which includes a group or groups of undifferentiated or differentiated cells, embryos, embryoid bodies, tissues, or organs) is maintained outside the body (ex vivo and / or in vitro) under conditions suitable for survival, proliferation, differentiation, and / or avoidance of senescence. In other words, the cultured cell or group of cells can be kept alive, and culturing results in cell growth, differentiation, and division. In this context, the terms "growth" and "culturing" can be used interchangeably, and these terms can refer to maintaining living cells in culture under certain conditions. The above terms do not imply that all cells in the culture survive, grow, or divide, since some may naturally senesce. Cells are typically cultured in a medium, which can be changed during the culture period. So-called two-dimensional (2D) cell cultures are grown on flat surfaces, typically in plastic vessels that can be coated with a substrate (e.g., vitronectin, laminin 521, Matrigel, Geltrex). Three-dimensional (3D) cultures are cultures in which biological cells are allowed to grow in three dimensions or interact with their surrounding environment. 3D cultures can be grown in a variety of artificial environments (e.g., but not limited to, plates, flasks, bioreactors, or small capsules), within which cells can develop into spheroids, spheres, or neurospheres. 3D cultures include so-called scaffold-free and scaffold-based technologies. Scaffold-free methods use, but are not limited to, low-attachment plates, hanging drop plates, micropatterned surfaces, rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting. A scaffold is a structure or material that provides structural support for cell attachment and, in some cases, cell differentiation.Scaffolds include solid scaffolds, sponges (e.g., cellulose sponges), protein-based scaffolds (e.g., collagen- or gelatin-based scaffolds), hydrogels, nanofiber scaffolds, and synthetic polymer scaffolds (e.g., polycaprolactone or polystyrene scaffolds). Generally, the culture environment includes consideration of factors such as the substrate for cell growth, cell density and cell shrinkage, gas phase, culture medium, and temperature. Cells in culture are generally maintained under conditions known to be optimal for cell growth. Such conditions can include, for example, a temperature of approximately 37°C and a humidified atmosphere containing approximately 5% CO2. The duration of incubation can vary widely depending on the desired results.

[0122] As used herein, the terms "culture medium," "cultivation medium," "growth medium," and related terms and phrases refer to a medium that supports the survival and / or growth of cells (including single cells and multiple cells), tissues, organoids, organs or portions thereof, or embryonic structures (e.g., but not limited to, morula, blastocoel, blastocyst, or embryo). Culture media are typically isotonic and can be liquid, colloidal liquid, gel, solid, and / or semi-solid. Culture media may be configured to provide a matrix for cell attachment or support, or may provide a separate support (e.g., a culture vessel surface or scaffold). Culture media can contain components for nutritional, chemical, and structural support necessary for the culture of a cell(s). A chemically defined medium (or "defined medium") is a medium in which the concentrations of all of its chemical components are known. On the other hand, an undefined medium can contain complex biological components (e.g., serum albumin or serum) that do not have a completely defined composition. Conditioned medium is understood to be a previously used medium from cultured cells. Conditioned media contain metabolites, growth factors, and extracellular matrix proteins secreted into the medium by cultured cells, which may be beneficial in subsequent use of such conditioned media. Culture media may be provided in powder form for preparation before use, as a concentrate for dilution before use, or in a form for use without further dilution. For example, culture media may be a sterile liquid provided as a "working solution" for use without further dilution (in the case of culture media). A working solution of culture media may contain one or more additives in effective amounts or concentrations. In another example, a culture medium may be a gel containing effective amounts of one or more additives. When a culture medium is provided in a form requiring further preparation (e.g., a powder or concentrate), one or more may be included in an amount or concentration intended to provide an effective amount(s) after the medium is prepared. For example, a 2x concentrated medium may contain twice the effective amount(s) of one or more additives intended to be included in the final "working" form of the medium.Culture media typically contain one or more appropriate nutrient sources for the growth and / or maintenance of the cells (e.g., mammalian cells, including human cells) they are intended to support. Culture media maintain an appropriate pH and osmolality. Culture media can contain natural, artificial, and / or synthetic components. Examples of natural components include biological fluids (e.g., plasma, serum, lymph, or amniotic fluid), tissue extracts (e.g., extracts of liver, spleen, tumor, white blood cells, bone marrow, or animal embryos). Some examples of culture media composed of artificial components ("artificial media") include MEM and DMEM. Artificial culture media can be serum-containing, serum-free (which can include purified growth factors of defined quality, lipoproteins, and other components provided by serum), chemically defined, or protein-free. Culture media can include one or more of a buffer, one or more inorganic salts, essential amino acids, one or more carbohydrates (e.g., glucose), fatty acids, lipids, vitamins, and trace elements. One example of a buffering agent is the so-called natural buffer system, in which gaseous CO2 balances the CO32- / HCO3- content in the culture. Another example is a chemical buffer system, such as a buffer system using the zwitterionic buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The culture medium can contain a pH indicator (e.g., phenol red) to allow pH monitoring during cell growth. Inorganic salts in the culture medium provide sodium, potassium, and calcium ions, providing osmotic balance and helping to regulate the cell membrane potential. The culture medium contains essential amino acids (amino acids that cannot be synthesized by cells), but nonessential amino acids can also be included to improve cell growth and viability. Carbohydrates (e.g., glucose, galactose, maltose, or fructose) are included as an energy source. Proteins and peptides (e.g., albumin, transferrin, or fibronectin), as well as fatty acids and lipids, can also be included, especially in serum-free media. Vitamins essential for cell growth and proliferation (e.g., B vitamins) can also be included. Examples of trace elements added to culture media, particularly serum-free media, include copper, zinc, and selenium.Some examples of culture media include commercially available media, such as, but not limited to, Essential 8 Medium, CTS Essential 8 Medium, Essential 6 Medium, StemFlex Medium, CTS KnockOut SR Xeno-free Medium, KnockOut Serum Replacement, StemPro, mTeSR, mTeSR1, StemFit, Nutristem, L7 Medium, iPS-Brew, Neurobasal, or BrainPhys.

[0123] In the context of cell culture, the term "dissociation," as used herein, can refer to the process of isolating cells from other cells or from a surface (e.g., a culture plate surface). For example, cells can be dissociated from an organ or tissue by mechanical or enzymatic methods. In another example, cells that have aggregated in vitro can be dissociated from each other. In yet another example, adherent cells are dissociated from a culture plate or other surface. Dissociation can include disrupting the extracellular matrix (ECM) and cell-cell interactions with the substrate (e.g., the culture surface) or disrupting the ECM between cells.

[0124] Stem cells are cells characterized by the ability to self-renew by mitosis and the potential to differentiate into tissues or organs. Among stem cells, embryonic stem cells and somatic stem cells can be distinguished. For example, mammalian embryonic stem cells are present in blastocysts and can give rise to embryonic tissues, while somatic stem cells can be present in adult tissues for the purpose of tissue regeneration and repair.

[0125] "Adult stem cells," also known as "somatic stem cells," are stem cells found within the differentiated cells of a tissue or organ in an organism and can differentiate to give rise to some or all of the specialized cell types of that tissue or organ. Somatic stem cells can be grown in culture. Somatic stem cells typically generate intermediate somatic cells called "precursor" or "progenitor" cells as they differentiate into specialized cells. Somatic stem cells and progenitor cells are sometimes described as "multipotent" or "oligopotent" depending on their degree of potency. Some examples of somatic stem cells include: hematopoietic stem cells (which give rise to all types of blood cells (erythrocytes, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, and macrophages)); mesenchymal stem cells (which include bone marrow stromal stem cells and skeletal stem cells and can give rise to bone cells (osteoblasts and osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and stromal cells that support blood formation); neural stem cells (which can give rise to nerve cells (neurons), astrocytes, and oligodendrocytes); epithelial stem cells of the lining of the digestive tract (which can give rise to absorptive cells, goblet cells, Paneth cells, and enteroendocrine cells); skin stem cells (which reside in the basal layer of the epidermis (which can give rise to keratinocytes) or at the base of hair follicles (which can give rise to both hair follicles and epidermis)). Tissue-specific progenitor cells are cells that are committed to differentiate into cells of a specific organ or tissue and lack the potential for self-renewal. Certain somatic stem cells can differentiate into cell types found in organs or tissues other than those predicted by the somatic stem cell's origin. This phenomenon is called "transdifferentiation."

[0126] As used herein, the term "stem cell" and related terms and phrases refer to animal cells that are capable of division and self-renewal over long periods of time, are unspecialized, and can give rise to specialized cell types. Stem cells are capable of division and self-renewal over long periods of time. Stem cells, unlike cells that do not normally self-renew, such as muscle cells, blood cells, or nerve cells, can replicate or proliferate many times. If the resulting cells remain unspecialized like the parent stem cells, they are said to be capable of long-term self-renewal.

[0127] As used herein, the term "cell line" refers to a cell culture typically developed from a single cell of a multicellular organism. Cells of a cell line have a relatively homogeneous genetic makeup. Some cell lines originate from stem cells. Some cell lines originate from naturally occurring cancerous cells that have undergone genetic modification (e.g., one or more mutations or the introduction of viral genes) leading to uncontrolled proliferation. Some cell lines originate from cells that have been artificially immortalized by various methods.

[0128] As used herein, the term "self-renewal," when used with respect to a cell, describes the ability to divide and generate at least one daughter cell that has the self-renewal properties of the parent cell, although one or more of the other daughter cells may be committed to a particular differentiation pathway. For example, a self-renewing hematopoietic stem cell can divide to form one daughter stem cell and another daughter cell that is committed to differentiation along the myeloid or lymphoid pathway. A non-self-renewing cell can continue to undergo cell division to produce daughter cells, none of which have the differentiation potential of the parent cell type, but instead generate differentiated daughter cells.

[0129] As used herein, the terms "pluripotency," "pluripotency," and related terms and phrases refer to an animal cell or cell population that has the capacity, under appropriate conditions, to differentiate and give rise to progeny that can become cell types that collectively exhibit characteristics associated with cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm). For example, the phrase "characteristics of pluripotent stem cells" refers to characteristics of a cell or cell population that distinguish a pluripotent stem cell or population from other cells. The ability to differentiate, under appropriate conditions, to give rise to progeny that can become cell types that collectively exhibit characteristics associated with cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm) is a characteristic of pluripotent stem cells. In addition to cell morphology, the expression or non-expression of certain combinations of molecular markers also characterize pluripotent stem cells. Pluripotent stem cells (PSCs) include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Embryonic stem cells (ESCs) are derived from embryos and can remain undifferentiated (unspecialized) under appropriate conditions. Embryonic stem cell lines are lines of ESCs cultured under conditions that allow them to grow for months to years without differentiation. Under other conditions, for example, when cells are aggregated together to form embryoid bodies, they will spontaneously differentiate.

[0130] As used herein, "radial glia cells" (singular "radial glia cell") refer to specific cells that exist transiently in vertebrate embryos during the neurogenic and gliogenic stages of brain development. These cells are also called "radial glial cells" or "radial glial progenitor cells" and are sometimes considered multipotent stem cells. During embryonic development, the body of radial glial cells is found in the ventricular region of the developing neural tube. In vivo, radial glial cells give rise to all neurons of the cerebral cortex and also to certain lineages of glial cells, including astrocytes and oligodendrocytes. Radial glial cells exist transiently during development and are generally not considered somatic stem cells.

[0131] As used herein, "astrocytes" (singular - "astrocyte") (sometimes collectively referred to as "astroglia") are glial cells within the central nervous system of vertebrates. Astrocytes have a characteristic star shape. Astrocytes are known to perform many functions, including structural, biochemical, and cytoprotective (e.g., detoxification) support for other cells in the central nervous system, energy supply to neurons, maintenance of ionic balance, immune function, critical components of the blood-brain barrier, and a role in central nervous system repair (e.g., scar formation). Astrocytes are also known to propagate calcium ion waves between cells and release transmitters in response to stimuli. Thus, astrocytes may have a neural signaling function.

[0132] As used herein, the term "induced pluripotent stem cells" (iPSCs) refers to pluripotent stem cells artificially derived from non-pluripotent cells. For example, human iPSCs are artificially derived from human non-pluripotent cells. iPSCs can be derived by introducing the products of a specific set of pluripotency-associated genes, i.e., "reprogramming factors," into a given cell type and / or by exposing non-pluripotent cells to specific conditions.

[0133] As used herein, the term "non-pluripotent cells" refers to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells, somatic stem cells, and progenitor cells. Some non-pluripotent cells retain some ability, and some examples are somatic stem cells and progenitor cells.

[0134] As used herein, the term "cell potential" describes the ability of a cell to differentiate into other cell types. Cells can be referred to as pluripotent cells, multipotent cells (cells that can differentiate into some, but not all, cell types, e.g., umbilical cord blood stem cells and mesenchymal stem cells), or oligopotent cells (cells that can differentiate into a few cell types, e.g., lymphocytes or vascular cells). Current understanding is that potential exists on a continuum. Thus, the boundaries between cell divisions based on potential can be fluid and are not necessarily definitive.

[0135] As used herein, the term "progenitor cell" or "precursor cell" typically refers to a cell that can differentiate to form one or more cell types. A "progenitor cell" or "precursor cell" can be any cell in a cell differentiation pathway that can differentiate into a more mature cell. Progenitor cells can be primary cells obtained from an organism, cells grown in culture, or cells derived from stem cells. Progenitor cells can be early descendants of pluripotent stem cells or pluripotent cells themselves. Progenitor cells can also be partially differentiated multipotent cells or reversibly differentiated cells. The term "progenitor cell population" refers to a group of cells that can develop into more mature or differentiated cell types. Progenitor cell populations can include pluripotent cells, stem cell lineage-restricted cells (cells that cannot develop into all lineages, e.g., only cells of the neural lineage), and reversibly stem cell lineage-restricted cells. Therefore, the terms "progenitor cell" or "progenitor cell" can refer to "pluripotent cells" or "multipotent cells."

[0136] As used herein, the term "astrocytic precursor" or "astrocytic progenitor" refers to a cell that can generate progeny that are mature astrocytes. Generally, the cell expresses some of the phenotypic markers characteristic of the astrocyte lineage. Astrocyte markers can be expressed on the cell surface or internally. Examples of astrocyte markers include S100 beta, glial fibrillary acidic protein (GFAP), glutamine synthetase, GLAST, and GLT1.

[0137] "Differentiation" is the process by which less specialized cells become more specialized cell types. For example, early development of multicellular animals is characterized by the rapid proliferation of embryonic cells, which then differentiate to produce the many specialized cell types that make up the multicellular animal's tissues and organs. As cells differentiate, the rate of proliferation typically decreases. While some types of differentiated cells never divide again, many differentiated cells can resume proliferation as needed to replace cells lost as a result of damage or cell death. Some cells divide continuously throughout life, replacing cells with high turnover rates in adult multicellular animals. Examples of differentiated cells include, but are not limited to, cells from tissues selected from bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Exemplary differentiated cell types include, but are not limited to, fibroblasts, tissue and hepatocytes, cardiomyocytes, myoblasts, neurons, osteoblasts, osteoclasts, and lymphocytes.

[0138] As used herein, the term "modified cells" and related terms and phrases encompass all cells derived or derived from artificially modified cells by any method, compared to the original or derived cell. Modified cells can be produced from primary cells, secondary cells, stem cells, cultured cells, and / or other modified cells. Modifications include, but are not limited to, genetic modification or genetic engineering, in which case the modified cells can be referred to as "genetically modified" or "genetically engineered." Genetic modification can be accomplished by a variety of methods that result in the incorporation of foreign or heterologous nucleic acid into the modified cell. Some examples of such methods include transduction with a virus or viral vector, or transfection of an isolated nucleic acid into a cell through transient pores in the cell membrane. Other modifications include exposing the source cell to biological and non-biological molecules or factors or culture conditions. Some examples of modified cells include iPSCs and genetically modified cells, including those used in gene therapy, such as gene-edited cells, e.g., cells modified using CRISPR / Cas9, TALEN, or ZFN.

[0139] As used herein, "vessel" refers to a container, dish, plate, flask, bottle, cell culture tube, bioreactor, etc., that can be used to culture, maintain, or grow cells, cell groups, tissues, or organs ex vivo or in vitro. Suitable vessels include, for example, multiwell plates, wells of multiwell plates, dishes, tubes, flasks, bottles, and reactors.

[0140] As used herein, the term "stabilize" when used with respect to cells, as well as related terms and phrases (e.g., "stabilizing a cell"), refers to the reduction of a negative cellular response (e.g., cell death or senescence). For example, stem cells and other cells may die in response to cell passaging, dissociation, separation, freezing, and / or thawing. In other words, these conditions may reduce cell viability. Embodiments of the compositions, methods, and kits described herein can mitigate the reduction in cell viability and improve cell survival, which may be expressed as stabilizing a cell.

[0141] As used herein, the terms "passage," "passaging," and related terms and phrases used in the context of cell culture refer to subculture, which typically involves transferring cells from a previous culture to fresh growth medium. Passage is performed to ensure the proliferation of cells in culture. Cell growth in culture is reduced or halted when the cells reduce the capacity of the culture vessel and / or medium to support further cell growth. For example, cells in adherent cultures may occupy all available substrate and run out of room for expansion, while cells in suspension cultures may exceed the medium's capacity to support further growth. To maintain cells in culture at an optimal density for continued growth and stimulate further proliferation, the culture must be expanded and replenished with fresh medium. To split a culture of adherent cells, e.g., a monolayer culture of cells, e.g., a culture of differentiated PSCs described herein, the cells are first dissociated, e.g., by enzymatic dissociation. Enzymatic dissociation can be performed by removing the incubation medium from the plate, adding a buffer such as PBS and an enzymatic dissociation reagent (e.g., Accutase, TrypLE, or trypsin, available from Thermo Fisher Scientific) to the plate, incubating the cells with the buffer and dissociation reagent under appropriate conditions, and harvesting the resulting dissociated cells by centrifugation, sedimentation, filtration, or other appropriate method. The dissociated cells are transferred to a similar or equivalent reaction vessel (e.g., a flask) containing fresh medium, thereby reducing the cell density.

[0142] As used herein, "marker" refers to any molecule that can be observed or detected. For example, markers can include, but are not limited to, nucleic acids, such as the transcription product of a particular gene, the polypeptide product of a gene, non-gene product polypeptides, glycoproteins, carbohydrates, lipids, lipoproteins, or small molecules (e.g., molecules with a molecular weight of less than 10,000 AMU). If the presence, absence, or amount of a marker can be experimentally observed or detected, such a marker or its amount can be described as "observable" or "detectable."

[0143] As used herein, in the context of observable or detectable markers of cell development or differentiation, "expression" refers to the production of a gene product (which may be a nucleic acid such as RNA or a protein) and the level or amount of the production of the gene product. Thus, quantifying the expression of a particular marker refers to detecting either the relative or absolute amount of the expressed marker (which may also refer to detecting the expression of RNA or protein), or simply detecting the presence or absence of the marker (which may also refer to detecting the expression of RNA or protein). If expression of the RNA or protein corresponding to the marker is detected, the marker can be said to be "detectably expressed." For most markers described herein, the symbols shown are symbols developed or recognized by the HUGO Gene Nomenclature Committee of the European Bioinformatics Institute.

[0144] As used herein, the term "cryopreservation," as well as related terms and phrases, refers to the process(es) of preserving cells, cell populations, or cell cultures by cooling them below freezing and the results of such process(es).

[0145] Embodiments of the present invention are envisioned based, at least in part, on the discoveries discussed below. The inventors have discovered a procedure for converting human pluripotent stem cells in culture into cells resembling radial glia-like cells of the central nervous system by manipulating key cell signaling pathways at defined time points through the use of various additives and their combinations. The radial glia-like cells generated by the inventors were subjected to further differentiation to produce a homogenous cell population resembling human astrocytes in a highly reproducible manner. Extensive morphological, molecular, and electrophysiological characterization experiments confirmed the astrocyte-like nature of the resulting differentiated cells, including the expression of typical astrocyte markers.

[0146] Astrocytes play critical roles in normal brain development, synaptic function, neurodegenerative diseases, brain injury, and various other pathologies (e.g., but not limited to, Alzheimer's disease, amyotrophic lateral sclerosis (ALS) (also known as Lou Gehrig's disease), Down syndrome, autism, intellectual disability, epilepsy, opioid addiction, and aging). Among other things, the discoveries made by the inventors and described in this disclosure have provided a process for culturing human astrocyte-like cells from scalable sources, such as induced pluripotent stem cells (iPSCs). While such a process is highly desirable for biomedical research and the development of novel therapeutics, the mechanisms underlying astrogliogenesis, the process by which astrocytes are generated in the human brain, remain unclear, and previously available processes for culturing astrocyte-like cells have been variable, inefficient, and time-consuming (up to several months). Based on the discoveries described in this disclosure, the inventors have devised processes (methods) for producing in culture cells that can be differentiated into cells exhibiting at least some characteristics of radial glial cells (radial glial-like cells) (including human radial glial-like cells), processes (methods) for producing in culture cells that exhibit at least some characteristics of vertebrate astrocyte cells (astrocytic-like cells) (including human astrocyte-like cells), and various compositions and kits related to the processes.

[0147] The processes described herein enable the production of desired cell populations (e.g., radial glia-like cells and / or astrocyte cells) in culture in a highly efficient, controlled, and stepwise manner. The processes described herein overcome the chemical and technical limitations of previously published methods, such as low efficiency, long durations (up to six months or more), cell sorting requirements, genetic manipulation, and the use of animal products (e.g., fetal bovine serum (FBS)). Some embodiments of the processes described herein produce cultures of radial glia-like cells from iPSCs. Some other embodiments of the processes described herein produce cultures of astrocyte-like cells from radial glia-like cells. The processes described herein are highly advantageous and superior to previously known processes for a variety of reasons. For example, the processes described herein produce substantially pure populations of radial glia-like cells and astrocyte-like cells in culture without any genetic manipulation. In another example, the processes described herein produce substantially pure populations of radial glia-like cells and astrocyte-like cells in culture using chemically defined conditions. Some embodiments of the processes described herein do not require the use of undefined culture medium components (e.g., fetal bovine serum (FBS)). Such embodiments can be performed under chemically defined conditions compatible with Good Manufacturing Practice (GMP) approaches, clinical translation, and cell therapy. In another example, the processes described herein produce substantially pure populations of radial glia-like cells and astrocyte-like cells in culture in a shorter period of time than previously known methods. In yet another example, the processes described herein produce cultures of radial glia-like cells and astrocyte-like cells with a higher percentage of the desired cell type (radial glia-like cells and astrocyte-like cells) than previously known processes.

[0148] Embodiments of the processes described in this disclosure can be combined to produce cultures of astrocyte-like cells from iPSCs. By identifying and simultaneously manipulating key developmental pathways, some embodiments of the processes described in this disclosure achieved derivation of astrocyte-like cells from iPSCs with greater than 90% efficiency (meaning that 90 out of 100 total cells obtained detectably expressed one or more astrocyte markers (e.g., S100B and / or NFIA)) in less than 30 days. Notably, the processes described in this disclosure produce cultures of astrocyte-like cells from iPSCs by largely bypassing the production of neurons (neurogenesis). Astrogliogenesis (the production of astrocyte-like cells) from iPSCs in culture without preceding neurogenesis has not previously been achieved. Using various methods, iPSC-derived astrocyte-like cells produced by the methods described in this disclosure were extensively characterized (e.g., based on their morphology, gene expression, protein expression, electrophysiology, and biochemistry) and compared to their in vivo counterparts. This confirms that the iPSC-derived astrocyte-like cells produced by the methods described herein are similar to naturally occurring astrocytes. The inventors have automated procedures for generating vertebrate radial glia-like cells and astrocyte-like cells from pluripotent stem cells using a robotic cell culture system. The inventors have also devised various applications and uses of these processes (methods), compositions, and kits, including high-throughput applications and uses requiring large quantities of high-quality, standardized cells. Various embodiments of the invention described in this disclosure can be used in, among other things, drug discovery and development, toxicity screening, disease modeling and research (e.g., directed toward better understanding the molecular mechanisms of neurodegenerative diseases), cell and tissue engineering, cell replacement therapy (e.g., cell replacement due to central, peripheral, and autonomic nervous system injury, stroke, neonatal hypoxia / ischemia, and other chronic diseases), cellular delivery of enzymes, compounds, or genes for the treatment of genetic diseases or cancer (e.g., enzyme replacement therapy for lysosomal storage diseases, migratory astrocytes for delivering chemotherapeutic drugs or genes to brain tumor cells), and regenerative medicine.

[0149] method Various methods (processes) are contemplated and encompassed by embodiments of the present invention. Among the methods according to embodiments of the present invention are methods for producing cells or cell cultures comprising cells in culture that have at least some defined characteristics. Such methods may also be referred to as "cell production methods," "cell culture production methods," "generation methods," "culturing methods," "differentiation methods," "differentiation methods," "differentiation processes," and other related terms and phrases, which may be used interchangeably with respect to methods for producing cells or cell cultures. One example of such a method is a method for producing or generating multipotent cells, which in turn can be differentiated into cells that exhibit at least some characteristics of astrocyte cells. Multipotent cells produced by such methods exhibit at least some characteristics of radial glial cells (e.g., expression of one or more of brain lipid-binding protein (BLBP), CD133 (prominin 1), ASPM, BIRC5 (survivin), FAT1, HES5, SOX21, or PAX6). Accordingly, such multipotent cells may be referred to as "cells exhibiting at least some characteristics of radial glial cells," "radial glial-like cells," "cells similar to radial glial cells," and other related terms and phrases. Cells exhibiting at least some characteristics of radial glial cells, i.e., radial glial-like cells, are discussed further in this disclosure, along with related characteristics. Another example of a method according to an embodiment of the present invention is a method for producing or generating cells exhibiting at least some characteristics of astrocyte cells, such as flattened and / or stellate morphology, expression of one or more of S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), glial fibrillary acidic protein (GFAP), vimentin, or hepatic glial cell adhesion molecule (HEPACAM). Cells exhibiting at least some characteristics of astrocyte cells produced according to an embodiment of the method of the present invention may be referred to as "astrocyte-like cells," "astrocyte-similar cells," and other related terms and phrases. Cells exhibiting at least some characteristics of astrocyte cells are discussed further in this disclosure, along with related characteristics.Methods according to the above embodiments of the invention, as well as other embodiments related to cell production, are carried out in culture and may be referred to as "methods of culturing" or "culturing." Such methods typically progress from less differentiated cells with higher potential (e.g., pluripotent, progenitor, multipotent, or oligopotent cells) as the starting material or intermediate product, to more differentiated cells with lower potential (multipotent, progenitor, oligopotent, or differentiated cells) as the intermediate and / or final product. Thus, the methods may be referred to as "methods of differentiating cells," even if the final product is or includes cells that are not fully differentiated.

[0150] In some exemplary embodiments, the present methods use pluripotent stem cells (PSCs) as starting materials. Such PSCs may be vertebrate PSCs, including mammalian PSCs or human PSCs (hPSCs). The PSCs used in methods according to embodiments of the present invention may be isolated from natural sources or artificially derived PSCs (e.g., induced PSCs (iPSCs)). Accordingly, the present methods may be referred to as "methods of differentiating PSCs," e.g., methods of differentiating hPSCs, methods of differentiating PSCs, etc. PSCs can be maintained and expanded in defined media (e.g., E8, E8 Flex, StemFlex, StemPro, mTeSR, mTeSR1, StemFit, Nutristem, L7 medium, or iPS-Brew), in monolayer culture, or in a suitable 3D culture system (e.g., using microcarriers). The maintenance and / or expansion of PSCs described above may be performed as part of or independently of methods according to embodiments of the present invention. In other words, cell production methods according to embodiments of the present invention are not limited by the steps or processes used to provide PSCs for further steps, unless such limitations are explicitly stated. For example, if PSCs are simply listed as a starting material or "provided" without further limitation, the processes for obtaining, culturing, expanding, or growing the PSCs are not intended to be incorporated into the method. PSCs may be provided, for example, in the form of monolayer cultures exhibiting typical PSC morphology (e.g., prominent nucleoli and / or a high nuclear / cytoplasmic ratio, cell growth in colonies, and expression of pluripotency-associated markers (e.g., but not limited to, OCT3 / 4, NANOG, SSEA-4, TRA-1-60, TRA-1-81, and / or alkaline phosphatase)). In another example, PSCs may be provided in the form of 3D cultures or attached to microcarriers.

[0151] The cell production method according to an embodiment of the present invention involves culturing vertebrate pluripotent stem cells (PSCs) (which may be ESCs or iPSCs), for example, human PSCs, for example, human iPSCs, on a vitronectin-coated surface of a culture vessel at approximately 5,000 to 50,000 cells / cm. 2 Density of, for example, but not limited to, approximately 5,000 to 40,000 cells / cm 2 Approximately 5,000 to 20,000 cells / cm 2 Approximately 10,000 to 50,000 cells / cm 2 Approximately 10,000 to 40,000 cells / cm 2 , or approximately 10,000-20,000 cells / cm 2 Some embodiments of the cell production method may include plating at a plating density of approximately 5,000-50,000 cells / cm. Some embodiments of the cell production method may not include a plating step. In such embodiments, the PSCs may be provided at the start of the method as an adherent monolayer culture at a specified density, for example, approximately 5,000-50,000 cells / cm. 2 The density is, for example but not limited to, approximately 5,000 to 40,000 cells / cm. 2 Approximately 5,000 to 20,000 cells / cm 2 Approximately 10,000 to 50,000 cells / cm 2 Approximately 10,000 to 40,000 cells / cm 2 , or approximately 10,000-20,000 cells / cm 2 The culture medium can be provided at a plating density of 0.1%.

[0152] A cell production method according to an embodiment of the present invention can include a step of incubating plated vertebrate PSCs (which may be ESCs or iPSCs) (e.g., human PSCs, e.g., human iPSCs) in a culture medium containing at least one Rho-associated protein kinase (ROCK) inhibitor. It should be understood that the above incubation step is optional and may not be included in some embodiments of the cell production method according to an embodiment of the present invention. The culture medium for the above incubation step (which may be referred to as a "first culture medium" or "incubation medium") can be a defined culture medium (in which case it may be referred to as a "first defined culture medium" or "defined incubation medium"), although the use of other types of media is also contemplated. Some non-limiting examples of defined media suitable for incubating PSCs include E8, E8 Flex, StemFlex, mTeSR, StemFit, or mouse embryonic fibroblast (MEF) conditioned medium. The first culture medium comprises an effective amount or concentration of at least one (one or more) ROCK inhibitor compound. Some non-limiting examples of ROCK inhibitors include Chroman 1 or a derivative thereof, Y27632, blebbistatin, or thiazovivin. In some embodiments, the medium for the incubation step described above contains Chroman 1 and can further contain an effective concentration of Emricasan or a derivative thereof, an effective concentration of trans-ISRIB, and one or more of a polyamine, including effective concentrations of putrescine, spermine, and spermidine. In an exemplary embodiment, the medium contains about 4 nM to about 80 μM of Chroman 1 or a derivative thereof, about 100 nM to about 80 μM of Emricasan or a derivative thereof, about 50 nM to about 80 μM of trans-ISRIB, and about 0.5 nM to 1 mM of each of putrescine, spermine, and spermidine (collectively referred to as "polyamines"). The combination of Chroman 1 or a derivative thereof, Emricasan or a derivative thereof, trans-ISRIB, and a polyamine may be referred to as "CEPT." In an exemplary embodiment, the medium is E8.The period for which the plated PSCs are incubated is from about 12 to about 24 hours, for example, 12 hours ± 1.2 hours to 24 hours ± 2.4 hours, for example, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.

[0153] In an embodiment of the cell production method including the incubation step described above, after the incubation step, the first culture medium in the PSC culture is replaced with a culture medium (sometimes referred to as a "second culture medium" or "first differentiation medium") containing an effective amount or concentration of one or more BMP pathway inhibitors, an effective amount or concentration of one or more Notch pathway activators, an effective amount or concentration of one or more interleukin-6 (IL-6) family cytokines, and an effective amount or concentration of a platelet-derived growth factor (PDGF) protein. Some examples of suitable BMP pathway inhibitors include LDN-193189, dorsomorphin, noggin, chordin, follistatin, or gremlin. In one example, the second culture medium contains about 2 nM to 40 μM LDN-193189. In some other examples, the second culture medium can include one or more of about 2 nM to 40 μM LDN-193189, about 2 nM to 40 μM LDN-214117, about 2 nM to 40 μM LDN-212854, about 2 nM to 40 μM DMH2, about 2 nM to 40 μM ML347, about 2 nM to 40 μM UK383367, about 2 nM to 40 μM K02288, about 5 nM to 40 μM dorsomorphin, about 5 ng / ml to 500 ng / ml noggin, about 5 ng / ml to 500 ng / ml chordin, about 5 ng / ml to 500 ng / ml follistatin, or about 5 ng / ml to 500 ng / ml gremlin. Some examples of suitable Notch pathway activators include Jagged 1 protein, Jagged 2 protein, and Delta-Like protein 1 (DLL1), Delta-Like protein 2 (DLL2), or Delta-Like protein 3 (DLL3). In one example, the second culture medium contains 1 ng / mL to 800 ng / mL of Jagged 1 protein and / or 1 ng / mL to 800 ng / mL of Delta-Like protein 1 (DLL1). Some examples of suitable IL-6 family cytokines include oncostatin M protein, ciliary-derived neurotrophic factor protein (CNTF), and leukemia inhibitory factor protein (LIF).In one example, oncostatin M protein, ciliary-derived neurotrophic factor protein (CNTF), and leukemia inhibitory factor protein (LIF) are each present in the second culture medium at a concentration of 1 to 800 ng / mL. Examples of suitable PDGF proteins include platelet-derived growth factor-AA protein (PDGF-AA), platelet-derived growth factor-AB protein (PDGF-AB), or platelet-derived growth factor-BB protein (PDGF BB). The second culture medium may be a defined culture medium (in which case it may be referred to as a "second defined culture medium" or a "first defined differentiation medium"), although other types of media are also contemplated. Some non-limiting examples of suitable defined media include DMEM-F12, E6, Neurobasal medium, or Minimum Essential Medium (MEM). In some embodiments, the second defined culture medium includes an N2 supplement and a B27 supplement without vitamin A. In some embodiments, the second culture medium includes CEPT.

[0154] Vertebrate PSCs (which may be ESCs or iPSCs), for example, human PSCs, for example, human iPSCs, are cultured in the second culture medium for approximately 168 to 360 hours, for example, 168 hours ± 17 hours to 360 hours ± 36 hours, for example, 168 to 396 hours, 192 to 396 hours, 192 to 396 hours, 240 to 396 hours, 264 to 396 hours, 288 to 396 hours, 312 to 396 hours, 336 to 396 hours, 360 to 396 hours, 151 to 360 hours, 192 to 360 hours, 192 to 360 hours, 240 to 360 hours, 264 to 360 hours, 288 to 360 hours, 312 to 360 hours, or 336 to 360 hours. During culture in the second culture medium, the medium can be changed approximately every 20 to 28 hours, for example, every 20±2 hours to every 28±3 hours, for example, approximately every 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours. During culture in the second culture medium, the cultured cells can be passaged when they become confluent. Passage can be performed at a ratio of 1:3 to 1:5 (e.g., 1:3, 1:3.5, 1:4, 1:4.5, or 1:5) of confluent cell culture to fresh medium. Culturing in the second culture medium can include 3 to 7 (e.g., 3, 4, 5, 6, or 7) passage steps.

[0155] Culturing vertebrate PSCs (which may be ESCs or iPSCs), e.g., human PSCs, e.g., human iPSCs, in a second culture medium induces or initiates differentiation of the vertebrate PSCs. At the end of culture in the second culture medium, the cell culture contains approximately 50% to 100% radial glia-like cells (approximately 50 to 100 out of 100 cells expressing the radial glia marker BLBP). For example, at the end of culture in the second culture medium, the culture can contain approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or greater than 90% (e.g., approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) radial glia-like cells. Thus, during culture in the second culture medium, the cells are appropriately referred to as differentiating (e.g., differentiating vertebrate PSCs). During subsequent step(s) (discussed elsewhere in this disclosure), radial glia-like cells produced by culturing vertebrate PSCs in the second culture medium can produce astrocyte-like cells when subjected to method steps further described in this disclosure. Radial glia-like cells emerge in cultures cultured in the second culture medium at various time points after initiation of culture. Radial glia-like cells emerging in cultures can be characterized by detectable expression of one or more radial glial cell markers. Expression of one or more radial glial cell markers can be detected in differentiating cells cultured in the second culture medium at approximately 120 to 216 hours (e.g., approximately 120 hours, approximately 144 hours, approximately 168 hours, approximately 192 hours, or approximately 216 hours after initiation of culture). One example of a radial glial cell marker is brain lipid-binding protein (BLBP). Other examples of radial glial cell markers include CD133 (prominin 1), abnormal fusiform cerebellum-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), and SRY-Box transcription factor 21 (SOX21). Another example of a radial glial cell marker is the PAX6 protein.In one embodiment, the radial glia-like cells detectably express each of BLBP, CD133 (prominin 1), ASPM, BIRC5 (survivin), FAT1, HES5, and SOX21, and PAX6.

[0156] Neural stem cells emerge from cells cultured according to some embodiments of the methods described herein approximately five days after the initiation of culturing the pluripotent cells in the second culture medium. The neural stem cells emerging in the culture can be characterized by the detectable expression of one or more neural stem cell markers. "Neural stem cells" is a broad term that includes early neuroepithelial stem cells that express only PAX6, which then transition to radial glial cells and express PAX6, BLBP, CD133 (prominin 1), ASPM, BIRC5 (survivin), FAT1, HES5, and SOX21. In other words, differentiation of cells cultured in the second culture medium can be described as first progressing from pluripotent cells to neural stem cells, and then to radial glia-like cells. Expression of one or more neural stem cell markers can be detected in differentiating cells cultured in the second culture medium at approximately 72 to 168 hours (e.g., approximately 72 hours, approximately 96 hours, approximately 120 hours, approximately 144 hours, or approximately 168 hours after initiation of culture). One example of a neural stem cell marker is PAX6. At the end of culture in the second culture medium, the cultures can contain varying proportions of radial glia-like cells (which can be characterized by expression of BLBP) and astrocyte-like cells (which can be characterized by expression of S100 calcium-binding protein B (S100B)). At the end of culture in the second culture medium, the cell cultures also contain a detectable proportion of S100B-positive glial progenitor cells, and S100B expression increases as the cells acquire astrocyte-like characteristics. For example, at the end of culturing in the second culture medium, the culture can contain approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or greater than 90% cells that detectably express S100B.

[0157] Cells exhibiting at least some characteristics of radial glial cells (radial glia-like cells) that can be differentiated into cells exhibiting at least some characteristics of astrocyte cells (astrocyte-like cells), as well as mixtures of cells comprising radial glia-like cells and one or both of neural stem cells, can be the end product of some (but not all) of the methods according to embodiments of the present invention. Radial glia-like cells, or cell mixtures comprising radial glia-like cells and neural stem cells, can be intermediates in some methods according to embodiments of the present invention, and can also be starting materials in some other methods according to embodiments of the present invention. Radial glia-like cells, or cell mixtures comprising radial glia-like cells and neural stem cells, can be prepared for cryopreservation and cryopreserved. Method steps related to cryopreservation can be incorporated into methods of cell generation according to embodiments of the present invention. Some methods and compositions related to cryopreservation are further described in the "Cryopreservation" section of this application, but it should be understood that the description in that section is not limiting and that other compositions and methods may be used for cryopreservation.

[0158] Cells exhibiting at least some characteristics of radial glial cells can be differentiated into cells exhibiting at least some characteristics of astrocyte cells under appropriate conditions. Methods for producing cells exhibiting at least some characteristics of astrocyte cells (astrocyte-like cells) in culture are included in embodiments of the present invention. Radial glial-like cells, or cultures containing such cells, can be the starting material or intermediate for such methods. In one example, cells containing cells exhibiting at least some characteristics of radial glial cells (radial glial-like cells) are cultured under conditions that induce differentiation into cells exhibiting at least some characteristics of astrocyte cells (astrocyte-like cells). Thus, embodiments of methods for producing astrocyte-like cells can include one or more steps of producing radial glial-like cells in culture according to embodiments of the present invention. Alternatively, embodiments of methods for producing astrocyte-like cells need not include a step of producing radial glial-like cells, but may simply be provided at the start of the method for producing astrocyte-like cells.

[0159] In one embodiment of a method for producing astrocyte-like cells, including one or more steps of producing radial glia-like cells in culture according to embodiments of the present invention, after culturing in the second culture medium, the second culture medium in the culture is replaced with a culture medium (sometimes referred to as a "third culture medium" or "second differentiation medium") containing an effective amount or concentration of one or more Notch pathway activators and an effective amount or concentration of one or more interleukin-6 (IL-6) family cytokines. Alternatively, an embodiment of the method for producing astrocyte-like cells may begin with culturing radial glia-like cells in the third culture medium. Some examples of suitable Notch pathway activators include Jagged 1 protein, Jagged 2 protein, and Delta-Like Protein 1 (DLL1), Delta-Like Protein 2 (DLL2), or Delta-Like Protein 3 (DLL3). In one example, the second culture medium contains 1 ng / mL to 800 ng / mL of Jagged 1 protein and / or 1 ng / mL to 800 ng / mL of Delta-like protein 1 (DLL1). Some examples of suitable IL-6 family cytokines include oncostatin M protein, ciliary-derived neurotrophic factor protein (CNTF), and leukemia inhibitory factor protein (LIF). In one example, oncostatin M protein, ciliary-derived neurotrophic factor protein (CNTF), and leukemia inhibitory factor protein (LIF) are each present in the third culture medium at a concentration of 1 to 800 ng / mL. The third culture medium may be a defined culture medium (in which case it may be referred to as a "third defined culture medium" or a "second defined differentiation medium"), although other types of media are also contemplated. Some non-limiting examples of suitable defined media include DMEM-F12, E6, Neurobasal medium, Minimum Essential Medium (MEM), or BrainPhys Neuronal Medium. In some embodiments, the third defined culture medium includes N2 supplement and B27 supplement. The third culture medium may or may not also include fetal bovine serum albumin, depending, for example, on the initial PSC line used.In some embodiments, the third culture medium comprises a chemically defined lipid concentrate comprising one or more (e.g., each) of arachidonic acid, cholesterol, DL-α-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid. In some embodiments, the third culture medium comprises CEPT. The cells are cultured in the third culture medium for approximately 120 to 720 hours, for example, 120 hours ± 12 hours to 720 hours ± 72 hours, for example, 120 to 720 hours, 144 to 720 hours, 120 to 360 hours, or 144 to 720 hours, for example, approximately 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384, 408, 432, 456, 480, 504, 528, 552, 576, 600, 624, 648, 672, 696, or 720 hours. During culture in the third culture medium, the cultured cells can be passaged when they become confluent. Passage can be performed at a ratio of 1:2 between confluent cell culture and fresh medium. Culturing in the second culture medium can include one to three, for example, one, two, or three, passage steps.

[0160] During culture in the third culture medium, the cells differentiate into astrocyte-like cells. At the end of culture in the third culture medium, the cell culture contains a detectable proportion of astrocyte-like cells, e.g., approximately 50-100% cells expressing the astrocyte markers S100B and NF-IA. For example, at the end of culture in the second culture medium, the culture can contain approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or more than 90% (e.g., approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) astrocyte-like cells. Radial glia-like cells develop (emerge) in cultures cultured in the second culture medium at various times after initiation of culture. The astrocyte-like cells that emerge in the culture can be characterized by the detectable expression of one or more astrocyte markers. In differentiating cells cultured in the third culture medium, expression of one or more astrocyte markers can be detected approximately 0 to 360 hours after initiation of culture in the third culture medium (e.g., approximately 0, 6, 120, 180, 240, 300, or 360 hours after initiation of culture). One example of an astrocyte marker is S100 calcium-binding protein B (S100B). Another example of an astrocyte marker is nuclear factor 1 type A protein (NFIA). Another example of an astrocyte marker is glial fibrillary acidic protein (GFAP). Another example of an astrocyte marker is vimentin. In one embodiment, astrocyte-like cells detectably express S100B, NFIA, GFAP, and vimentin. Astrocyte-like cells that emerge in culture are also characterized by a characteristic morphology (e.g., flattened and / or stellate). In one embodiment, the astrocyte-like cells detectably express S100B, NFIA, GFAP, and vimentin, and also exhibit a flattened and / or stellate morphology. During culture in the third culture medium, the cells differentiate into astrocyte-like cells with high efficiency.For example, at the end of culture in the third culture medium, detectable neuron-like cells characterized by expression of MAP2 and / or TUJ1 (beta-III tubulin) are present in less than 10% or less than 5% of the total cells in culture.

[0161] In some embodiments, differentiation of human pluripotent stem cells (hPSCs) (including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)) is performed in a stepwise manner (see, e.g., Example 6; Figures 13A-C), which allows for the generation of radial glial cells that can differentiate into functional astrocytes. The present inventors have shown and demonstrated for the first time that the combined use of gliogenic factors and appropriate cell culture conditions promotes the efficient, rapid, and targeted generation of radial glial cells and astrocytes.

[0162] The surprising and unexpected finding that this stepwise process largely avoids the generation of neuronal cells (neurogenesis), which always precedes the appearance of astrocytes (astrogliogenesis) during in vivo brain development, has great relevance for basic and translational research, including fundamental neurobiology studies (e.g., the neurotrophic support astrocytes provide to neurons in co-culture models, their support of neuronal maturation, and their promotion of synapse formation and electrical activity), disease modeling (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Alexander disease, etc.), high-throughput screening, drug discovery, cell therapy, and regenerative medicine.

[0163] The sphere formation procedure for promoting astrocyte maturation is shown schematically in Figure 13. In some embodiments, the entire astrocyte differentiation procedure was performed as a monolayer. In some embodiments, the entire astrocyte differentiation procedure was performed to include a sphere formation step. During these procedures, the sphere formation step on day 14 served to mature the cells and reduce the cell passaging step. Specifically, single cell dissociation was performed on day 14, and the cells were maintained in suspension in Astro-2 medium containing CEPT for 24 hours to form spheres (100,000 cells / well (U-bottom 96-well plate)). One day later, the spheres were then transferred to a container with a low cell-adhesion surface containing Astro-2 medium. Medium changes were performed every other day. After 1 week in Astro-2 medium, we introduced Astro-3 medium, which contained DMEM / F12 supplemented with N2B27 complete, chemically defined lipid concentrate (2%), LIF (10 ng / ml), and CNTF (10 ng / ml). Concentrated Astro-3 medium contained DMEM / F12 supplemented with N2B27 complete, chemically defined lipid concentrate (2%), LIF (10 ng / ml), and CNTF (10 ng / ml), Jagged 1 (10 ng / ml), DLL-1 (10 ng / ml), triiodothyronine (also known as T3; thyroid hormone) (40 ng / ml), phorbol ester (200 nM), forskolin (2 μM), neuregulin-1 (20 ng / ml), and ascorbic acid (200 μM). Spheres were cultured for an additional week in Astro-3 or enriched Astro-3 medium, with medium changes every other day. On day 28, spheres were dissociated into single cells by Accutase treatment, and astrocytes were maintained as monolayers in Astro-3 or enriched Astro-3 medium until day 50.

[0164] In some embodiments of the method for producing astrocyte-like cells, after the step of culturing in the third culture medium, the third culture medium in the culture is replaced with a culture medium (sometimes referred to as a "fourth culture medium" or "third differentiation medium") containing an effective amount or concentration of one or more interleukin-6 (IL-6) family cytokines. Some examples of suitable IL-6 family cytokines include ciliary-derived neurotrophic factor protein (CNTF) and leukemia inhibitory factor protein (LIF). In one example, each of the ciliary-derived neurotrophic factor protein and the leukemia inhibitory factor protein is present in the fourth culture medium at a concentration of 1 to 800 ng / mL. The fourth culture medium may be a defined culture medium (in which case it may be referred to as a "fourth defined culture medium" or "third defined differentiation medium"), although other types of media are also contemplated. Some non-limiting examples of suitable defined media include DMEM-F12, E6, Neurobasal medium, Minimum Essential Medium (MEM), or BrainPhys Neuronal Medium. In some embodiments, the third defined culture medium includes N2 supplement and B27 supplement. In some embodiments, the fourth culture medium includes CEPT. The fourth culture medium may or may not include fetal bovine serum (FBS), and if included, contains FBS at a concentration of approximately 2%. Cells are cultured in the fourth culture medium for up to 1,200 hours. During culture in the fourth culture medium, the medium can be changed approximately every 24 to 96 hours, e.g., every 20±2.4 hours to 96±9.6 hours, e.g., approximately every 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96 hours.

[0165] During culture in the fourth culture medium, a significant proportion of cells in the culture continue to emerge as astrocyte-like cells. For example, the culture may contain approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or greater than 90% astrocyte-like cells. The astrocyte-like cells emerging in the culture can be characterized by detectable expression of one or more astrocyte markers. In differentiating cells cultured in the third culture medium, expression of one or more astrocyte markers can be detected approximately 0 to 1200 hours after initiation of culture in the fourth culture medium. One example of an astrocyte marker is S100 calcium-binding protein B (S100B). Another example of an astrocyte marker is nuclear factor 1 type A protein (NFIA). Another example of an astrocyte marker is glial fibrillary acidic protein (GFAP). Another example of an astrocyte marker is vimentin. Another example of an astrocyte marker is hepatic glial cell adhesion molecule (HEPACAM). Another example of an astrocyte marker is CD44 protein. In one embodiment, the astrocyte-like cells detectably express S100B, NFIA, GFAP, vimentin, and HEPACAM. In another embodiment, the astrocyte-like cells detectably express CD44, GFAP, vimentin, and HEPACAM. The astrocyte-like cells that emerge in culture are also characterized by a characteristic morphology (e.g., flattened and / or stellate morphology). In one embodiment, the astrocyte-like cells detectably express one or more of the above-mentioned markers and also exhibit a flattened and / or stellate morphology.

[0166] The efficiency of the methods described in this disclosure in accordance with embodiments of the present invention can be adjusted by varying certain parameters, including, but not limited to, cell growth conditions, additive concentrations, and step timing. The method steps described herein can result in about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% conversion of less differentiated cells with high potential (e.g., pluripotent, progenitor, multipotent, or oligopotent cells) to more differentiated cells with low potential (e.g., multipotent, progenitor, oligopotent, or differentiated cells). Examples of conversion steps that can be characterized by the above-mentioned degree of efficiency include conversion of PSCs to radial glia-like cells, conversion of radial glia-like cells to astrocyte-like cells, or conversion of PSCs to astrocyte-like cells. As an example, starting with 1 million PSCs on day 1, it is possible to generate 10 million to 100 million radial glia-like cells by day 7 of culture in the second culture medium. As another example, starting with 1 million PSCs on day 1, it is possible to generate a mixture of 100 million to 1 billion astrocyte-like cells by day 30.

[0167] automation Embodiments of the present invention include automated cell culture methods. Embodiments of the present invention also include systems for implementing or partially implementing automated method embodiments of the present invention. Systems according to embodiments of the present invention can include various stations and / or components, some examples of which are described below. As used herein, the term "station" is broadly defined to include any suitable device or assembly, collection or assemblage of devices or components, suitable for carrying out methods according to embodiments of the present invention. Stations need not be integrally connected or located relative to one another in any particular manner. Systems according to embodiments of the present invention can include any suitable arrangement of stations relative to one another. For example, the stations need not even be in the same room. However, in some embodiments, the stations are integrally connected to one another.

[0168] Automated cell culture methods and systems for performing various methods according to embodiments of the present invention can be used to optimize conditions for various method steps and / or scale up the production yield of cells (e.g., radial glia-like cells and / or astrocyte-like cells) produced by the methods. Generally, automated methods and systems according to embodiments of the present invention can minimize the human intervention required during cell culture procedures, such as cell cultivation, passaging, or harvesting. In addition to reducing laboratory staff requirements, the disclosed automated methods and systems can perform these techniques in a reliable and reproducible manner. For example, systems for performing various methods according to embodiments of the present invention can include a robotic or automated cell culture station, such as the CompacT SelecT® (Sartorius, Wilmington, DE) system. Automated cell culture systems can grow, expand, and differentiate cells by performing methods according to embodiments of the present invention. Automated cell culture systems can also perform one or more steps required for cell cryopreservation. An automated cell culture system can perform one or more cell culture processes, such as, but not limited to, seeding cell culture flasks or plates, maintaining cell cultures, e.g., in cell culture flasks or plates, harvesting cells, pooling cells from harvested flasks or plates, diluting cells for subculture or plating, performing cell counts, performing cell viability assays, etc. An automated cell culture system can include various stations, such as, but not limited to, a station for incubating cells (which is exemplified by an automated flask incubator that maintains a controlled environment (including a controlled temperature, a controlled gas composition, and / or maintaining a sterile environment)), a station for handling flasks and other cell culture equipment (e.g., pipettes) (which may be exemplified by a robotic arm or other type of robotic handler), a station for reagent dispensing (e.g., a robotic low-volume dispenser), etc.

[0169] An automated cell culture system can include various computer components. Embodiments of an automated cell culture system, or portions of the system, may be controlled by a computer. For example, an automated cell culture system can include a computer base station for generating reports. An automated cell culture system can include a computer base station or components for data analysis. An automated cell culture system can include a computer, a processor, electronic memory, software instructions, etc. An automated cell culture system can include software instructions for one or more of system operation, workflow optimization, auditing and / or tracking of cell culture flasks or plates, etc. For example, an automated cell culture system can include an application software program for executing a programmed protocol on a robotic liquid handling system. The software program can run on an external device (e.g., a portable computer, e.g., a tablet computer or smartphone) in communication with a controller integrated into the robotic liquid handling system, and in some embodiments, the software program can coordinate control of the robotic liquid handling system, and also control of external robotic systems, if present, to implement at least some steps of methods according to embodiments of the present invention. The software program can be programmed to alert a user when intervention is required, either due to a fault / error or completion of a procedure, for example, using sound, light, vibration, email alert, or text alert.

[0170] Computer-based calculations and tools The methods described herein may include computer-based calculations and tools. The tools may be advantageously provided in the form of a computer program executable on a conventionally designed general-purpose computer system (sometimes referred to as a "host computer"). The host computer may be configured with many different hardware components and may be produced in many sizes and styles (e.g., desktop PCs, laptops, tablet PCs, handheld computers, servers, workstations, mainframes). Standard components may include a monitor, keyboard, disk drive, CD and / or DVD drive. If the host computer is connected to a network, the connection may be provided via any suitable transport medium (e.g., wired, optical, and / or wireless) and any suitable communication protocol (e.g., TCP / IP), and the host computer may include suitable network hardware (e.g., a modem, an Ethernet card, a WiFi card). The host computer may implement any of a variety of operating systems, including UNIX, Linux, Microsoft Windows, MacOS, or any other operating system.

[0171] Computer code for implementing aspects of the invention can be written in a variety of languages, including PERL, C, C++, Java, JavaScript, VBScript, AWK, or any other scripting or programming language that can be executed on a host computer or compiled to run on a host computer. Code may also be written and distributed in lower level languages ​​such as assembly language or machine code.

[0172] The host computer system advantageously provides an interface through which the user can control the operation of the tool. In the examples described herein, the software tool is implemented as a script (e.g., using PERL), and its execution can be initiated by the user from the standard command line interface of an operating system such as Linux or UNIX. The commands can be adapted to the OS as needed. In other embodiments, a graphical user interface can be provided, allowing the user to control the operation using a pointing device. Thus, the present invention is not limited to any particular user interface.

[0173] Scripts or programs incorporating various features of the present invention may be encoded on a variety of computer-readable media for storage and / or transmission. Examples of suitable media include magnetic disks or tapes, optical recording media such as compact disks (CDs) or digital versatile disks (DVDs), flash memory, and carrier signals adapted for transmission over wired, optical, and / or wireless networks conforming to various protocols, including the Internet.

[0174] additive Various additives can be used in cell production methods and related compositions and kits according to embodiments of the present invention. Some additives and / or additive components are discussed below for clarity. It is understood that other additives and / or additive components can also be used, even if not discussed below. In the context of embodiments of the present invention, each component separately or combinations of components may be referred to in the singular or plural as an "additive," "supplement," "active agent," or other related term. Various additive formulations are contemplated. For example, an additive can be formulated to include an amount of one or more active agents sufficient to provide an effective concentration or amount of each active agent(s) when added to a culture medium. In the context of embodiments of the present invention, an effective concentration or amount is the concentration or amount, respectively, of one or more active agents that elicits a desired effect in cells exposed to the composition (e.g., but not limited to, improved survival time (viability), stabilization of cells, improved growth, reduced cell death, reduced senescence, improved growth, improved differentiation, etc.). Additives are typically formulated to be easily incorporated into a culture medium. For example, the culture medium additive may be provided in powder form, tablets, or capsules that can be readily dissolved in the aqueous culture medium. In another example, the additive may be provided as a concentrated solution or suspension that is added to the culture medium.

[0175] N-2 supplement is a chemically defined serum-free supplement based on Bottenstein, J.E. Cell Culture in the Neurosciences, Bottenstein, J.E. and Harvey, A.L., editors, pp. 3-43, Plenum Press: New York and London (1985).

[0176] B-27 supplement is an optimized serum-free supplement described, for example, in Brewer et al. Journal of Neuroscience Research 35:567-76, 1993.

[0177] As used herein, the term "Chroman 1" refers to (3S)-N-{2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl}-6-methoxy-3,4-dihydro-2H-1-benzopyran-3-carboxamide. Chroman-related compounds or derivatives are structurally related compounds (Chroman moiety-containing ROCK inhibitors), some of which are described in Chen et al., "Chroman-3-amides as potent Rho kinase inhibitors," Bioorganic and Medicinal Chemistry Letters 18:6406-6409 (2008) and LoGrasso et al., "Rho Kinase (ROCK) Inhibitors and Their Application to Inflammatory Disorders," Current Topics in Medicinal Chemistry 9:704-723 (2009). Chroman 1, its derivatives, or related compounds may be provided as a salt or as a solution.The effective concentration of Chroman 1 (or an active derivative or related compound thereof) is about 4 nM to about 80 μM, about 10 nM to about 20 μM, about 20 nM to about 10 μM, or about 30 nM to about 500 nM, for example, about 4 nM, 5 nM, 30 nM, 55 nM, 80 nM, 105 nM, 130 nM, 155 nM, 180 nM, 205 nM, 230 nM, 240 nM, 250 nM, 260 nM, 270 nM, 280 nM, 290 nM, 300 nM, 310 nM, 320 nM, 330 nM, 340 nM, 350 nM, 360 nM, 370 nM, 380 nM M, 255nM, 280nM, 305nM, 330nM, 355nM, 380nM, 405nM, 430nM, 455nM, 480nM, 500nM, 525 nM, 550nM, 575nM, 600nM, 625nM, 650nM, 675nM, 700nM, 725nM, 750nM, 775nM, 800nM, 825 nM, 850nM, 875nM, 900nM, 925nM, 950nM, 975nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 11μM, 12μM, 13μM, 14μM, 15μM, 16μM, 17μM, 18μM, 19μM, 20μM, 21μM, 22μM, 23μM M, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 45 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, or 80 μM.

[0178] As used herein, the term "Emricasan" refers to 3-(2-(2-tert-butylphenylaminooxalyl)aminopropionylamino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid, the structure of which is shown in Figure 1. Emricasan-related compounds or derivatives are structurally related compounds (e.g., Q-VD-OPh hydrate), some of which are described in Linton et al., "First-in-Class Pan Caspase Inhibitor Developed for the Treatment of Liver Disease," J. Med. Chem. 48:6779-6782, (2005). Emricasan, its derivatives, or related compounds can be supplied as a salt or solution. The effective concentration of Emricasan (or its active derivative or related compound) is about 5 nM to about 100 μM, about 5 nM to about 80 μM, about 200 nM to about 30 μM, about 300 nM to about 20 μM, for example, about 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 1μM, 1.5μM, 2μM, 2.5μM, 3μM, 3.5μM, 4μM, 4.5μM, 5μM, 5.5μM, 6μM, 6.5μM, 7μM, 7.5μM, 8μM, 8.5μM, 9μM, 9.5μM, 10μM, 10.5μM, 1 1μM, 11.5μM, 12μM, 12.5μM, 13μM, 13.5μM, 14μM, 14.5μM, 15μM, 15.5μM, 16μM, 16.5μM, 1 7μM, 17.5μM, 18μM, 18.5μM, 19μM, 19.5μM, 20μM, 21μM, 22μM, 23μM, 24μM, 25μM, 26μM, 27μM M, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 45 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, or 100 μM.

[0179] As used herein, the term "trans-ISRIB" can be used interchangeably with the terms "ISRIB" or "ISRIB (trans isomer)" and refers to N,N'-((1r,4r)-cyclohexane-1,4-diyl)bis(2-(4-chlorophenoxy)acetamide), the structure of which is shown in Figure 2. As described by Sidrauski et al., "Pharmacological brake-release of mRNA translation enhances cognitive memory," eLIFE 2:e00498 (2013), trans-ISRIB is 100-fold more potent (IC50 = 5 nM) than cis-ISRIB (IC50 = 600 nM), suggesting a stereospecific interaction with its cellular target. trans-ISRIB can be supplied as a salt or in solution.The effective concentration of trans-ISRIB is about 5 nM to about 80 μM, about 5 nM to about 50 μM, about 100 nM to about 6.25 μM, or about 200 nM to about 6.25 μM, for example, about 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 70 0nM, 750nM, 800nM, 850nM, 1μM, 1.25μM, 1.5μM, 1.75μM, 2μM, 2.25μM, 2.5μM, 2.75μM, 3μM, 3.25 μM, 3.5 μM, 3.75 μM, 4 μM, 4.25 μM, 4.5 μM, 4.75 μM, 5 μM, 5.25 μM, 5.5 μM, 5.75 μM, 6 μM, 6.25 μM, 6.5 μM M, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM, 12.5 μM, 13 μM, 13.5 μM M, 14μM, 14.5μM, 15μM, 15.5μM, 16μM, 16.5μM, 17μM, 17.5μM, 18μM, 18.5μM, 19μM, 19.5μM, 20μM, It can be 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 45 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, or 80 μM.

[0180] As used herein, the term "polyamine" refers to one or more of the polycations putrescine, spermidine, and spermine, which are known to interact with negatively charged macromolecules such as DNA, RNA, and proteins. Effective concentrations of spermine range from about 0.5 nM to 1 mM, for example, about 0.5 nM, 20.5 nM, 40.5 nM, 60.5 nM, 80.5 nM, 100.5 nM, 120.5 nM, 140.5 nM, 160.5 nM, 180.5 nM, 200.5 nM, 220.5 nM, 240.5 nM, 260.5 nM, 280.5 nM, 300.5 nM, 320.5 nM, and the like. 340.5nM, 360.5nM, 380.5nM, 400.5nM, 420.5nM, 440.5nM, 460.5nM, 480.5nM, 0.5μM, 20.5μM, 40 .5μM, 60.5μM, 80.5μM, 100.5μM, 120.5μM, 140.5μM, 160.5μM, 180.5μM, 200.5μM, 220.5μM, 240. 5μM, 260.5μM, 280.5μM, 300.5μM, 320.5μM, 340.5μM, 360.5μM, 380.5μM, 400.5μM, 420.5μM, 440 .5μM, 460.5μM, 480.5μM, 500.5μM, 520.5μM, 540.5μM, 560.5μM, 580.5μM, 600.5μM, 620.5μM, 64 It can be 0.5 μM, 660.5 μM, 680.5 μM, 700.5 μM, 720.5 μM, 740.5 μM, 760.5 μM, 780.5 μM, 800.5 μM, 820.5 μM, 840.5 μM, 860.5 μM, 880.5 μM, 900.5 μM, 920.5 μM, 940.5 μM, 960.5 μM, 980.5 μM, or 1 mM.The effective concentration of spermidine is about 0.5 μM to 1 mM, for example, about 0.5 nM, 20.5 nM, 40.5 nM, 60.5 nM, 80.5 nM, 100.5 nM, 120.5 nM, 140.5 nM, 160.5 nM, 180.5 nM, 200.5 nM, 220.5 nM, 240.5 nM, 260.5 nM, 280.5 nM, 300.5 nM, 320.5 nM, 330.5 nM, 340.5 nM, 350.5 nM, 360.5 nM, 370.5 nM, 380.5 nM, 390.5 nM, 400.5 nM, 410.5 nM, 420.5 nM, 430.5 nM, 440.5 nM, 450.5 nM, 460.5 nM, 470.5 nM, 480.5 nM, 490.5 nM, 500.5 nM, 510.5 nM, 520.5 nM, 530.5 nM, 540.5 nM, 550.5 nM, 560.5 nM, 570.5 nM, 580.5 nM, 590.5 nM, 600.5 nM, 610.5 nM, 620.5 nM, 630.5 nM, 640.5 nM, 650.5 nM, 660.5 nM, 670.5 nM, 680.5 nM, 690.5 nM, 700.5 nM nM, 340.5nM, 360.5nM, 380.5nM, 400.5nM, 420.5nM, 440.5nM, 460.5nM, 480.5nM, 0.5μM, 20.5μM 24 0.5μM, 260.5μM, 280.5μM, 300.5μM, 320.5μM, 340.5μM, 360.5μM, 380.5μM, 400.5μM, 420.5μM, 4 40.5μM, 460.5μM, 480.5μM, 500.5μM, 520.5μM, 540.5μM, 560.5μM, 580.5μM, 600.5μM, 620.5μM, 6 It can be 40.5 μM, 660.5 μM, 680.5 μM, 700.5 μM, 720.5 μM, 740.5 μM, 760.5 μM, 780.5 μM, 800.5 μM, 820.5 μM, 840.5 μM, 860.5 μM, 880.5 μM, 900.5 μM, 920.5 μM, 940.5 μM, 960.5 μM, 980.5 μM, or 1 mM.The effective concentration of putrescine is about 0.5 μM to 1 mM, for example, about 0.5 nM, 20.5 nM, 40.5 nM, 60.5 nM, 80.5 nM, 100.5 nM, 120.5 nM, 140.5 nM, 160.5 nM, 180.5 nM, 200.5 nM, 220.5 nM, 240.5 nM, 260.5 nM, 280.5 nM, 300.5 nM, 320.5 nM, 330 nM, 340 nM, 350 nM, 360 nM, 370 nM, 380 nM, 390 nM, 400 nM, 410 nM, 420 nM, 430 nM, 440 nM, 450 nM, 460 nM, 470 nM, 480 nM, 490 nM, 500 nM, 510 nM, 520 nM, 530 nM, 540 nM, 550 nM, 560 nM, 570 nM, 580 nM, 590 nM, 600 nM, 610 nM, 620 nM, 630 nM, 640 nM, 650 nM, 660 nM, 670 nM, 680 nM, 690 nM, 700 nM, 710 nM, 720 nM, 730 nM, 740 nM, 750 nM, 760 nM, 770 nM, 780 nM, 790 nM, 800 nM, 81 M, 340.5nM, 360.5nM, 380.5nM, 400.5nM, 420.5nM, 440.5nM, 460.5nM, 480.5nM, 0.5μM, 20.5μM, 40.5μM, 60.5μM, 80.5μM, 100.5μM, 120.5μM, 140.5μM, 160.5μM, 180.5μM, 200.5μM, 220.5μM, 240 .5μM, 260.5μM, 280.5μM, 300.5μM, 320.5μM, 340.5μM, 360.5μM, 380.5μM, 400.5μM, 420.5μM, 44 0.5μM, 460.5μM, 480.5μM, 500.5μM, 520.5μM, 540.5μM, 560.5μM, 580.5μM, 600.5μM, 620.5μM, 6 It can be 40.5 μM, 660.5 μM, 680.5 μM, 700.5 μM, 720.5 μM, 740.5 μM, 760.5 μM, 780.5 μM, 800.5 μM, 820.5 μM, 840.5 μM, 860.5 μM, 880.5 μM, 900.5 μM, 920.5 μM, 940.5 μM, 960.5 μM, 980.5 μM, or 1 mM.

[0181] As used herein, the terms "CEPT," "CEPT cocktail," or "CEPT small molecule cocktail" refer to a combination of effective amounts or concentrations of Chroman 1 or a derivative thereof, Emricasan or a derivative thereof, trans-ISRIB, and a polyamine.

[0182] As used herein, the term "Y27632" refers to trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride. The effective concentration of Y27632 can be 1-100 μM.

[0183] As used herein, the term "blebbistatin" refers to (±)-1,2,3,3a-tetrahydro-3a-hydroxy-6-methyl-1-phenyl-4H-pyrrolo[2,3-b]quinolin-4-one. The effective concentration of blebbistatin can be from 5 nM to 500 μM.

[0184] As used herein, the term "thiazovivin" refers to N-benzyl-[2-(pyrimidin-4-yl)amino]thiazole-4-carboxamide. The effective concentration of thiazovivin can be from 5 nM to 200 μM.

[0185] As used herein, the term "LDN-193189" refers to 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride. The effective concentration of LDN-193189 can be approximately 2 nM to 40 μM.

[0186] As used herein, the term LDN-214117 refers to 1-[4-[6-methyl-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenyl]piperazine. The effective concentration of LDN-214117 can be from 2 nM to 40 μM.

[0187] As used herein, the term DMH2 refers to 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline. The effective concentration of DMH2 can be between 2 nM and 40 μM.

[0188] As used herein, the term LDN-212854 refers to 5-(6-(4-(1-piperazinyl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline. The effective concentration of LDN212854 can be from 2 nM to 40 μM.

[0189] As used herein, the term ML347 refers to 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline. The effective concentration of ML347 can be from 2 nM to 40 μM.

[0190] As used herein, the term UK383367 refers to 3-(aminocarbonyl)-β-(3-cyclohexylpropyl)-N-hydroxy-1,2,4-oxadiazole-5-propanamide. The effective concentration of UK383367 can be between 2 nM and 40 μM.

[0191] As used herein, the term K02288 refers to 3-[(6-amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol. The effective concentration of K02288 is 2 nM to 40 μM.

[0192] As used herein, the term "dorsomorphin" refers to 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride. Effective concentrations of dorsomorphin can range from about 2 nM to 100 μM.

[0193] As used herein, the term "Noggin" refers to the protein Noggin. An effective concentration of Noggin can be from about 1 ng / mL to 100 μg / mL.

[0194] As used herein, the term "chordin" refers to the protein chordin. An effective concentration of chordin can be from about 1 ng / mL to 100 μg / mL.

[0195] As used herein, the term "follistatin" refers to the glycoprotein follistatin. Effective concentrations of follistatin can be from about 1 ng / mL to 100 μg / mL.

[0196] As used herein, the term "gremlin" refers to the protein gremlin. An effective concentration of gremlin can be from about 1 ng / mL to 100 μg / mL.

[0197] As used herein, the term "PDGF-AA" refers to the protein platelet-derived growth factor-AA. An effective concentration of PDGF-AA can be from about 1 ng / mL to 20 μg / mL.

[0198] As used herein, the term "PDGF-AB" refers to the protein platelet-derived growth factor-AB. An effective concentration of PDGF-AB can be about 1 ng / mL to 20 μg / mL.

[0199] As used herein, the term "PDGF-BB" refers to the protein platelet-derived growth factor-BB. An effective concentration of PDGF-BB can be about 1 ng / mL to 20 μg / mL.

[0200] As used herein, the term "Jagged 1 protein" refers to a Jagged 1 protein ligand that can activate a Notch receptor. An effective concentration of Jagged 1 protein can be about 1 ng / mL to 1000 ng / mL.

[0201] As used herein, the term "Jagged 2 protein" refers to a Jagged 2 protein ligand that can activate a Notch receptor. An effective concentration of Jagged 2 protein can be from about 1 ng / mL to 800 ng / mL.

[0202] As used herein, the term "Delta-Like Protein 1 (DLL1)" refers to a Delta-Like 1 protein ligand that can activate the Notch receptor. An effective concentration of Jagged 1 protein can be about 1 ng / mL to 1000 ng / mL.

[0203] As used herein, the term "Delta-Like Protein 2 (DLL2)" refers to a Delta-Like 2 protein ligand that can activate the Notch receptor. An effective concentration of Jagged 1 protein can be about 1 ng / mL to 1000 ng / mL.

[0204] As used herein, the term "Delta-Like Protein 3 (DLL3)" refers to a Delta-Like 3 protein ligand that can activate the Notch receptor. An effective concentration of Jagged 1 protein can be about 1 ng / mL to 800 ng / mL.

[0205] As used herein, the term "oncostatin M" refers to oncostatin M, a pleiotropic cytokine that belongs to the interleukin-6 family of cytokines. Effective concentrations of oncostatin M protein can be from about 1 ng / mL to 1000 ng / mL.

[0206] As used herein, the term "ciliary-derived neurotrophic factor protein" refers to the polypeptide hormone and neurotrophic factor ciliary-derived neurotrophic factor protein (CNTF). An effective concentration of ciliary-derived neurotrophic factor protein (CNTF) can be from about 1 ng / mL to 800 ng / mL.

[0207] As used herein, the term "leukemia inhibitory factor protein" refers to leukemia inhibitory factor (LIF), an interleukin-6 family cytokine. An effective concentration of leukemia inhibitory factor protein can be from about 1 ng / mL to 1000 ng / mL.

[0208] As used herein, the term "triiodothyronine" refers to a thyroid hormone. Triiodothyronine plays an important role in metabolic regulation in the body. Effective concentrations of triiodothyronine can be approximately 1 ng / mL to 1000 ng / mL.

[0209] As used herein, the term "phorbol ester" refers to any phorbol ester in which two hydroxyl groups on adjacent carbon atoms are esterified with a fatty acid. Phorbol and phorbol esters are members of the tigliane family of diterpenes, defined by polycyclic compounds. Effective concentrations of phorbol esters can be from about 1 nM to 1000 nM.

[0210] As used herein, the term "forskolin" refers to a compound that inhibits the activity of adenylate cyclase (IC), an enzyme that produces cyclic adenosine monophosphate (cAMP). 50 Forskolin is a cell-permeable diterpene that directly activates cAMP (cAMP = 41 nM), resulting in increased intracellular cAMP levels. The effective concentration of forskolin can be approximately 1 μM to 200 μM.

[0211] As used herein, the term "neuregulin-1" refers to a protein or peptide capable of binding to and activating ErbB2, ErbB3, ErbB4, or a combination thereof, including, but not limited to, all neuregulin isoforms, the neuregulin EGF domain alone, polypeptides containing the neuregulin EGF-like domain, neuregulin variants or derivatives, and any type of neuregulin-like gene product that also activates the above receptors (discussed in more detail below). Neuregulin also includes NRG-1, NRG-2, NRG-3, and NRG-4 proteins, peptides, fragments, and compounds that mimic the activity of neuregulin. The effective concentration of neuregulin-1 can be approximately 1 ng / mL to 1000 ng / mL.

[0212] As used herein, the term "ascorbic acid" is the name recognized by the IUPAC-IUB Commission on Biochemical Nomenclature for Vitamin C. Other names include L-ascorbic acid, L-xyloascorbic acid, and L-threo-hex-2-enoic acid gamma lactone. The pure vitamin is CHO with a molecular weight of 176.13. There are four possible stereoisomers of ascorbic acid: L-ascorbic acid, D-araboascorbic acid (erythorbic acid) (which exhibits vitamin C activity), L-araboascorbic acid, and D-xyloascorbic acid. Ascorbic acid intermediates or "pathway intermediates" are biochemicals that can be converted to ASA by enzymatic or chemical means, including, but not limited to, gluconic acid, 2-keto-D-gluconic acid, 2,5-diketo-D-gluconic acid, 2-keto-L-gluconic acid, idonic acid, gluconic acid, sorbitol, sorbose, sorbosone, and sorbose diacetone. Effective concentrations of ascorbic acid can be about 1 μM to 1000 μM.

[0213] Cells, compositions, and kits Some embodiments of the cell production methods described herein include, as starting materials or intermediates, pluripotent or progenitor cells, or populations of pluripotent or progenitor cells, that can selectively (and sometimes reversibly) develop into specified cell lineages when cultured under appropriate conditions. As used herein, the term "population" refers to a cell culture of multiple cells with the same distinguishing characteristic. The term "cell lineage" refers to all developmental stages of a cell type, from the earliest progenitor cells to fully mature (specialized) cells. One example of a progenitor cell population that can be involved in the cell production methods described herein is a culture of pluripotent stem cells (PSCs), which may be cultured embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Some embodiments of the cell production methods described herein include human PSCs (hPSCs) or populations thereof as starting materials for deriving radial glia-like cells and astrocyte-like cells. It should be understood that embodiments of the cell production methods described herein may include modified PSCs, including hPSCs. Some examples of PSCs that can be used in methods according to embodiments of the present invention include various ESCs (e.g., WA01, WA09, WA14 from WiCell) and iPSC lines (LiPSC-GR1.1, NCRM-1, NCRM-2, NCRM-5; all available from the National Institutes of Health (USA)).

[0214] Another example of a progenitor cell population that can be involved in the cell production methods described herein is a population of radial glia-like cells, which can be produced from PSCs according to some embodiments of the methods described herein. Radial glia-like cells, as discussed herein, are cells that exhibit at least some properties of radial glia cells that arise during vertebrate embryonic development. Radial glia-like cells according to embodiments of the present invention can express at least one marker of natural radial glia cells: brain lipid-binding protein (BLBP), CD133 (prominin 1), ASPM, BIRC5 (survivin), FAT1, HES5, SOX21, and PAX6. For example, radial glia-like cells involved in methods according to embodiments of the present invention can express at least one marker of natural radial glia cells: brain lipid-binding protein (BLBP), CD133 (prominin 1), ASPM, BIRC5 (survivin), FAT1, HES5, and SOX21. In another example, radial glia-like cells involved in methods according to embodiments of the present invention can express all of brain lipid-binding protein (BLBP), CD133 (prominin 1), ASPM, BIRC5 (survivin), FAT1, HES5, SOX21, and PAX6.

[0215] As used herein, "astrocyte-like cells" are defined as a cell population that expresses glial fibrillary acidic protein (GFAP) and that is differentiated from embryonic stem cells. Astrocyte-like cells are cells that contain at least one astrocyte phenotype that enables them to mediate astrocytic activity, i.e., support neurons, in vivo.

[0216] As used herein, the phrase "astrocyte phenotype" refers to structural and / or functional parameters typical (e.g., specific) of astrocytes. An astrocyte phenotype can include a single characteristic or multiple characteristics. Examples of structural astrocyte phenotypes include cell size, cell shape, organelle size, and organelle number. Thus, structural astrocyte phenotypes can include a round nucleus, a "stellate" body, and expression of astrocyte markers.

[0217] As used herein, the phrase "astrocyte marker" refers to a polypeptide that is selectively or non-selectively expressed in astrocytes. Astrocyte markers can be expressed on the cell surface or internally. Examples of astrocyte markers include S100 beta, glial fibrillary acidic protein (GFAP), glutamine synthetase, GLAST, and GLT1.

[0218] As discussed throughout this disclosure, some embodiments of the methods of the present invention produce astrocyte-like cells or populations thereof. Astrocyte-like cells, as discussed throughout this disclosure, are cells that exhibit some properties of natural astrocyte cells. Astrocyte-like cells according to embodiments of the present invention can express one or more markers expressed by natural astrocytes, such as S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), CD44, HEPACAM, glial fibrillary acidic protein (GFAP), or vimentin. In one example, astrocyte-like cells according to embodiments of the present invention can express all of S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), CD44, HEPACAM, glial fibrillary acidic protein (GFAP), or vimentin. Astrocyte-like cells according to embodiments of the present invention can exhibit a flattened and / or stellate morphology. In one example, astrocyte-like cells according to embodiments of the present invention may exhibit a flattened and / or stellate morphology and express all of S100 calcium binding protein B (S100B), nuclear factor 1 type A protein (NFIA), CD44 protein, HEPACAM, glial fibrillary acidic protein (GFAP), or vimentin (VIM).

[0219] The presence or absence of a marker, as applied to embodiments of the present invention, refers to the detectable presence or absence of a marker as detected by an applicable method for detecting such marker, and may also refer to a particular detectable or undetectable level of such marker. In other words, presence may refer to presence above a particular detectable level, while absence may refer to absence below a particular detectable level, and not necessarily a zero detectable level. It should also be understood that astrocyte-like cells can include a range of cells on a continuum, with varying levels of the presence or absence of a particular detectable marker.

[0220] Compositions according to embodiments of the present invention include in vitro or ex vivo compositions comprising at least one radial glia-like cell or at least one astrocyte-like cell. The cells included in such compositions can be vertebrate cells (meaning cells derived from vertebrate PSCs), including mammalian cells (meaning cells generated from mammalian PSCs) or human cells (meaning cells generated from mammalian PSCs). The cells included in such compositions can be modified cells. The compositions can include multiple cells of the same or different types. For example, the multiple cells can include one or more of pluripotent stem cells, multipotent stem cells, progenitor cells, differentiated cells, and modified cells. The multiple mammalian cells can be a plurality of cells, a cell culture, a cell aggregate, a spheroid, or a tissue. The at least one cell or multiple cells can be cryopreserved or thawed after cryopreservation. It should be understood that some compositions according to embodiments of the present invention can further include a culture medium, one or more additives, a container containing the culture medium, such as a culture flask, a culture dish, a tube, or a reactor, and can also include a support or scaffold for the cells.

[0221] The described methods can be used to produce compositions containing various mixtures of pluripotent stem cells and other multipotent or differentiated cells. Such compositions are included within embodiments of the present invention. In some embodiments, compositions can be produced that contain at least about 5 multipotent or differentiated cells for every 95 pluripotent cells. In other embodiments, compositions can be produced that contain at least about 95 multipotent or differentiated cells for every 5 pluripotent cells. Additionally, compositions containing other ratios of multipotent or differentiated cells:pluripotent cells are also contemplated. For example, compositions comprising at least about 1 multipotent or differentiated cell for about 1,000,000 pluripotent cells, at least about 1 multipotent or differentiated cell for about 100,000 pluripotent cells, at least about 1 multipotent or differentiated cell for about 10,000 pluripotent cells, at least about 1 multipotent or differentiated cell for about 1000 pluripotent cells, at least about 1 multipotent or differentiated cell for about 500 pluripotent cells, at least about 1 multipotent or differentiated cell for about 100 pluripotent cells, at least about 1 multipotent or differentiated cell for every 5 to a maximum of 1 pluripotent cell, and about 1,000,000 multipotent or differentiated cells for every 1 pluripotent cell are contemplated. Some embodiments of the composition may be a cell culture or cell population comprising at least about 5% to at least about 99% multipotent or differentiated cells. In some embodiments, the cell culture or cell population comprises mammalian cells. In preferred embodiments, the cell culture or cell population comprises human cells. For example, certain specific embodiments relate to cell cultures comprising human cells, wherein at least about 5% to at least about 99% of the human cells are multipotent or differentiated cells.Other embodiments relate to cell cultures comprising human cells, wherein at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more than 99% of the human cells are multipotent or differentiated cells.

[0222] The progression of pluripotent cells to multipotent cells and then to further differentiated cells (e.g., the progression of PSCs to radial glia-like cells or the progression of radial glia-like cells to astrocyte-like cells) can be monitored by detecting markers characteristic of the particular cell type. Identification of cell types related to embodiments of the present invention can also be performed by detecting markers characteristic of the particular cell type. For example, expression of a particular marker can be detected. Expression of a particular marker can be determined by detecting the presence or absence of the marker in a cell, cell culture, or cell population. Expression of a particular marker can also be determined by measuring the level of the marker present in a cell, cell culture, or cell population. In some embodiments of the present invention, expression of one or more markers characteristic of radial glia-like cells, such as BLBP, CD133 (prominin 1), ASPM, BIRC5 (survivin), FAT1, HES5, SOX21, or PAX6, can be determined. In some embodiments, the expression of one or more markers characteristic of astrocyte-like cells, such as S100B, NFIA, CD44, HEPACAM, GFAP, or vimentin, can be determined. Quantitative, qualitative, or semi-quantitative techniques can be used to measure marker expression. For example, marker expression can be detected and / or quantified using techniques that detect nucleic acids (e.g., PCR-based detection or RNA detection using RNA sequencing (e.g., real-time reverse transcriptase PCR), RNA sequencing (RNA-seq), or nucleic acid sequence-based techniques). In another example, immunochemistry can be used to detect and / or quantify marker proteins. For example, the expression of marker gene products can be detected using antibodies specific for the marker gene product of interest using Western blotting, immunocytochemical characterization, flow cytometry analysis, and the like. Various marker detection techniques can be used in conjunction to effectively and accurately characterize and identify cell types and to quantify both the amount and relative proportion of such markers in a cell type of interest.The expression of a particular marker can be determined by measuring the level of the marker present in cells of a cell culture or cell population compared to a standardized or normalized control marker. Identification and characterization of cells, cell cultures, and cell populations can be based on the expression of a particular marker, or on different expression levels and patterns of multiple markers (including the presence or absence, high or low expression, of one or more markers). Also, a particular marker may have transient expression, where it shows high expression at one or more stages of the processes described herein and low expression at other stage(s).

[0223] Kits for culturing cells, tissues, or organs are included in embodiments of the present invention. The kit is a set containing at least some components for culturing cells, including single cells and cell populations. The kit may include one or more additives discussed in the corresponding sections of this disclosure. The kit may further include one or more of the following: a culture medium configured to support at least one cell or one or more culture medium components in vitro or ex vivo; a container for holding the culture medium; a culture vessel, such as a flask, dish, plate (including a multiwell plater), or reactor; or a support or scaffold for cell or tissue culture. The kit may also include one or more mammalian cells, such as human cells. The cells included in the kit may be one or more of PSCs (including embryonic stem cells and / or induced pluripotent stem cells), radial glia-like cells, or astrocyte-like cells. The one or more cells may be provided in frozen or non-frozen form (which may be thawed).

[0224] cryopreservation Methods, compositions, and kits involving cryopreservation (including processes, tools, and / or compositions for cryopreserving, thawing, and culturing previously cryopreserved cells, cell populations, or cell cultures) are included in embodiments of the present invention. Some compositions for preservation can include a cryopreservation medium used to cryopreserve cells or cell populations (e.g., radial glia-like cells and astrocyte-like cells) described herein. Some compositions can include a cryopreservation medium and one or more cells described herein. For example, an embodiment of a composition can include one or more radial glia-like cells and a cryopreservation medium. In another example, a composition can include one or more astrocyte-like cells and a cryopreservation medium. The cryopreservation medium can be the liquid medium in which the cells are found before freezing and / or in the frozen state. Some examples of cryopreservation media include PSC Cryopreservation Kit (Thermo Fisher Scientific), FreezIS (Irving Scientific), NutriFreez (Biological Industries USA), CryoStor, HypoThermosol, mFreSR, mFreSR-S, and STEMdiff Neural Progenitor Freezing Medium (all from Stem Cell Technologies). Cryopreservation media can contain one or more cryoprotectants (meaning compounds that protect cells from freezing damage). Cryoprotectants can be permeable or non-permeable. An example of a suitable permeable cryoprotectant that can permeate cell membranes is dimethyl sulfoxide (DMSO). Some examples of suitable non-permeable cryoprotectants include sucrose, glycerol, dextran, trehalose, Percoll, polyethylene glycol, polyvinylpyrrolidone, serum albumin, Ficoll, maltose, and polyvinyl alcohol (PVA). Cryopreservation media can further contain one or more additives described in the "Additives" section of this disclosure.For example, the cryopreservation medium may contain one or more of Chroman-1 or its derivatives, Emricasan or its derivatives, trans-ISRIB, or polyamines, each in an effective combination. The combination of all four of the above additives is sometimes referred to as "CEPT."

[0225] Methods involving the cryopreservation of cells, cell populations, or cell cultures are included in embodiments of the present invention. Such methods can include contacting one or more cells (e.g., radial glia-like cells or astrocyte-like cells) with a cryopreservation medium. This can involve adding the cryopreservation medium to the one or more cells, or vice versa, and mixing the cells with the medium. In some embodiments, 0.5 mL to 5 mL of cryopreservation medium per million cells, e.g., about 1 mL per million cells, can be added. However, it is contemplated that greater or lesser amounts of cryopreservation medium may be used in certain embodiments. In some embodiments, the cryopreservation medium can be added to the cells in incremental amounts of increasing concentration, which can reduce the risk of osmotic shock to the cells associated with adding it in a single step. The temperature of the cryopreservation medium when added to the cells can range from about 15°C to about 40°C. For example, the temperature of the cryopreservation medium added to the cells can be about 37°C. The contacting step of the methods of the present invention can result in cells suspended in the cryopreservation medium, which may be referred to as a "mixture." The cells before the contacting step or the cell suspension after the contacting step can be provided in a container or vessel. The volume of the container can be 1 mL to 50 mL, for example, the container can be a 15 mL tube.

[0226] Methods involving the cryopreservation of cells can include freezing a composition comprising one or more cells (e.g., radial glia-like cells or astrocyte-like cells) and a cryopreservation medium, thereby obtaining a frozen or cryopreserved composition. The mixture of cells and cryopreservation medium can be equilibrated before freezing. During equilibration, water can be removed from the cells and replaced with a medium containing a cryoprotectant, which enters the cells after incubation with the cryopreservation medium. To avoid damage to the cells, the equilibration time is limited. For example, the mixture can be equilibrated for a period of 10 seconds to 5 minutes, 20 seconds to 1.5 minutes, or 30 seconds to 1 minute. Before freezing, the mixture can be transferred to a freezing container or vessel, or the mixture can remain in the same container. Water can be removed from the cells and replaced with a medium containing a cryoprotectant, which enters the cells after incubation with the cryopreservation medium. The containers used for freezing are typically provided as stacked tubes, and placing the containers in the freezer ensures a consistent cooling rate.

[0227] Freezing results in cells being placed in a low temperature or cryopreserved state (sometimes simply referred to as "frozen"), where they can remain for days, weeks, months, or years, ready for retrieval when needed. When needed, the cryopreserved cells are retrieved and thawed. Thus, methods involving cryopreservation can include thawing the cryopreserved composition (more specifically, under conditions that maintain cell viability). For example, a container containing cryopreserved cells can be thawed in a water bath at a temperature of 42°C or below, e.g., 10°C to 40°C, e.g., about 37°C. To improve cell viability after thawing, a thawing rate of about 10°C to about 40°C per minute, e.g., about 20°C to about 40°C per minute, e.g., approximately 30°C per minute, can be used.

[0228] The described methods and / or method steps can increase the post-thaw viability of cryopreserved cells. As used herein, the term "viability" refers to the number of living cells based on the presence of DNA and an intact cell membrane system. Viability can be measured by various tests, such as trypan blue internalization or measuring propidium iodide uptake. The viability of cells (e.g., thawed radial glia-like cells or thawed astrocyte cells) after cryopreservation can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. After thawing, cells may exhibit limited amounts of necrosis and apoptosis. In certain embodiments, necrosis and / or apoptosis is observed in less than 25%, more particularly less than 15%, and most particularly less than 10% of the cells. The methods described herein can further ensure that radial glia-like cells maintain their ability to differentiate into astrocyte-like cells. After thawing, the cryopreserved cells can be used for further culture, differentiation (in the case of radial glia-like cells), therapeutic purposes (e.g., regenerative medicine), and other uses.

[0229] Exemplary embodiments of the present invention include methods for producing radial glia-like cells in culture. For example, in some embodiments disclosed herein, methods for producing radial glia-like cells in culture are disclosed herein, the methods comprising: (a) Vertebrate pluripotent stem cells are cultured on the substrate-coated surface of a culture vessel at a density of 1,000 to 100,000 cells / cm. 2 and plating at a density of (b) incubating the plated vertebrate pluripotent stem cells in a first culture medium; (c) replacing the first culture medium with a second culture medium, the second culture medium comprising: (i) an effective amount or concentration of one or more BMP pathway inhibitors; (ii) an effective amount or concentration of one or more Notch pathway activators; (iii) one or more cytokines of the interleukin-6 family; (d) culturing the plated vertebrate pluripotent stem cells in a second culture medium; This produces radial glia-like cells.

[0230] Some methods for producing radial glia-like cells in culture according to embodiments of the present invention include culturing vertebrate pluripotent stem cells in a second culture medium containing an effective amount or concentration of one or more BMP pathway inhibitors, an effective amount or concentration of one or more Notch pathway activators, an effective amount or concentration of one or more interleukin-6 family cytokines, and an effective amount or concentration of one or more platelet-derived growth factor proteins, and culturing the plated vertebrate pluripotent stem cells in the second culture medium for approximately 168 to 360 hours, thereby producing radial glia-like cells. The vertebrate pluripotent stem cells may be induced pluripotent stem cells or embryonic pluripotent stem cells. The vertebrate pluripotent stem cells may be human pluripotent stem cells.

[0231] In some embodiments of the methods disclosed herein, the substrate comprises vitronectin, laminin 521, Matrigel, and / or Geltrex.

[0232] In some embodiments of the methods disclosed herein, plating the vertebrate pluripotent stem cells comprises plating at a density of 2,000 to 90,000 cells / cm. 2 , 3,000~80,000 cells / cm 2 , 4,000~70,000 cells / cm 2 , 5,000~50,000 cells / cm 2 , and / or 10,000 to 30,000 cells / cm 2 This involves plating at a cell density of

[0233] In some embodiments of the methods disclosed herein, incubating the plated vertebrate pluripotent stem cells in the first culture medium comprises incubating for 12 to 48 hours.

[0234] In some embodiments of the methods disclosed herein, culturing the plated vertebrate pluripotent stem cells in the second culture medium comprises culturing for at least 5 to 20 days.

[0235] In some embodiments of the methods disclosed herein, the first culture medium is a first defined culture medium, and the first defined culture medium is E8, E8 Flex, StemFlex, mTeSR, StemFit, or mouse embryonic fibroblast (MEF) conditioned medium. In some embodiments of the methods disclosed herein, 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 effective concentrations of polyamines including putrescine, spermine, and spermidine. In some embodiments of the methods disclosed herein, the effective concentration of Chroman 1 or a derivative thereof is about 4 nM to about 80 μM, the effective concentration of Emricasan or a 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 the putrescine, spermine, and spermidine are each at a concentration of about 0.5 nM to 1 mM. In some embodiments of the methods disclosed herein, the first culture medium further comprises at least one inhibitor of Rho-associated protein kinase (ROCK). In some embodiments of the methods disclosed herein, the one or more ROCK inhibitors comprise one or more of Chroman 1 or a derivative thereof, Y27632, blebbistatin, or thiazovivin.

[0236] In some embodiments of the methods disclosed herein, during culture in the second culture medium, the cultured cells detectably express one or more radial glial cell markers approximately 4 to 10 days after initiation of culture in the second culture medium. In some embodiments of the methods disclosed herein, for example, the radial glial-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 proteins. Radial glial-like cells are multipotent stem cells that can differentiate into neuron-like cells, oligodendrocyte-like cells, and / or astrocyte-like cells. In some embodiments of the methods disclosed herein, during culture in the second culture medium, the cultured cells detectably express one or more astrocyte markers approximately 5-20 days after initiation of culture. In some embodiments of the methods disclosed herein, the one or more astrocyte markers include S100 calcium-binding protein B (S100B). In some embodiments of the methods disclosed herein, during culture in the second culture medium, the cultured cells detectably express one or more neural stem cell markers approximately 2-10 days after initiation of culture. In some embodiments of the methods disclosed herein, the one or more neural stem cell markers may include PAX6.

[0237] In some embodiments of the methods disclosed herein, the radial glia-like cells are multipotent stem cells that can differentiate into neuron-like cells, oligodendrocyte-like cells, and / or astrocyte-like cells.

[0238] In some embodiments of the methods disclosed herein, the vertebrate pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells. In some embodiments of the methods disclosed herein, the vertebrate pluripotent stem cells are human pluripotent stem cells.

[0239] In some embodiments of the methods disclosed herein, the second culture medium may be a second defined culture medium (e.g., but not limited to, DMEM-F12, E6, Neurobasal medium, or Minimum Essential Medium (MEM)). In some embodiments of the methods disclosed herein, the second defined culture medium may include an N2 supplement and / or a B27 supplement without vitamin A. In some embodiments of the methods disclosed herein, the one or more BMP pathway inhibitors included in the second culture medium may include one or more of LDN-193189, LDN-214117, LDN-212854, DMH2, ML347, UK383367, K02288, dorsomorphin, noggin, chordin, follistatin, or gremlin. For example, in some embodiments of the methods disclosed herein, the effective amount or concentration of the one or more BMP pathway inhibitors may include 2 nM to 40 μM LDN-193189. In some embodiments of the methods disclosed herein, the second culture medium further comprises an effective amount or concentration of one or more platelet-derived growth factor proteins. In some embodiments of the methods disclosed herein, the one or more platelet-derived growth factor proteins included in the second culture medium can be platelet-derived growth factor-AA (PDGF-AA), platelet-derived growth factor-BB (PDGF-BB), or platelet-derived growth factor-AB (PDGF-AB). In some embodiments of the methods disclosed herein, the effective amount or concentration of the one or more platelet-derived growth factor proteins is about 1 ng / mL to 800 ng / mL. In some embodiments of the methods disclosed herein, the effective amount or concentration of one or more Notch pathway activators included in the second culture medium can include Jagged 1 protein, Jagged 2 protein, and one or more of Delta-Like Protein 1 (DLL1), Delta-Like Protein 2 (DLL2), or Delta-Like Protein 3 (DLL3).For example, in some embodiments of the methods disclosed herein, the one or more Notch pathway activators in the second culture medium comprise one or both of Jagged 1 protein and Delta-Like protein 1 (DLL1) at 1 ng / mL to 800 ng / mL. In some embodiments of the methods disclosed herein, the one or more cytokines of the 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). For example, in some embodiments of the methods disclosed herein, each of one or more of oncostatin M protein, ciliary-derived neurotrophic factor protein (CNTF), and leukemia inhibitory factor protein (LIF) is present in the second culture medium at a concentration of 1 ng / mL to 800 ng / mL. In some embodiments of the methods disclosed herein, the second culture medium can comprise 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 effective concentrations of polyamines including putrescine, spermine, and spermidine. In some embodiments of the methods disclosed herein, the effective concentration of Chroman 1 or a derivative thereof can be about 4 nM to about 80 μM, the effective concentration of Emricasan or a derivative thereof can be about 100 nM to about 80 μM, the effective concentration of trans-ISRIB can be about 50 nM to about 80 μM, and the putrescine, spermine, and spermidine can each be present at a concentration of about 0.5 nM to 1 mM. In some embodiments of the methods disclosed herein, the step of culturing in the second culture medium can include changing the second culture medium approximately every 20 to 28 hours. In some embodiments of the methods disclosed herein, the step of culturing in the second culture medium can include one or more passaging steps when the cultured cells become confluent. For example, in some embodiments of the methods disclosed herein, one or more passaging steps can be performed at a ratio of 1:3 to 1:5 of confluent cell culture:fresh medium.The step of culturing in the second culture medium can include 3 to 7 passage steps.

[0240] An exemplary embodiment of the present invention includes a method for producing astrocyte-like cells in culture from radial glia-like cells produced according to a method in accordance with an embodiment of the present invention.

[0241] In one embodiment, a method for producing astrocyte-like cells in culture includes performing at least one of the methods disclosed herein, and after the step of generating radial glia-like cells, culturing the radial glia-like cells for approximately 5 to 30 days in a third culture medium, an effective amount or concentration of one or more Notch pathway activators, and an effective amount or concentration of one or more interleukin-6 (IL-6) family cytokines, thereby generating a culture of astrocyte-like cells. In some embodiments of the methods disclosed herein, the third culture medium can be a third defined culture medium (e.g., but not limited to, DMEM-F12, Neurobasal medium, Minimum Essential Medium (MEM), or BrainPhys Neuronal Medium). In some embodiments of the methods disclosed herein, the third defined culture medium can include N2 supplement and / or complete B27 supplement.

[0242] In some embodiments of the methods disclosed herein, the one or more Notch pathway activators included in the third culture medium can include 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). For example, in some embodiments of the methods disclosed herein, the effective amount or concentration of the one or more Notch pathway activators in the third culture medium can include 1 ng / mL to 800 ng / mL of Jagged 1 protein and / or 1 ng / mL to 800 ng / mL of Delta-Like protein 1 (DLL1). In some embodiments of the methods disclosed herein, the one or more cytokines of the interleukin-6 family in the third culture medium include one or more of oncostatin M protein, ciliary-derived neurotrophic factor protein (CNTF), and leukemia inhibitory factor protein (LIF). For example, in some embodiments of the methods disclosed herein, an effective amount or concentration of each of oncostatin M protein, ciliary-derived neurotrophic factor (CNTF) protein, and leukemia inhibitory factor (LIF) protein can be present in the third culture medium at a concentration of 1 to 800 ng / mL.

[0243] In some embodiments of the methods disclosed herein, the third culture medium can comprise 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 effective concentrations of polyamines including putrescine, spermine, and spermidine. In some embodiments of the methods disclosed herein, the effective concentration of Chroman 1 or a derivative thereof can be about 4 nM to about 80 μM, the effective concentration of Emricasan or a derivative thereof can be about 100 nM to about 80 μM, the effective concentration of trans-ISRIB can be about 50 nM to about 80 μM, and the putrescine, spermine, and spermidine can each be present at a concentration of about 0.5 nM to 1 mM. In some embodiments of the methods disclosed herein, the step of culturing in the third culture medium can include changing the third culture medium approximately every 24 to 72 hours. In some embodiments of the methods disclosed herein, the step of culturing in the third culture medium can include one or more passaging steps when the cultured cells become confluent. In some embodiments of the methods disclosed herein, the one or more passaging steps can be performed at a ratio of 1:2 of confluent cell culture to fresh medium. In some embodiments of the methods disclosed herein, the step of culturing in the third culture medium can include one to three passaging steps. In some embodiments of the methods disclosed herein, during the step of culturing in the third culture medium, detectable neuron-like cells are present at 10% or less of the total cells in the culture.

[0244] In some embodiments of the methods disclosed herein, the entire astrocyte differentiation procedure was performed as a monolayer. In some embodiments, the entire astrocyte differentiation procedure was performed to include a sphere formation step. During these procedures, the sphere formation step on day 14 served to mature the cells and reduce the cell passaging step. Specifically, single cell dissociation was performed on day 14, and the cells were maintained in suspension in Astro-2 medium containing CEPT for 24 hours to form spheres (100,000 cells / well (U-bottom 96-well plate)). One day later, the spheres were then transferred to a container with a low cell attachment surface containing Astro-2 medium. Medium changes were performed every other day. After one week in Astro-2 medium, Astro-3, i.e., concentrated Astro-3 medium, was introduced, which contained DMEM / F12 medium supplemented with N2 B27 complete chemically defined lipid concentrate (2%), LIF (10 ng / ml), and CNTF (10 ng / ml). In some embodiments of the methods disclosed herein, enriched Astro-3 medium was prepared by supplementing DMEM / F12 medium with N2B27 complete, chemically defined lipid concentrate (2%), LIF (10 ng / ml), and CNTF (10 ng / ml), Jagged 1 (10 ng / ml), DLL-1 (10 ng / ml), triiodothyronine (also known as T3; thyroid hormone) (40 ng / ml), phorbol ester (200 nM), forskolin 2 μM, neuregulin-1 (20 ng / ml), and ascorbic acid (200 μM). Spheres were cultured in Astro-3 or enriched Astro-3 medium for an additional week, with medium changes every other day. On day 28, spheres were dissociated into single cells by Accutase treatment, and astrocytes were maintained as monolayer cultures in Astro-3 or enriched Astro-3 medium for up to day 50.

[0245] Astrocyte-like cells produced by methods according to embodiments of the present invention detectably express one or more astrocyte markers. In some embodiments of the methods disclosed herein, the one or more astrocyte markers can include S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), glial fibrillary acidic protein (GFAP), and vimentin (VIM). In some embodiments of the methods disclosed herein, astrocyte-like cells produced by methods according to embodiments of the present invention can exhibit a flattened and / or stellate shape.

[0246] In some embodiments disclosed herein, the third culture medium further comprises a chemically defined lipid concentrate 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 at a concentration of approximately 2%, or further comprises fetal bovine serum at a concentration of approximately 2%.

[0247] As discussed throughout this disclosure, some embodiments of the methods of the present invention produce astrocyte-like cells exhibiting stellate and / or spherical morphology. For example, in another embodiment of the present invention, an exemplary method for culturing astrocyte-like cells comprises performing at least one of the methods disclosed herein and further culturing the astrocyte-like cells in a fourth culture medium and an effective amount or concentration of one or more interleukin-6 family cytokines, thereby enhancing maturation of the astrocyte-like cells. In some embodiments of the methods disclosed herein, the fourth culture medium may be a fourth defined culture medium (e.g., but not limited to, DMEM-F12, E6, Neurobasal medium, or Minimum Essential Medium (MEM)). In some embodiments of the methods disclosed herein, the fourth defined culture medium may include an N2 supplement and / or a B27 supplement. In some embodiments of the methods disclosed herein, the one or more interleukin-6 family cytokines include one or both of ciliary-derived neurotrophic factor protein (CNTF) and leukemia inhibitory factor protein (LIF). For example, in some embodiments of the methods disclosed herein, an effective amount or concentration of one or both of ciliary-derived neurotrophic factor protein (CNTF) and leukemia inhibitory factor protein (LIF) is present at a concentration of 1 to 800 ng / mL. In some embodiments disclosed herein, the fourth culture medium is optionally an enriched, defined fourth culture medium containing an effective amount or concentration of one or more Notch pathway activators, and / or one or more thyroid hormones, phorbol esters, forskolin, neuregulin, and ascorbic acid. In some embodiments of the methods disclosed herein, the thyroid hormone is triiodothyronine, and the one or more Notch pathway activators in the fourth culture medium include one or more of Jagged 1 protein and Delta-Like protein 1 (DLL1).In some embodiments of the methods disclosed herein, the one or more Notch pathway activators are about 1 ng / mL to about 800 ng / mL of Jagged 1 protein and 1 ng / mL to about 800 ng / mL of Delta-Like Protein 1 (DLL1), the thyroid hormone concentration is about 1 ng / mL to about 1000 ng / mL, the phorbol ester concentration is about 1 nM to about 1000 nM, the forskolin concentration is about 1 μM to about 200 μM, the neuregulin concentration is about 1 ng / mL to about 1000 ng / mL, and the ascorbic acid concentration is about 1 μM to about 1000 μM.

[0248] In some embodiments of the methods disclosed herein, the fourth culture medium can comprise 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 effective concentrations of polyamines including putrescine, spermine, and spermidine. In some embodiments of the methods disclosed herein, the effective concentration of Chroman 1 or a derivative thereof can be about 4 nM to about 80 μM, the effective concentration of Emricasan or a derivative thereof can be about 100 nM to about 80 μM, the effective concentration of trans-ISRIB can be about 50 nM to about 80 μM, and the putrescine, spermine, and spermidine can each be present at a concentration of about 0.5 nM to 1 mM. In some embodiments of the methods disclosed herein, culturing in the fourth culture medium is carried out for at least approximately 40 to 60 hours. In some embodiments of the methods disclosed herein, culturing in the fourth culture medium can include changing the fourth culture medium approximately every 24 to 96 hours. In some embodiments of the methods disclosed herein, during culture in the fourth culture medium, the astrocyte-like cells detectably express one or more of hepatic glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, and vimentin (VIM). As discussed above, during culture in the fourth culture medium, the astrocyte-like cells exhibit a stellate and / or spherical morphology.

[0249] In some embodiments of the methods disclosed herein, one or more steps of the method are performed by an automated system. In some embodiments disclosed herein, the fourth culture medium further comprises a chemically defined lipid concentrate 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 at a concentration of approximately 2%, or further comprises fetal bovine serum at a concentration of approximately 2%.

[0250] In another embodiment, the composition comprises radial glia-like cells that detectably express one or more of brain lipid-binding protein (BLBP), CD133 (prominin 1), abnormal spindle-like microcephaly-associated protein (ASPM), baculovirus 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 proteins. In some embodiments of the compositions disclosed herein, the at least one cultured radial glia-like cell is or has been cryopreserved in a cryopreservation medium comprising, for example, Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine. In some embodiments of the compositions disclosed herein, the concentration of Chroman 1 and / or its derivatives can be about 4 nM to about 80 μM, the concentration of Emricasan and / or its derivatives can be about 100 nM to about 80 μM, the concentration of trans-ISRIB can be about 50 nM to about 80 μM, and the concentrations of each of putrescine, spermine, and spermidine can be about 0.5 μM to 1 mM in the cryopreservation medium.

[0251] In some embodiments of the compositions disclosed herein, the composition comprises at least one cultured radial glia-like cell that detectably expresses 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. In some embodiments of the compositions disclosed herein, the at least one cultured radial glia-like cell is or has been cryopreserved. In some embodiments of the compositions disclosed herein, at least one cultured radial glia-like cell is or has been cryopreserved in a cryopreservation medium comprising Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and a polyamine comprising putrescine, spermine, and spermidine. In some embodiments of the compositions disclosed herein, the concentration of Chroman 1 and / or a derivative thereof is or has been about 4 nM to about 80 μM, the concentration of Emricasan and / or a derivative thereof is or has been about 100 nM to about 80 μM, the concentration of trans-ISRIB is or has been about 50 nM to about 80 μM, and the concentrations of each of putrescine, spermine, and spermidine are or have been about 0.5 μM to 1 mM in the cryopreservation medium.

[0252] In another embodiment, the composition comprises at least one cultured radial glia-like cell produced by the methods disclosed herein 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.

[0253] In another embodiment, the cell culture comprises 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), and PAX6 protein. In some embodiments of the cell cultures disclosed herein, the cell culture can be grown from previously cryopreserved cells, for example, from cells cryopreserved in a cryopreservation medium comprising Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine. In some embodiments of the cell cultures disclosed herein, the previously cryopreserved cells may be vertebrate pluripotent stem cells (e.g., induced pluripotent stem cells or embryonic pluripotent stem cells). In some embodiments of the cell cultures disclosed herein, the vertebrate pluripotent stem cells may be human pluripotent stem cells. In some embodiments of the cell cultures disclosed herein, the previously cryopreserved cells may be cultured radial glia-like cells that detectably express 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 (HES5), SRY-Box transcription factor 21 (SOX21), and PAX6 proteins.

[0254] In another embodiment, the cell culture is produced by the methods disclosed herein and comprises at least one cultured radial glia-like cell that expresses 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.

[0255] In another embodiment, the composition comprises at least one cultured astrocyte-like cell exhibiting a flat, stellate, and / or spherical morphology and detectably expressing at least one marker, wherein the at least one marker is S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM). In some embodiments of the compositions disclosed herein, the at least one cultured astrocyte-like cell is or has been cryopreserved in a cryopreservation medium comprising, for example, Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine. In some embodiments of the compositions disclosed herein, in the cryopreservation medium, the concentration of Chroman 1 and / or its derivatives is or was about 4 nM to about 80 μM, the concentration of Emricasan and / or its derivatives is or was about 100 nM to about 80 μM, the concentration of trans-ISRIB is or was about 50 nM to about 80 μM, and the concentrations of each of putrescine, spermine, and spermidine are or was about 0.5 μM to 1 mM.

[0256] In another embodiment, the composition comprises at least one cultured astrocyte-like cell produced by the methods disclosed herein, expressing at least one marker and exhibiting a flat, stellate, and / or spherical morphology, wherein the at least one marker is S100 calcium binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic-glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM).

[0257] In another embodiment, the cell culture comprises at least one cultured astrocyte-like cell exhibiting a flat, stellate, and / or spherical morphology and detectably expressing at least one marker, wherein the at least one marker is S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM). In some embodiments of the cell cultures disclosed herein, detectable neuron-like cells may be present at 10% or less of the total cells in culture. In some embodiments of the cell cultures disclosed herein, the cell cultures may be grown from previously cryopreserved cells, for example, from cells cryopreserved in a cryopreservation medium containing Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine. In some embodiments of the cell cultures disclosed herein, the previously cryopreserved cells may be vertebrate pluripotent stem cells (e.g., induced pluripotent stem cells or embryonic pluripotent stem cells). In some embodiments of the cell cultures disclosed herein, the vertebrate pluripotent stem cells may be human pluripotent stem cells. In some embodiments of the cell cultures disclosed herein, the previously cryopreserved cells may be cultured radial glia-like cells that detectably express 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 proteins.In some embodiments of the cell cultures disclosed herein, the previously cryopreserved cells are astrocyte-like cells that exhibit flat, stellate, and / or spherical morphology and detectably express one or more of S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), CD44, HEPACAM, glial fibrillary acidic protein (GFAP), and vimentin (VIM).

[0258] In another embodiment, the cell culture produced by the methods disclosed herein comprises at least one cultured astrocyte-like cell that detectably expresses at least one marker and exhibits a flat, stellate, and / or spherical morphology, wherein the at least one marker is S100 calcium binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepato-glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM).

[0259] The present invention is further described in the following examples, which do not limit the scope of the invention as defined in the claims. [Example]

[0260] The following examples illustrate certain embodiments of the present invention and various uses thereof. The examples are provided for illustrative purposes only and should not be construed as limiting the invention.

[0261] Example 1: Differentiation of human pluripotent stem cells into radial glia-like cells and astrocyte-like cells The procedure for differentiating human pluripotent stem cells into radial glia-like cells and astrocyte-like cells is shown schematically in Figures 1 and 2. Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), were maintained and expanded in defined E8 medium. Human ESC lines were purchased from WiCell (Madison, Wisconsin), and iPSCs were generated by the NIH. hPSCs were grown as adherent monolayer cultures. At the start of the differentiation procedure ("day -1"), a defined number of hPSCs (approximately 10,000 cells / cm) were cultured. 2 ) were plated on vitronectin-coated surfaces in culture vessels and allowed to recover for 1 day in E8 medium supplemented with CEPT to ensure consistent cell protection and optimal cell survival after cell dissociation. To initiate cell differentiation on "Day 0," the supplemented E8 medium was replaced with Astro 1 medium (see Figure 2), which was changed daily for 15 days. Once cells reached confluence (typically occurring 4–5 times during the 15-day differentiation period), they were passaged (at a 1:3 ratio). On "Day 15," the culture medium was switched to "Astro 2 medium" (see Figure 2). From "Day 15" to "Day 30," the cells were cultured in Astro 2 medium with daily changes. Around "Day 23," the cells were passaged once (at a 1:2 ratio) due to a decline in cell proliferation activity. On "Day 30," the cells were switched to Astro 3 medium (see Figure 2) for maturation, with medium changes every 3 days. During the above culture procedure, cells were passaged by exposing them to Accutase for 7 minutes at each passage. Exemplary images of cells at different time points in the above differentiation procedure are shown in Figure 1B.

[0262] Example 2: Immunochemical characterization of radial glia-like and astrocyte-like cells derived from human iPSCs Cells produced by the procedure described in Example 1 exhibited highly efficient differentiation into astrocyte-like cells, as demonstrated by the figures discussed below. Figure 3 shows representative images of cells at different time points during the differentiation procedure discussed in Example 1. In Figure 3, images labeled "Phase" are phase-contrast microscopy images. Images labeled with specific protein names, discussed below, are photomicrographs of cells immunochemically stained with antibodies (both monoclonal and polyclonal) specific for the indicated proteins. The procedure was performed using human iPSCs. The images demonstrate highly efficient and controlled differentiation of human iPSCs into specific cell types. As shown in Figure 3A, differentiating cells expressed the neural stem cell marker paired box protein Pax-6 (PAX6) at "day 5," followed by the radial glia marker brain lipid-binding protein (BLBP) at "day 7." At "day 15," there was widespread expression of the astrocyte marker S100 calcium-binding protein B (S100B). As shown in Figure 3B, at "day 30," the cultures consisted essentially of large cells with a flat morphology that expressed typical astrocyte markers S100B, nuclear factor type 1 A (NFIA), CD44, HEPACAM, glial fibrillary acidic protein (GFAP), and vimentin (VIM). Antibodies against the neuronal marker beta-III tubulin (TUJ1) detected only a small percentage of cells in the culture. As shown in Figure 3C, astrocyte-like cells generated by the differentiation procedure were cryopreserved at "day 30" or cultured for an additional 20 days and passaged twice, leading to further cell maturation, as indicated by stellate morphology and expression of hepatic-glial cell adhesion molecule (HEPACAM), CD44, glial fibrillary acidic protein (GFAP), and NFIA.

[0263] Quantitative analysis of cell cultures generated from hPSCs according to the procedure described in Example 1 demonstrates highly efficient differentiation in such cultures, as shown in Figure 4. At "day 30," cells were stained for the astrocyte markers NFIA and S100B and the neuronal marker TUJ1. It was found that the majority of cells in the cultures were astrocyte-like cells expressing NFIA and S100B, with only sporadic production of neuron-like cells.

[0264] Western blot analysis of differentiated cells generated from hPSCs according to the procedure described in Example 1 demonstrated the superiority of the described procedure over previously described procedures relying on a dual-SMAD inhibition strategy (Chambers et al., "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling," Nat. Biotechnol. 27(3):275-280 (2009) and Tchieu et al., "NFIA is a gliogenic switch enabling rapid derivation of functional human astrocytes from pluripotent stem cells," Nat. Biotechnol. 37:267-275 (2019)). The results of the Western blot analysis are shown in Figure 5A. To compare the two procedures, hPSCs were exposed to Dual-SMADi or Astro 1 medium for 7 days and then analyzed for the expression of the radial glia marker BLBP. The "housekeeping" protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control to demonstrate similar protein loading in the lanes of the gel used to generate Western blots. Three replicate experiments demonstrated that Astro-1 medium induces much stronger BLBP expression in cultured cells than the dual-SMAD inhibition strategy. The multipotency of radial glia-like cells was confirmed by differentiation of BLBP-positive cells into OLIG2-positive precursors of oligodendrocytes, S100B-positive astrocytes, and MAP2-positive neurons, as shown in Figure 5B. For differentiation into OLIG2-positive precursors of oligodendrocytes, radial glia-like cells were expanded for three passages in DMEM / F12 medium supplemented with N2 and B27 (without vitamin A), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).For differentiation into MAP2-positive neurons, radial glial cells were maintained under high cell density conditions in ASTRO1 medium for 5 days and then subcultured at high density for an additional 15 days in N2 and B27 (with vitamin A) supplemented DMEM medium supplemented with brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), ascorbic acid (AA), and cyclic adenosine monophosphate (cAMP). Differentiation into astrocytes was performed using methods and embodiments of the present invention.

[0265] Example 3: Time course gene expression profiling Cells produced according to the procedure described in Example 1 were characterized by time-course gene expression profiling (shown in the figures discussed below) performed by RNA sequencing ("RNA-seq") analysis. Time-course gene expression profiling by RNA-seq for hPSC differentiation is shown in Figure 6. Systematic analysis of gene expression from days 0 to 30 demonstrated the gradual and controlled differentiation of hPSCs into radial glia-like and astrocyte-like cells. Native astrocytes are known to support neuronal cell function and survival by secreting important neurotrophic and synaptogenic proteins. As shown in the bottom of Figure 6, transcription of several important astrocyte-secreted proteins (e.g., BDNF, SEMA3A, and THBS1) was induced in cell cultures from days 21 to 30.

[0266] The results of time-course gene expression profiling by RNA-seq of cells produced according to the procedure described in Example 1 were compared with results available in public databases. Figure 7 shows the results of this comparison. For gene ontology analysis, the top 200 upregulated genes at each time point shown in Figure 7 ("Day 0," "Day 7," "Day 14," "Day 21," and "Day 30") were compared using the web-based tool EnrichR, available online from the Mount Sinai Center for Bioinformatics, Icahn School of Medicine at Mount Sinai (New York, New York, USA), and the ARCHS tool, also available online from the Mount Sinai Center for Bioinformatics, Icahn School of Medicine at Mount Sinai. 4 The results were compared with the human RNA-seq database. The top 10 gene up-regulation profiles at each time point are plotted in Figure 7. For the cultured cells generated according to the procedure described in Example 1, "astrocytic" was the top at both "day 21" and "day 30," confirming the astrocyte-like nature of the generated cells.

[0267] Comparative single-cell analysis and gene expression profiling of cells generated from iPSCs using the procedures described in Example 1 was performed, and the results are shown in Figure 8. Single-cell RNA-seq and comparison of the results with other cell types shown in Figure 8 (pluripotent stem cells, neuroectoderm, neurons, oligodendrocytes, microglia, and endothelial cells) confirmed that at "day 30," iPSC-derived cells exhibited a gene expression signature characteristic of human astrocytes.

[0268] Example 4: Functional characterization of astrocyte-like cells derived from iPSCs Functional analysis of astrocyte cells derived from iPSCs according to the procedure described in Example 1 was performed. The results are shown in Figures 9-11 and discussed below. Figure 9A shows exemplary microscopic images demonstrating comparable glycogen accumulation capacity between iPSC-derived astrocyte-like cells derived according to the procedure described in Example 1 ("SCTL iPSC Astro") and commercially available iPSC-derived astrocyte-like cells ("Commercial iPSC Astro" obtained from Fujifilm Cellular Dynamics International). Figure 9B shows a bar graph illustrating the basal level of glutamate in the culture medium and the reduction in glutamate levels in the culture medium after 3 hours of incubation with astrocytes. Glutamate concentration was quantified by an enzymatic assay that produced a colorimetric product in an amount proportional to glutamate levels. The data shown in Figure 9B demonstrate that iPSC-derived astrocyte-like cells derived according to the procedure described in Example 1 are capable of glutamate uptake, consistent with the functional role of native astrocytes in the human brain.

[0269] Figure 10 illustrates experimental results demonstrating that iPSC-derived astrocyte-like cells derived according to the procedure described in Example 1 promoted neuronal maturation and synaptic activity. To generate the data shown in Figure 10A, neuronal cells were derived from a human ESC reporter cell line (SYN1:GFP; green fluorescent protein expressed under the control of the synapsin 1 promoter) and cultured for 13 days with or without iPSC-derived astrocyte-like cells derived according to the procedure described in Example 1. The neurons showed high levels of synapsin 1 expression when cocultured with iPSC-derived astrocyte-like cells, demonstrating their ability to promote synaptic maturation. Figure 10B illustrates the results of a multielectrode array experiment (Axion Biosystems), demonstrating that glutamatergic neurons procured from Fujifilm Cellular Dynamics International exhibited increased spike counts and functional activity when cocultured with iPSC-derived astrocyte-like cells for 72 hours.

[0270] Figure 11 illustrates the neuroprotective effect of iPSC-derived astrocyte-like cells derived according to the procedure described in Example 1. Multielectrode array experiments were performed using the Maestro APEX system (Axion Biosystems). The bars in the graph in the lower panel of Figure 11 represent representative data points. It is well known that high concentrations of glutamate in the extracellular space can damage and kill neuronal cells (excitotoxicity). Indeed, excitotoxicity is considered an important contributing factor in various neurodegenerative diseases, such as amyotrophic lateral sclerosis (also known as Lou Gehrig's disease). To model this aspect of neurodegenerative disease, motor neurons procured from Fujifilm Cellular Dynamics International were cultured with or without iPSC-derived astrocyte-like cells for 7 days until they achieved electrical activity, as measured by spike counts (baseline). On day 7, treatment with 100 μM glutamate was administered for 1 hour, and spike counts were measured again (100 μM glutamate). The activity of motor neurons cultured without astrocytes was reduced when treated with 100 μM glutamate. Multielectrode array experiments demonstrated that iPSC-derived astrocyte-like cells protected motor neurons from the toxic effects of glutamate.

[0271] Example 5: Automated Procedure The procedure described in Example 1 was used as the basis for an automated procedure (shown in Figure 12) by using the CompacT SelecT® system (Sartorius, Wilmington, USA). Highly efficient, standardized, and scalable production of astrocyte-like cells from iPSCs was achieved using the automated procedure. Figure 12A shows a schematic of the automated protocol. Figure 12B shows a representative microscopic image of the cell culture at "day 30" of the automated procedure.

[0272] Example 6: Sphere formation enhances astrocyte maturation As shown schematically in Figure 13A-C, a sphere formation procedure was used to enhance astrocyte maturation. The entire astrocyte differentiation procedure was performed as a monolayer or, as described below, included a sphere formation step. The sphere formation step on day 14 resulted in cell maturation and reduced cell passaging steps. Single cell dissociation was performed on day 14, and the cells were maintained in suspension in Astro-2 medium containing CEPT for 24 hours to form spheres (100,000 cells / well (U-bottom 96-well plate)). One day later, the spheres were then transferred to a container with a low cell attachment surface containing Astro-2 medium. Medium changes were performed every other day. After one week in Astro-2 medium, Astro-3, i.e., concentrated Astro-3 medium, was introduced, which contains DMEM / F12 medium supplemented with N2 B27 complete chemically defined lipid concentrate (2%), LIF (10 ng / ml), and CNTF (10 ng / ml). Enriched Astro-3 medium was prepared by supplementing DMEM / F12 medium with N2B27 complete, chemically defined lipid concentrate (2%), LIF (10 ng / ml), and CNTF (10 ng / ml), Jagged 1 (10 ng / ml), DLL-1 (10 ng / ml), triiodothyronine (also known as T3; thyroid hormone) (40 ng / ml), phorbol ester (200 nM), forskolin (2 μM), neuregulin-1 (20 ng / ml), and ascorbic acid (200 μM). Spheres were cultured in Astro-3 or enriched Astro-3 medium for an additional week, with medium changes every other day. On day 28, spheres were dissociated into single cells by Accutase treatment, and astrocytes were maintained as monolayer cultures in Astro-3 or enriched Astro-3 medium until day 50.

[0273] While the present invention has been described in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention as defined in the appended claims. More specifically, while certain aspects of the invention have been identified herein as particularly advantageous, it is intended that the invention not necessarily be limited to these particular aspects.

Claims

1. 1. A method for producing radial glia-like cells in culture, said method comprising: (a) Vertebrate pluripotent stem cells are cultured on the substrate-coated surface of a culture vessel at a density of 1,000 to 100,000 cells / cm. ２ and plating at a density of (b) incubating the plated vertebrate pluripotent stem cells in a first culture medium; (c) replacing the first culture medium with a second culture medium, the second culture medium comprising: (i) an effective amount or concentration of one or more BMP pathway inhibitors; (ii) an effective amount or concentration of one or more Notch pathway activators; and (iii) the replacing comprises one or more cytokines of the interleukin-6 family; (d) culturing the plated vertebrate pluripotent stem cells in the second culture medium; This method thereby produces radial glia-like cells.

2. 10. The method of claim 1, wherein the substrate comprises vitronectin, laminin 521, Matrigel, and / or Geltrex.

3. Plating vertebrate pluripotent stem cells to a density of 2,000-90,000 cells / cm ２ , 3,000-80,000 cells / cm ２ , 4,000-70,000 cells / cm ２ , 5,000-50,000 cells / cm ２ and / or 10,000 to 30,000 cells / cm ２ 2. The method of claim 1, comprising plating at a cell density of

4. 10. The method of claim 1, wherein incubating the plated vertebrate pluripotent stem cells in the first culture medium comprises incubating for 12 to 48 hours.

5. 10. The method of claim 1, wherein culturing the plated vertebrate pluripotent stem cells in the second culture medium comprises culturing for at least 5 to 20 days.

6. 2. The method of claim 1, wherein the first culture medium is a first defined culture medium, and the first defined culture medium is E8, E8 Flex, StemFlex, mTeSR, StemFit, or mouse embryonic fibroblast (MEF) conditioned medium.

7. 7. The method of claim 1, 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 effective concentrations of polyamines including putrescine, spermine, and spermidine.

8. 8. The method of claim 7, wherein the effective concentration of Chroman 1 or a derivative thereof is about 4 nM to about 80 μM, the effective concentration of Emricasan or a 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 putrescine, spermine, and spermidine are each at a concentration of about 0.5 nM to 1 mM.

9. The method of claim 1 , wherein the first culture medium further comprises at least one inhibitor of Rho-associated protein kinase (ROCK).

10. 10. 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.

11. 11. The method of any one of claims 1 to 10, wherein during the culturing in the second culture medium, the cultured cells detectably express one or more radial glial cell markers approximately 4 to 10 days after initiation of the culturing in the second culture medium.

12. 12. The method of any one of claims 1 to 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), baculovirus 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 proteins.

13. 13. The method of any one of claims 1 to 12, wherein during the culturing in the second culture medium, the cultured cells detectably express one or more astrocyte markers approximately 5 to 20 days after initiation of the culturing.

14. 14. The method of claim 13, wherein the one or more astrocyte markers include S100 calcium binding protein B (S100B).

15. 15. The method of any one of claims 1 to 14, wherein during the culturing in the second culture medium, the cultured cells detectably express one or more neural stem cell markers approximately 2 to 10 days after initiation of the culturing.

16. 16. The method of claim 15, wherein the one or more neural stem cell markers include PAX6.

17. The method according to any one of claims 1 to 16, wherein the radial glia-like cells are multipotent stem cells that can differentiate into neuron-like cells, oligodendrocyte-like cells, and / or astrocyte-like cells.

18. The method according to any one of claims 1 to 17, wherein the vertebrate pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells.

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

20. 20. The method of any one of claims 1 to 19, wherein the second culture medium is a second defined culture medium, and the second defined culture medium is DMEM-F12, E6, Neurobasal medium, or Minimum Essential Medium (MEM).

21. 21. The method of claim 20, wherein the second defined culture medium comprises an N2 supplement and / or a B27 supplement that does not contain vitamin A.

22. 22. The method of any one of claims 1-21, wherein the one or more BMP pathway inhibitors comprise one or more of LDN-193189, LDN-214117, LDN-212854, DMH2, ML347, UK383367, K02288, dorsomorphin, noggin, chordin, follistatin, or gremlin.

23. 23. The method of any one of claims 1 to 22, wherein the effective amount or concentration of the one or more BMP pathway inhibitors comprises 2 nM to 40 μM LDN-193189.

24. The method of any one of claims 1 to 23, wherein the second culture medium further comprises an effective amount or concentration of one or more platelet-derived growth factor proteins.

25. 25. The method of claim 24, wherein the one or more platelet-derived growth factor proteins are platelet-derived growth factor-AA (PDGF-AA), platelet-derived growth factor-BB (PDGF-BB), or platelet-derived growth factor-AB (PDGF-AB).

26. 26. The method of any one of claims 1 to 25, wherein the effective amount or concentration of the one or more platelet-derived growth factor proteins is from about 1 ng / mL to 800 ng / mL.

27. 27. The method of any one of claims 1-26, wherein the effective amount or concentration of the one or more Notch pathway activators in the second culture medium comprises 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).

28. 28. The method of any one of claims 1-27, wherein the one or more Notch pathway activators in the second culture medium comprise one or both of 1 ng / mL to 800 ng / mL Jagged 1 protein and 1 ng / mL to 800 ng / mL Delta-Like protein 1 (DLL1).

29. 29. The method of any one of claims 1 to 28, wherein the one or more interleukin-6 family cytokines 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).

30. 30. The method of claim 29, wherein each of the one or more of oncostatin M protein, ciliary-derived neurotrophic factor protein (CNTF), and leukemia inhibitory factor protein (LIF) is present in the second culture medium at a concentration of 1 ng / mL to 800 ng / mL.

31. 31. The method of any one of claims 1 to 30, wherein said culturing in said second culture medium comprises changing said second culture medium approximately every 20 to 28 hours.

32. The method according to any one of claims 1 to 31, wherein the culturing in the second culture medium comprises one or more steps of passaging the cultured cells when they become confluent.

33. 33. The method of claim 32, wherein the one or more steps of passaging the cells are performed at a ratio of 1:3 to 1:5 of confluent cell culture:fresh medium.

34. The method of claim 32 or 33, wherein said culturing in said second culture medium comprises 3 to 7 of said passage steps.

35. 35. A method for producing a culture of astrocyte-like cells, comprising performing the method of any one of claims 1 to 34, and after the step of generating radial glia-like cells, culturing the radial glia-like cells for approximately 5 to 30 days in a third culture medium, an effective amount or concentration of one or more Notch pathway activators, and an effective amount or concentration of one or more interleukin-6 (IL-6) family cytokines, thereby generating the culture of astrocyte-like cells.

36. 36. The method of claim 35, wherein the third culture medium is a third defined culture medium.

37. 37. The method of claim 36, wherein the third defined culture medium is DMEM-F12, Neurobasal medium, Minimum Essential Medium (MEM), or BrainPhys neuronal medium.

38. 38. The method of claim 36 or 37, wherein the third defined culture medium comprises N2 supplement and / or complete B27 supplement.

39. 39. The method of any one of claims 37-38, wherein the one or more Notch pathway activators 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 protein 3 (DLL3).

40. 40. The method of any one of claims 35-39, wherein the effective amount or concentration of the one or more Notch pathway activators in the third culture medium comprises between 1 ng / mL and 800 ng / mL of Jagged 1 protein and between 1 ng / mL and 800 ng / mL of Delta-Like protein 1 (DLL1).

41. 41. The method of any one of claims 35 to 40, wherein the one or more interleukin-6 family cytokines 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).

42. 42. The method of claim 41, wherein the effective amount or concentration of each of the one or more of oncostatin M protein, ciliary-derived neurotrophic factor protein (CNTF), and leukemia inhibitory factor protein (LIF) is present in the third culture medium at a concentration of 1 to 800 ng / mL.

43. 43. The method of any one of claims 35 to 42, wherein said culturing in said third culture medium comprises changing said third culture medium approximately every 24 to 72 hours.

44. The method according to any one of claims 35 to 43, wherein the culturing in the third culture medium comprises one or more steps of passaging the cultured cells when they become confluent.

45. 45. The method of claim 44, wherein the one or more passaging steps are performed at a ratio of 1:2 of confluent cell culture:fresh medium.

46. 46. ​​The method of claim 44 or 45, wherein said culturing in said third culture medium comprises 1 to 3 passage steps.

47. 47. The method of any one of claims 35 to 46, wherein the astrocyte-like cells detectably express one or more astrocyte markers.

48. 48. The method of claim 47, wherein the one or more astrocyte markers comprise S100 calcium binding protein B (S100B), nuclear factor 1 type A protein (NFIA), glial fibrillary acidic protein (GFAP), and vimentin (VIM).

49. The method according to any one of claims 35 to 48, wherein the astrocyte-like cells exhibit a flattened and / or stellate morphology.

50. 50. The method of any one of claims 35 to 49, wherein during said culturing in said third culture medium, detectable neuron-like cells are present at 10% or less of the total cells in culture.

51. 51. The method of any one of claims 35 to 50, wherein the third culture medium further comprises a chemically defined lipid concentrate 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 at a concentration of approximately 2%, or further comprises fetal bovine serum at a concentration of approximately 2%.

52. 52. A method for culturing astrocyte-like cells, comprising performing the method of any one of claims 35 to 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 the interleukin-6 family, thereby enhancing maturation of the astrocyte-like cells.

53. 53. The method of claim 52, wherein the fourth culture medium is a fourth defined culture medium.

54. 54. The method of claim 53, wherein the fourth defined culture medium is DMEM-F12, E6, Neurobasal medium, or Minimum Essential Medium (MEM).

55. 55. The method of claim 53 or 54, wherein the fourth defined culture medium comprises an N2 supplement and / or a B27 supplement.

56. 56. The method of any one of claims 52 to 55, wherein the one or more cytokines of the interleukin-6 family comprise one or both of ciliary-derived neurotrophic factor protein (CNTF) and leukemia inhibitory factor protein (LIF).

57. 57. The method of claim 56, wherein the effective amount or concentration of one or both of ciliary-derived neurotrophic factor protein (CNTF) and leukemia inhibitory factor protein (LIF) is present at a concentration of 1 to 800 ng / mL.

58. 53. The method of claim 52, wherein the fourth culture medium is an enriched fourth defined culture medium optionally comprising an effective amount or concentration of one or more Notch pathway activators, and / or one or more thyroid hormones, phorbol esters, forskolin, neuregulin, and ascorbic acid.

59. 59. The method of claim 58, wherein the thyroid hormone is triiodothyronine and the one or more Notch pathway activators in the fourth culture medium comprise one or more of Jagged 1 protein and Delta-Like protein 1 (DLL1).

60. 59. The method of claim 58, wherein the one or more Notch pathway activators are from about 1 ng / mL to about 800 ng / mL of Jagged 1 protein and from 1 ng / mL to about 800 ng / mL of Delta-Like protein 1 (DLL1); the thyroid hormone is at a concentration of from about 1 ng / mL to about 1000 ng / mL; the phorbol ester is at a concentration of from about 1 nM to about 1000 nM; the forskolin is at a concentration of from about 1 μM to about 200 μM; the neuregulin is at a concentration of from about 1 ng / mL to about 1000 ng / mL; and the ascorbic acid is at a concentration of from about 1 μM to about 1000 μM.

61. 61. The method of any one of claims 52 to 60, wherein the culturing in the fourth culture medium is carried out for at least approximately 40 to 60 hours.

62. 62. The method of any one of claims 52-61, wherein said culturing in said fourth culture medium comprises changing said fourth culture medium approximately every 24 to 96 hours.

63. 63. The method of any one of claims 52 to 62, wherein during the culture in the fourth culture medium, the astrocyte-like cells detectably express one or more of hepatic glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, and vimentin (VIM).

64. 64. The method of any one of claims 52 to 63, wherein the astrocyte-like cells exhibit a stellate and / or spherical morphology during the culture in the fourth culture medium.

65. The method of any one of claims 1 to 64, wherein one or more steps of the method are performed by an automated system.

66. 66. The method of any one of claims 52-65, wherein the fourth culture medium further comprises a chemically defined lipid concentrate 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 at a concentration of approximately 2%; or further comprises fetal bovine serum at a concentration of approximately 2%.

67. 1. A composition comprising at least one cultured radial glia-like cell that detectably expresses 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), baculovirus 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.

68. 68. The composition of claim 67, wherein the at least one cultured radial glia-like cell is or has been cryopreserved.

69. 69. The composition of claim 68, wherein the at least one cultured radial glia-like cell is or has been cryopreserved in a cryopreservation medium comprising Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine.

70. 70. The composition of claim 69, wherein, in the cryopreservation medium, the concentration of Chroman 1 and / or its derivatives is or was about 4 nM to about 80 μM, the concentration of Emricasan and / or its derivatives is or was about 100 nM to about 80 μM, the concentration of trans-ISRIB is or was about 50 nM to about 80 μM, and the concentration of each of putrescine, spermine, and spermidine is or was about 0.5 μM to 1 mM.

71. 67. A composition comprising at least one cultured radial glia-like cell produced by the method of any one of claims 1 to 66, 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.

72. 1. A cell culture comprising at least one cultured radial glia-like cell that detectably expresses 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), baculovirus 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.

73. 73. The cell culture of claim 72, wherein the cell culture has been grown from previously cryopreserved cells.

74. 74. 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 a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine.

75. 75. The cell culture of Claim 73 or 74, wherein the previously cryopreserved cells are vertebrate pluripotent stem cells.

76. 76. The cell culture of claim 75, wherein the vertebrate pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells.

77. 77. The cell culture of claim 75 or 76, wherein the vertebrate pluripotent stem cells are human pluripotent stem cells.

78. 75. The cell culture of claim 73 or 74, wherein the previously cryopreserved cells are cultured radial glia-like cells that detectably express brain lipid-binding protein (BLBP), brain lipid-binding protein (BLBP), CD133 (prominin 1), abnormal spindle-like microcephaly-associated protein (ASPM), baculovirus 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 proteins.

79. 67. A cell culture comprising at least one cultured radial glia-like cell produced by the method of any one of claims 1 to 66, wherein the cell culture comprises at least one cultured radial glia-like cell 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.

80. A composition comprising at least one cultured astrocyte-like cell exhibiting a flat, stellate, and / or spherical morphology and detectably expressing at least one marker, wherein the at least one marker is S100 calcium-binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM).

81. 81. The composition of claim 80, wherein the at least one cultured astrocyte-like cell is or has been cryopreserved in a cryopreservation medium comprising Chroman 1 and / or a derivative thereof, Emricasan and / or a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine.

82. 82. The composition of claim 81, wherein, in the cryopreservation medium, the concentration of Chroman 1 and / or its derivatives is or was about 4 nM to about 80 μM, the concentration of Emricasan and / or its derivatives is or was about 100 nM to about 80 μM, the concentration of trans-ISRIB is or was about 50 nM to about 80 μM, and the concentration of each of putrescine, spermine, and spermidine is or was about 0.5 μM to 1 mM.

83. 67. A composition comprising at least one cultured astrocyte-like cell produced by the method of any one of claims 1 to 66, expressing at least one marker and exhibiting a flat, stellate, and / or spherical morphology, wherein the at least one marker is S100 calcium binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM).

84. A cell culture comprising at least one cultured astrocyte-like cell exhibiting a flat, stellate, and / or spherical morphology and detectably expressing at least one marker, wherein the at least one marker is S100 calcium binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM).

85. 85. The cell culture of claim 84, wherein detectable neuron-like cells are present in no more than 10% of the total cells in culture.

86. 86. The cell culture of claim 84 or 85, wherein the cell culture has been grown from previously cryopreserved cells.

87. 87. 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 a derivative thereof, trans-ISRIB, and polyamines including putrescine, spermine, and spermidine.

88. 88. The cell culture of Claim 86 or 87, wherein the previously cryopreserved cells are vertebrate pluripotent stem cells.

89. 89. The cell culture of claim 88, wherein the vertebrate pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells.

90. 90. The cell culture of claim 88 or 89, wherein the vertebrate pluripotent stem cells are human pluripotent stem cells.

91. 88. The cell culture of claim 86 or 87, wherein the previously cryopreserved cells are cultured radial glia-like cells that detectably express brain lipid-binding protein (BLBP), CD133 (prominin 1), abnormal spindle-like microcephaly-associated protein (ASPM), baculovirus 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 proteins.

92. 88. The cell culture of claim 86 or 87, wherein the previously cryopreserved cells are astrocyte-like cells that exhibit flat, stellate, and / or spherical morphology and detectably express one or more of S100 calcium binding protein B (S100B), nuclear factor 1 type A protein (NFIA), CD44, HEPACAM, glial fibrillary acidic protein (GFAP), and vimentin (VIM).

93. 67. A cell culture comprising at least one cultured astrocyte-like cell exhibiting a flattened, stellate, and / or spherical morphology, produced by the method of any one of claims 1 to 66, and detectably expressing at least one marker, wherein the at least one marker is S100 calcium binding protein B (S100B), nuclear factor 1 type A protein (NFIA), hepatic glial cell adhesion molecule (HEPACAM), glial fibrillary acidic protein (GFAP), CD44 protein, or vimentin (VIM).

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