Radial Glia and Astrocyte Differentiation from Human Pluripotent Stem Cells

A method of producing a culture of astrocyte-like cells, the method comprising:

• (a) plating vertebrate pluripotent stem cells on a substrate-coated surface of a culture vessel at a density of 1,000-100,000 cells/cm2;
• (b) incubating the plated vertebrate pluripotent stem cells in a first culture medium
• (c) replacing the first culture medium with a second culture medium comprising:
• (i) an effective amount or concentration of one or more inhibitors of BMP pathway, wherein the one or more inhibitors of the BMP pathway comprise one or more of LDN-193189, LDN-214117, LDN-212854, DMH2, ML 347, UK 383367, K 02288, Dorsomorphin, Noggin, Chordin, Follistatin, or Gremlin,
• (ii) an effective amount or concentration of one or more activators of Notch pathway wherein the effective amount or concentration of
• the one or more activators of Notch pathway in the second culture medium comprise one or more of Jagged 1 protein, Jagged 2 protein, and Delta-Like protein 1 (DLL1), Delta-Like protein 2 (DLL2), or Delta-Like protein 3 (DLL3), and
• (iii) one or more cytokines of interleukin-6 family;
• (d) culturing the plated vertebrate pluripotent stem cells in the second culture medium;
• wherein the resulting radial glia-like cells detectably express one or more of Brain Lipid Binding Protein (BLBP), 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), and SRY-Box Transcription Factor 21 (SOX21);
• and
• (e) culturing the resulting radial glia-like cells for approximately 5-30 days in a third culture medium, an effective amount or concentration of one or more activators of Notch pathway, and an effective amount or concentration of one or more cytokines of Interleukin-6 (IL-6) family, thereby generating the culture of the astrocyte-like cells.