Fig. 1

Differentiation of human embryonic stem cells into astrocyte progenitor cells and committed astrocytes. a Steps and timeline for differentiation of hESCs first into astrocyte progenitor cells (APCs) which can be stored frozen in APC banks. These APCs are further expanded with growth factors (bFGF, EGF and human serum), and then differentiated into astrocytes (hES-AS) by removal of growth factors for 7 days. b Representative images of different steps from hESCs to APCs (as in a, steps marked by asterisk). Arrows show selected neurospheres. c Representative spectral karyotyping analysis showing normal karyotype of APC cell bank at passage 12. d Flow cytometry analysis on nine batches of APC banks (grown with human serum, bFGF and EGF) versus 13 batches of astrocytes differentiated for 7 days showing expression of astrocytic markers (CD44, GLAST, GFAP, and Aquaporin-4) and neuroepithelial stem cell markers (Nestin, A2B5 and CXCR4). e Flow cytometry analysis of APCs and astrocytes differentiated for 7 days (same batches as in d) showing very low expression of pluripotent markers (below limit of detection, 0.1%). f Representative immunofluorescence images of astrocytes differentiated 7 days, showing expression of astrocyte markers (GFAP, GLAST, S100β and AQP-4) and very low proliferation marker (Ki-67, arrow). Scale bars = 100 μm. Error bars represent SD. hESC human embryonic stem cell, DAPI 4′,6-diamidino-2-phenylindole, GFAP Glial Fibrillary Acidic Protein, GLAST Glutamate Aspartate Transporter, RA Retinoic acid