Tms001 c

Human embryonic stem cell (ESC)-derived astrocytes [22 (link)] were expanded in Advanced DMEM/F12 (Thermo Fischer 12634-010) supplemented with 20 ng/ml Human Ciliary Neurotrophic Factor (CNTF) (Thermo Fischer), 1% penicillin streptavidin (Thermo Fischer 15140-122), 1% B27 supplement (Thermo Fischer 17504-044), 1% non-essential amino acids (Merc Millipore TMS001-C), and 1% l-glutamine (Thermo Fischer 25030-024). Cells were passaged using Trypsin-EDTA (Life technologies). Two weeks prior to the experiments, the media was changed to Neurobasal medium supplemented with 20 ng/ml CNTF, 1% penicillin streptavidin, 1% non-essential amino acids, 1% l-glutamine (Thermo Fischer 25030-024), and N2 supplements (Thermo Fischer 17502048). For experiments, the astrocytes were seeded at a concentration of 4500 cells/cm², resulting in a 40–50% confluence. Cells from 90 DIV to 120 DIV were used in the study.

Human ESC-derived astrocytes were generated as described previously (Holmqvist et al., 2015 (link); Stem Cell Research). The cells were cultured in Advanced DMEM/F12 (Thermo Fisher Scientific, 12634-010) supplied with 1% FBS (Thermo Fisher Scientific, 10082-147), 1% penicillin/streptavidin (Thermo Fisher Scientific, 15140-122), 2 μg/ml heparin (Sigma-Aldrich, H4784), 10% B27 supplement (Thermo Fisher Scientific, 17504-044), 1% nonessential amino acids (Merck Millipore, TMS001-C), and 1% l-glutamine (Thermo Fisher Scientific, 25030-024). Cells were passaged using Trypsin-EDTA (Life Technologies). Cells from 90 to 120 d in vitro (DIV) were used in the study. For experiments, the astrocytes were seeded at a concentration of 1500 cells/cm², resulting in a 20–30% confluence. TUNEL analysis showed that apoptotic cell death in the culture was <3% (Fig. 3-1B,C).