Generation of hiPSC derived cortical astrocytes
Minee L Choi
Abstract
This is a modified protocol that describes how to generate cortical region-specific astrocytes based on Gupta et al., 2012, Serio et al., 2013 and Seto-Salvia et al., 2021
Steps
hiPSC culture
hiPSCs are maintained on Geltrex in mTeSR (Stem Cell) and passaged using 0.5mM EDTA, as followed by Virdi et al., 2022.
Differentiation of hiPSC into Cortical astrocytes
Differentiation of cortical region-specific astrocytes is performed using a modified protocol based on Gupta et al., 2012, Serio et al., 2013 and Seto-Salvia et al., 2020.
Neural precursor cells (NPCs) are obtained from hiPSC using the established protocol of
First, NPCs are induced by dual SMAD inhibition for 10 days, followed by culturing with the N2B27 media for another 15 days.
Induction of glial precursor cells (GPCs),
NPCs are used to derive glial precursor cells (GPCs) by culturing in an N2B27 medium supplemented with 20 ng/ml of human FGF-2.
Cells are split twice per week (1:2 or 1:3) using Accutase (Cat No. A1110501, Thermo Fisher Scientific) by vigorously breaking pellets to remove neuronal cells.
Final differentiation into astrocytes
Upon the appearance of glial morphology (around day 90 from the neural induction), the GPCs are cultured for 7 days with 10 ng/ml Bone Morphogenetic Protein 4 (BMP4) and 20 ng/ml Leukemia inhibitory factor (LIF), which activates the JAK/STAT signalling pathway, refreshing the medium every other day
On the 8th day, BMP4 and LIF are withdrawn.
Cells are further differentiated for the final maturation in the N2B27 media without FGF2.
3-5 times further passes might be required (1:2) until the complete loss of the precursor property.
