Differentiation of hPSCs into Dopamine neurons
Beatrice Weykopf
Abstract
This protocol describes the differentiation of hPSCs into dopaminergic neurons modified from Kriks et al., 2011 and Ryan et al., 2013
Steps
Day -1: seed 200K cell/cm2 in mTeSR+ medium supplemented with 10µM Rock inhibitor (RI) onto Martigel coated TC plates
Day 0: Differentiation initiation. Aspirate mTeSR+medium and add SRM media supplemented with 200nM LDN193189 + 10µM SB431542
Day 1: SRM + LDN + SB supplemented with 100 ng/ml FGF8b, 100 ng/ml ShhC25II and 2µM Purmorphamine
Day 2: SRM + LDN + SB+ FGF8b + ShhC25II + Pur
Day 3: SRM + LDN + SB+ FGF8b + ShhC25II + Pur +3 µM CHIR99021
Day 4: no feed if medium is not consumed
Day 5: 75% SRM + 25% Neurobasal + LDN + SB+ FGF8b + ShhC25II + Pur +CHIR
Day 6: no feed if medium is not consumed
Day 7: 50% SRM + 50% Neurobasal + LDN + CHIR
Day 8: no feed if medium is not consumed
Day 9: 25% SRM + 75% Neurobasal + LDN + CHIR
Day 10: no feed if medium is not consumed
Day 11: 100% Dopa mat basal medium + CHIR
Day 12: no feed
Day 13: Passage cells in a ratio of 1:1 onto matrigel-coated dishes with 30-60min Accutase treatment. Spin down the cells in Dopa mat basal and resuspend in Dopa mat medium supplemented with 10µM Y27632 and CHIR.
Day 20-24: Cells are plated on Poly-D-lysine and laminin coated plates in a density of 2-2.5M cells / 6 well. Cells are dissociated using 60-90min Accutase supplemented with 10µM Y27632.
Day 25-27: 3µM cytosine arabinoside is added to Dopa mat media. Day 27 cells are washed twice with Dopa mat basal medium to remove any cytosine arabinoside residues.
Around Day 30: Cells are dissociated using Accutase supplemented with 10µM Y27632 and plated in onto Poly-D-lysine and laminin coated dishes. The final density depends on the assay.
