Biphasic activation of WNT signaling enhances the derivation of midbrain dopamine neurons for translational use [RNA-seq]

We report a novel midbrain dopamine neuron derivation protocol from human pluripotent stem cells (hPSC)s based on a two-step WNT signaling activation strategy. By comparing next-generation sequencing (NGS) derived transcriptome profiling (RNA-seq) of dopamine neuron differentiated cells derived from hPSC with different concentration of Chir-boosting (day4 – day9) measured at day 11 including hPSC, we find that CHIR-boosting improves expression of midbrain markers such as EN1 while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. Furthermore, gene expression analysis from RNA-seq study confirmed the progressive differentiation and maturation of Boost CHIR treated precursors into mDA neurons by day 30 of differentiation. The protocol presented here is the basis for clinical grade dopamine neuron production and preclinical safety and efficacy studies. Overall design: mRNA profiles of human pluripotent stem cells (hPSC)s, midbrain dopamine differentiated cells with different concentration of CHIR-boosting (day 4 - day 9) at day 11, and CHIR-BOOST (7.5uM) treated mDA neurons at day 20 and day 30 derived from hPSCs.