Fig. 2. Differentiation and purification of PD-TT hESC reporter cells.

a Eighty-days-old OPC culture expressing tdTomato driven by the endogenous PDGFRα promoter. Differentiation of the PD-TT reporter cells was performed ten times with similar results. Scale bar: 400 μm. b Schematic figure of MACS-based immunopurification of reporter cells using anti-Thy1.2 microbeads and a magnetic column. c Flow analysis indicating 23.1% of the total cells in differentiating culture are tdTomato⁺ at day 78, which is enriched to 90.1% after MACS purification. PE in X axis represents PDGFRα-tdTomato. A gate for flow analysis was set up using non-genome-edited hES cells differentiated to day 95 (left panel). Gating strategy is further detailed in Supplementary Fig. S1j and is also described in the “Methods” section. d Majority of the Thy1.2 immunopurified cells are tdTomato⁺ and have the OPC-like bipolar morphology. Scale bar: 200 μm. MACS-based purification and culture of the purified reporter OPCs were performed 10 times with similar result. e qPCR analysis shows enrichment of OPC markers in the MACS-based tdTomato enriched population vs the flow through. Two biological and three technical replicates each were used for qPCR analysis. Data are presented as mean ± SEM. Biological and technical replicates are distinguished by solid vs open symbols used for each data point. Source data for the qPCR are provided as a Source Data file. f–g Immunohistochemistry demonstrating that the MACS purified tdTomato⁺ cells express PDGFRα (f) and the OPC markers NKX2.2 and SOX10 (g). Scale bar: 200 μm. Immunohistochemistry was independently repeated twice with similar results.