Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture. Homo sapiens

We differentiated human iPSC to day 14 using our dopaminergic neuron protocol. At day 14, the cells were fixed, stained and FACS sorted in to the FOXA2/LMX1 double positive (wanted neural progenitor population) and double negative pools. We compared the RNA of the pools via microarray to identify surface markers in the double positive pool to be able to live purify the neural progenitors from the rest of the cells. Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can provide sources for midbrain dopaminergic (mDA) neural progenitors (NPCs) for cell therapy to treat Parkinson’s disease (PD) patients. However, the well-known line-to-cell line variability in the differentiation capacity of individual cell lines needs to be improved for the success of this therapy. To address this issue, we sought to identify mDA NPC specific cell surface markers for fluorescence activated cell sorting (FACS). Through RNA isolation after sorting for NPCs based on staining for cell-specific transcription factors followed by microarray, we identified two positive cell surface markers (CORIN and CD166) and one negative cell surface marker (CXCR4) for mDA NPC sorting. These three markers can enrich dopaminergic NPCs to 90% purity, and the sorted NPCs more efficiently differentiate to mature dopaminergic neurons compared to unsorted or CORIN+ alone mDA NPCs. This surface marker identification strategy can be used broadly to facilitate isolation of cell subtypes of interest from heterogeneous cultures. Complete processed data are not available Overall design: The cell line 18a was produced in biological triplicate for the experiment. The FACS sorted double positive was compared to the double negative pool of cells to identify markers upregulated in the double positive pool (neural progenitors)