Cell type determination for cardiac differentiation occurs after seeding of human induced pluripotent stem cells

Cardiac directed differentiation of human induced pluripotent stem cells consistently produces a mixed population of cardiomyocytes and non-cardiac cell types even when using very well-characterized protocols. We wondered whether differentiated cell types might result from intrinsic differences in hiPS cells prior to the onset of differentiation. By associating individual differentiated cells that share a common hiPS cell precursor, we were able to test whether expression variability in differentiated cells was pre-determined from the hiPS cell state. Although within a single experiment, differentiated cells that share an hiPS cell progenitor were more transcriptionally similar to each other than to other cells in the differentiated population, when the same hiPS cells were differentiated in parallel, we did not observe high transcriptional similarity across differentiations. Single cell RNA sequencing (10X Genomics single-cell RNA-seq kit v3) of barcoded hiPS cell-derived cardiomyocytes and hiPS cells. hiPS cells were lentivirally transduced with barcode libraries consisting of random 100-mers in the 3’ untranslated region of a green fluorescent protein (GFP) transgene after which they were permitted to divide approximately three times before being split and seeded for two parallel differentiations. On day 14 of differentiation (16 days after seeding), we used fluorescent activated cell sorting (FACS) gated for GFP positivity to enrich for barcode-containing cells on which we performed single cell RNA sequencing. As transcripts of the GFP transgene are captured by single cell RNA sequencing, we were also able to recover barcodes through a separate “side reaction” cDNA sequencing run and connect them to individual cells through analysis of GFP transcript 10x sequencing reads. Separately, we also transduced hiPS cells, allowed them to divide approximately six times before harvesting them for GFP sorting and sequencing. See Jiang et al. citation for additional details.