RNA-Seq of cKIT+ sorted cells from 16-16.5 week old fetal testes and ovaries and RNA-Seq of TRA-1-60+ H1 hESCs. Homo sapiens

Generation of research quality, clinically relevant cell types in vitro from human pluripotent stem cells (hPSCs) requires detailed understanding of the equivalent cell types in humans. Here we analyzed 130 human fetal samples at 6-20 weeks of development and identified the stages in which human cKIT+ primordial germ cells (PGCs), the precursors of gametes, undergo whole genome epigenetic reprogramming and ultimately initiation of imprint erasure with loss of both 5mC and 5-hydroxy-mC at differentially methylated regions. Using five alternate in vitro differentiation strategies combined with a single-cell microfluidic analysis, high throughput RNA sequencing and a bona fide human cKIT+ PGC signature, we show that hPSC differentiation generates a rare cKIT+ PGC subtype found in both the human fetal gonad and mouse embryo. Taken together, our study creates a resource of human germ line ontogeny that is absolutely essential for future studies aimed at interpreting in vitro differentiation of the human germ line. Overall design: cKIT+ cells analyzed from 2 biological samples for testes and 2 samples for ovaries at 16 and 16.5 weeks. 3 biological replicates of TRA-1-60+ cells sorted from H1 hESCs