Stabilization of hESCs in two distinct substates along the continuum of pluripotency

A detailed understanding of the developmental substates of human pluripotent stem cells (hPSCs) is needed to optimize their use in cell therapy and models of early development. Genetic instability and risk of tumorigenicity of primed hPSCs are well documented, but a systematic isogenic comparison between primed and naive substates has not been previously performed. We derived four hESC lines in naive conditions and generated isogenic pairs of naive and primed cultures. Through phenotypic, mRNA, miRNA, and DNA methylation profiling, we identified changes that arose during the naive-primed transition and over extended culture. Although early naive hESC cultures showed greater proliferation and clonogenic potential compared to primed cultures, they drifted toward a more primed-like substate over time, including accumulation of genetic abnormalities. Overall, we show that transcriptomic and epigenomic profiling can be used to place naive/primed cultures along a developmental continuum, and may inform their utility for clinical and research applications.