UC San Diego

Ectopic expression of reprogramming factors has been widely adopted to reprogram somatic nucleus into a pluripotent state to generate induced pluripotent stem cells (iPSCs). However, such cellular reprogramming process involves epigenetic reconfiguration, and the process has been lengthy and inefficient. Over the years, extensive studies have uncovered genetic and epigenetic aberrations in the reprogrammed iPSCs, which have raised concerns regarding their clinical utility.

To test whether genetic and epigenetic aberrations are primarily the by-products of current reprogramming methods, we reprogrammed embryonic fibroblasts of inbred C57BL/6 mice into either iPSCs or somatic cell nuclear transfer embryonic stem cells (SCNT-ESCs). Exome sequencing of these lines suggested a significantly lower mutation load in SCNT-ESCs than syngeneic iPSCs. Whole genome bisulfite sequencing analysis revealed significantly more aberrant hypomethylation in iPSCs that are enriched in regulatory regions or genes involved in developmental processes, suggesting potential functional consequences. However, further investigation on these differentially methylated regions suggested comparable transcriptional variation at population level. As pluripotent stem cell populations appear to proliferate asynchronously and exhibit strikingly heterogeneous gene expression profile, we performed single cell RNA-sequencing on cells lines cultured in both heterogeneous serum/LIF and ground state 2i/LIF media to investigate genome-wide transcriptome profile of iPSC and SCNT-ESC. Surprisingly, we observed comparable gene expression heterogeneity and population distribution among the two cell types, but they were in dynamic cellular states that were greatly influenced by extracellular culture environment. This suggested culture induced transcriptional heterogeneity masks potential reprogramming-induced transcriptional signature. Taken together, we highlight the need for improving reprogramming methods to generate genetically and epigenetically stable pluripotent cell lines for future clinical application.