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.

Human embryonic stem cells (hESCs) hold great promise for regenerative medicine as an unlimited source of cells for replacement of the tissues destroyed by injuries or diseases. One of the major impediments of fully realizing its poetical is the immune rejection from allogenic recipients. To overcome the histocompatibility, we established hESCs that lack the expression of Major Histocompatibility Complex (MHC) class I or II, which are the primary mediators for allograft rejection. [31] Briefly, CRIPR-Cas9n was used to delete exon 1 of beta-2 micro globulin (B2M), leading to a complete deficiency of MHC I on the cell surface of hESCs. In the same manner, exon 2 and 3 of Class II transactivator (CIITA) were deleted, aiming to abrogate any potential of inducible MHC II expression in hESCs. Despite of this genetic manipulation, MHC I-deficient hESCs maintained its self renewal capacity, expressed pluripotent genes and were pluripotent as indicated by their ability to form teratoma in NSG mice. Moreover, we demonstrated that the teratoma derived from MHC I - deficient hESCs were immune protected in Hu-mouse reconstituted with allogenic human immune system, while the teratoma from the parental hESCs showed apparent tissue necrosis and a mount of T cell infiltration, an indication of immune rejection. All these findings indicate that our MHC I-deficient hESCs are hypoimmunogenic, and might be employed as an appealing source of cells for tissue replacement in cell-based therapy without requiring the long-term systemic immune suppression.