Reproduction of reset cell

Functional-assay limitations are an emerging issue in characterizing human pluripotent stem cells (hPSCs). With rodent PSCs, chimera formation, using pre-implantation embryos, is the gold-standard assay of pluripotency. In hPSCs, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-animal chimera. To circumvent this issue, we established a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay). The assay uses mouse pre-implantation embryos and human PSCs to make interspecies chimeras cultured in vitro to the early egg cylinder stage. When hiPSCs, both conventional and naive type, which called “reset cell”, were injected into mouse embryos and cultured. The cells were never integrated into the epiblast of egg cylinder stage-embryo. These results suggest that hPSCs, including naïve type, are unable to form chimera with mouse embryo. Reset cells were converted from conventional human iPSC line PB004, and then compared their gene expression profile with or without transgene overexpression induced by doxycyclin treatment.