Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells.
Recent study has demonstrated that human pluripotent stem cells (PSCs) such as ES cells or iPS cells are not equivalent to mouse ES cells. They are a little more mature stem cells and, although they can turn into a variety of functional cells just like mouse ES cells, they appear to have lost the ability to form chimeras when injected into early embryos. It is important to tell if human PSCs are truly equivalent to mouse ES cells or not. However, we cannot inject human PSCs into human embryos because of obvious ethical reasons. In this study, we attempted to see if we can evaluate chimera forming ability of human PSCs by injecting into mouse embryos and culture them in vitro. However, when human induced PSCs were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike rodent PSCs, they never integrated into mouse embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species, as host embryos might be necessary to evaluate the chimera-forming ability of hPSCs.
Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra- and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naive-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs.