Abstract
BackgroundRecent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs.Methodology/Principal FindingsWe generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines.Conclusions/SignificanceOur data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras.
Highlights
Mouse embryonic stem cells (ESCs), first established in 1981, were originally generated from the inner cell mass of mouse blastocysts
Infected rat embryonic fibroblasts (REFs) were seeded onto mitomycin-C treated mouse embryonic fibroblasts (MEFs) and the medium was changed to serum-free medium (N2B27/F12) containing Dox and rat leukemia inhibitory factor (rLIF) on day 3
All ES-like colonies stained for alkaline phosphatase (ALP), indicating that generated rat iPSCs (riPSCs) were pluripotent (Fig. 1D)
Summary
Mouse embryonic stem cells (ESCs), first established in 1981, were originally generated from the inner cell mass of mouse blastocysts. In 2008, Ying et al reported establishment of rat ESCs (rESCs) with use of two or three kinds of kinase inhibitors, including glycogen synthase kinase 3 (GSK3) inhibitor, mitogen-activated protein kinase kinase (MEK) inhibitor, and fibroblast growth factor (FGF) receptor tyrosine kinase inhibitor in the culture medium. These inhibitors have been thought to maintain a ground state of pluripotency in mESCs [18] and can support efficient derivation and maintenance of rESCs, permitting generation of chimeric rats, with germline transmission [19] [20].
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