Abstract
The most stringent criterion for evaluating pluripotency is generation of chimeric animals with germline transmission ability. Because the quality of induced pluripotent stem cell (iPSC) lines is heterogeneous, an easy and accurate system to evaluate these abilities would be useful. In this study, we describe a simple but comprehensive system for generating and evaluating iPSCs by single transfection of multiple piggyBac (PB) plasmid vectors encoding Tet-inducible polycistronic reprogramming factors, a pluripotent-cell–specific reporter, a constitutively active reporter, and a sperm-specific reporter. Using this system, we reprogrammed 129 and NOD mouse embryonic fibroblasts into iPSCs, and then evaluated the molecular and functional properties of the resultant iPSCs by quantitative RT-PCR analysis and chimera formation assays. The iPSCs contributed extensively to chimeras, as indicated by the constitutively active TagRFP reporter, and also differentiated into sperm, as indicated by the late-spermatogenesis–specific Acr (acrosin)-EGFP reporter. Next, we established secondary MEFs from E13.5 chimeric embryos and efficiently generated secondary iPSCs by simple addition of doxycycline. Finally, we applied this system to establishment and evaluation of rat iPSCs and production of rat sperm in mouse–rat interspecific chimeras. By monitoring the fluorescence of Acr-EGFP reporter, we could easily detect seminiferous tubules containing rat iPSC–derived spermatids and sperm. And, we succeeded to obtain viable offspring by intracytoplasmic sperm injection (ICSI) using these haploid male germ cells. We propose that this system will enable robust strategies for induction and evaluation of iPSCs, not only in rodents but also in other mammals. Such strategies will be especially valuable in non-rodent species, in which verification of germline transmission by mating is inefficient and time-consuming.
Highlights
To evaluate the characteristics and qualities of pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells, several assays are used in combination
We demonstrate that our system can generate germline-competent induced pluripotent stem cell (iPSC) in mouse and rat, and that we can evaluate the formation of iPSC-derived male germ cells in the testes of chimeric animals using the Acr-EGFP reporter
This analysis showed that exogenous expression of the reprogramming factors in these iPSCs was efficiently downregulated to background levels following Dox withdrawal (Fig. 1H, I)
Summary
To evaluate the characteristics and qualities of pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), several assays are used in combination Among these assays, the most precise is verification of germline transmission by mating, because pluripotent stem cells are often utilized to generate genetically modified animal lines. The most precise is verification of germline transmission by mating, because pluripotent stem cells are often utilized to generate genetically modified animal lines This assay requires considerable effort and time: the process takes about 3 months in mouse, and longer in other animals. There has been no formal evidence that non-rodent pluripotent stem cells can contribute to the germline. An easy and accurate system for assaying germline competency without mating would provide a valuable tool for evaluation of the characteristics and qualities of iPSC lines in non-rodent species
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