A Comprehensive System for Generation and Evaluation of Induced Pluripotent Stem Cells Using piggyBac Transposition

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.

Xenogeneic chimera—Generated by blastocyst complementation—As a potential unlimited source of recipient‐tailored organs

“…More interestingly, similar results were obtained by Tsukiyama et al. using rat iPSCs, confirming the tremendous potential of this technology(Figure ).…”

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…A number of hyperactive transposase variants are available that approach the efficiency of retroviral or lentiviral gene transfer; the production costs are low and transposons are safe because they are noninfective and the removal of integrated transposons is possible. Transposons, specifically Sleeping Beauty (SB) and piggyBac (PB), have emerged as useful alternatives to virus-mediated reprogramming of somatic cells from different species, including human (Davis et al, 2013;Woltjen et al, 2009), murine Muenthaisong et al, 2012;Talluri et al, 2014;Tsukiyama et al, 2014), porcine (Kues et al, 2013), and equine somatic cells (Nagy et al, 2011). Until now, there are no reports on transposon-mediated reprogramming of bovine somatic cells to pluripotent iPSCs.…”

Discrimination of Stem Cell Status after Subjecting Cynomolgus Monkey Pluripotent Stem Cells to Naïve Conversion

“…. PB-EOS- GFP - puroR 20 and PB-CAG- Su9DsRediN were also transfected to confirm pluripotency and the ability to contribute to chimeric offspring, respectively. The EOS-containing vector also has a reverse tetracycline transactivator (rtTA) cassette for enhanced expression of the tet promoter21.…”

“…On the other hand, human naïve ESCs most resemble mouse ESCs and primate pre-implantation embryos but are only incorporated into mouse embryos as interspecific chimeras very inefficiently28. Interspecific chimera formation using PSCs has been achieved in the mouse and rat202632. On the other hand, if truly naïve PSCs from other mammalian species could be generated successfully, it is not certain whether they could contribute to interspecific chimeras efficiently or not.…”

“…To maintain their primed-state pluripotency, Cm ESCs were cultured in bFGF/KSR medium consisting of 78% Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F12) supplemented with 15% KSR, 2 mM GlutaMax (Invitrogen Life Sciences, Carlsbad, CA, USA), 1% non-essential amino acids, 0.1 mM mercaptoethanol, and 4 ng/ml human recombinant bFGF (Wako, Osaka, Japan). To convert Cm ESCs into a naïve-like state, PiggyBac (PB) vectors carrying Dox-inducible hKLF2 or hNANOG coupled to Venus were cotransfected with a PB-EOS- GFP/puroR reporter, which has an rtTA expression construct1520, and pCAG-PBase using an NEPA21 electroporator (Nepa Gene Co., Ltd., Chiba, Japan). To confirm the chimeric contribution of ESCs into mouse embryos as interspecific chimeras, cell lines transfected with PB-CAG- Su9DsRediN were established concurrently.…”