Preserving self-renewal of porcine pluripotent stem cells in serum-free 3i culture condition and independent of LIF and b-FGF cytokines

Yangyang Ma,Huayan Wang,Yuanxing Cai,Tong Yu

doi:10.1038/s41420-017-0015-4

Yangyang Ma, Huayan Wang + Show 2 more

Open Access

https://doi.org/10.1038/s41420-017-0015-4

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Abstract

Derivation of bona fide porcine pluripotent stem cells is still a critical issue because porcine embryonic stem cells (ESCs) are not available yet, and most of the culture conditions to maintain porcine induced pluripotent stem cells (piPSCs) are based on conditions for mouse and human iPS cells. In this study, we generated a doxycycline-inducible porcine iPS cell line (DOX-iPSCs) and used it to screen the optimal culture condition to sustain the self-renewal of piPSCs. We found that LIF and b-FGF were required for porcine cell reprogramming, but were not essential cytokines for maintaining the self-renewal and pluripotency of piPSCs. A serum-free 3i medium, which includes three inhibitors CHIR99021, SB431542, and PD0325901, three cytokines BMP4, SCF, and IL-6, and human platelet lysates (PL), was made through serious selections. In 3i condition, the doxycycline-inducible iPSCs could be passaged for a long term without the addition of doxycycline, and the flattened morphology of intermediate state piPSCs could convert to the naïve-like morphology with the increase in endogenous pluripotent gene expressions. Additionally, pPSC cell line isolated from 5.5 days blastocysts could be sustained in 3i medium and the expression of endogenous pluripotent genes OCT4, ESRRB, and STELLA was significantly increased. Our finding directed a new reprogramming strategy by using 3i condition to maintain and convert primed piPSCs into naïve-like pluripotent state. A combination of traditional LIF/b-FGF conditions and 3i condition may help us to find out an appropriate reprogramming approach to generate the naïve state of porcine iPSCs.

Highlights

Somatic cells can be reprogramed by the ectopic expression of defined transcription factors[1,2]
To set a doxycycline-inducible pluripotent stem cells (piPSCs), in which expression of the transgenes can be completely switched on/off by doxycycline (Dox), lentiviral particles of TetO-FUW-OSKM and FUW-M2rtTA were infected into porcine embryonic fibroblasts (PEFs) to reprogram the somatic cells into doxycycline-inducible porcine iPS cells (DOX-induced pluripotent stem cells (iPSCs)) (Supplementary Fig. 1A)
The results of immunofluorescence staining demonstrated that the pluripotent markers OCT4, SOX2, and SSEA-1 were highly expressed in all three piPS cell lines, but NANOG expression was low (Supplementary Fig. 1C)

Summary

Introduction

Somatic cells can be reprogramed by the ectopic expression of defined transcription factors[1,2]. Genetic individuality indicates that the resultant induced pluripotent stem cells (iPSCs) reserved from precursor cells enable personalized cell therapy and regenerative medicine[3]. With substantial improvement in the reprogramming approach, iPSCs were proved indistinguishable from embryonic stem cells (ESCs)[8,9,10,11]. The fundamental issue in animal species, including pig, is how to fully convert the somatic cells into ESC-like and germlinecompetent pluripotent stem cells (PSCs). Many efforts have been made to obtain the authentic porcine PSCs referring to the pluripotent criteria that were based on mouse ESCs or iPSCs, including gain of multiple differentiation capacities in vivo and in vitro[12,13], long term single-cell passages[13,14,15], double activated X

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