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Generation of Intermediate Porcine iPS Cells Under Culture Condition Favorable for Mesenchymal-to-Epithelial Transition

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Abstract

It has been demonstrated that naïve and primed pluripotency are determined by different extracellular signals. In this study, we investigated whether intermediate pluripotent states could be available by manipulating the culture condition during the process of generating pig induced pluripotent stem cells (piPSCs). By optimizing the culture condition that efficiently promotes mesenchymal-to-epithelial transition (MET), we found that combination of three growth factors (LIF, FGF2 and BMP4) and two inhibitors (2i: CHIR99021 and SB431542) could generate an intermediate pluripotent state of piPSCs, which were named as LFB2i-piPSCs. The LFB2i-piPSCs are stable and fulfill all the criteria of pluripotency, including expression of pluripotent genes, differentiation into three germ layers via embryoid bodies in vitro and teratoma in vivo. More importantly, the mRNA-sequencing data showed that LFB2i-piPSCs had a mixed transcriptome of naïve and primed pluripotency, which featured by expressing high levels of SOX2, L-MYC and ESRRB and relatively low levels of POU5F1, KLF4 and NANOG. Small RNA sequencing also demonstrated that LFB2i-piPSCs had a mixed microRNA profile of naïve and primed pluripotency, which featured by expressing high levels of miR-302b/367 cluster and miR-106a/363 cluster, and low levels of most let-7 family members and miR-17/92 cluster. Altogether, the LFB2i-piPSCs represent a stable intermediate pluripotent state with unique transcriptome and microRNA signatures. The LFB2i-piPSCs will provide a new tool to explore the mechanisms of pluripotency and reprogramming on pig species.

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Acknowledgments

We thank Dr. West for providing the RNA lysate of FGF2-piPSCs. This work was supported by the National Natural Science Foundation of China (No. 31371505, 31301218); the National Basic Research Program of China (2011CBA01002) and the Northwest A&F University research start-up grant (No. 201104050355).

Author Contribution

S.Z., Y.G.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; S.Z. and Y.G. contributed equally to this article. Y.L.: analysis of RNA-seq data; Y.C.: analysis of small RNA-seq data; T.Y.: collection and/or assembly of data; H.W.: conception and design, financial support, administrative support, data analysis and interpretation, manuscript writing, and final approval of manuscript.

Conflict of Interest

The authors have no conflicts of interest to declare.

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Authors and Affiliations

  1. Department of Animal Biotechnology, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China

    Shiqiang Zhang & Huayan Wang

  2. Shaanxi Center for Stem Cell Engineering and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China

    Shiqiang Zhang, Yanjie Guo, Yi Cui, Yajun Liu, Tong Yu & Huayan Wang

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  1. Shiqiang Zhang
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  2. Yanjie Guo
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Correspondence to Huayan Wang.

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Figure S1

(A) Genomic DNA PCR analysis of transgene integration in two piPSC lines LFB2i-piPSC-1 and LFB2i-piPSC-2. (B) Quantitative RT-PCR analysis of endogenous expression of POU5F1, SOX2, NANOG and ESRRB in LFB2i-piPSC-1 and LFB2i-piPSC-2. The y-axis represents the fold change vs. PEFs. (C) Karyotype analysis of LFB2i-piPSC-2. (D) Growth curve of LFB2i-piPSC-1 and PEFs. (GIF 74 kb)

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Figure S2

RT-PCR analysis of genes expression in embryoid bodies (EBs) derived from LFB2i-piPSCs. The undifferentiated cells were used as negative control. NESTIN is for ectoderm marker; AFP for endoderm marker; DESMIN for mesoderm marker. GAPDH is used as internal control. (GIF 20 kb)

High resolution image (TIFF 1072 kb)

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Zhang, S., Guo, Y., Cui, Y. et al. Generation of Intermediate Porcine iPS Cells Under Culture Condition Favorable for Mesenchymal-to-Epithelial Transition. Stem Cell Rev and Rep 11, 24–38 (2015). https://doi.org/10.1007/s12015-014-9552-x

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