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, 11 (6), e0158046
eCollection

Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig

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Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig

Kwang-Hwan Choi et al. PLoS One.

Abstract

Cellular reprogramming of committed cells into a pluripotent state can be induced by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. Reprogrammed cells can be maintained by activating endogenous pluripotent networks without transgene expression. Although various research groups have attempted to generate pig induced pluripotent stem cells (iPSCs), authentic iPSCs have not be obtained, instead showing dependence on transgene expression. In this study, iPSCs were derived from porcine fetal fibroblasts via drug-inducible vectors carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study investigated characteristics of iPSCs and reprogramming mechanisms in pig. The iPSCs were stably maintained over an extended period with potential in vitro differentiation into three germ layers. In addition, the pluripotent state of iPSCs was regulated by modulating culture conditions. They showed naive- or primed-like pluripotent states in LIF or bFGF supplemented culture conditions, respectively. However, iPSCs could not be maintained without ectopic expression of transgenes. The cultured iPSCs expressed endogenous transcription factors such as OCT4 and SOX2, but not NANOG (a known gateway to complete reprogramming). Endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated, as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occur in female iPSCs. Based on our results, expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu to achieve faithful pluripotency in pig. Accordingly, investigating iPSCs could help us improve and develop reprogramming methods by understanding reprogramming mechanisms in pig.

Conflict of interest statement

Competing Interests: The author JP is affiliated with a commercial company (LG Life Sciences). This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Characterization of three selected iPSC lines.
(A) Three cell lines, including one fussy type, piPS-9 and two naïve types, piPS-14 and 18, were selected. When the cells were cultured in suspension, the cells aggregated and subsequently formed embryoid bodies. (B) The formed EBs differentiated into three germ layers after being placed on gelatin-coated plates. Expression of three germ layer markers including Cytokeratin17 (endoderm), Vimentin (mesoderm), and Neurofilament (ectoderm) was confirmed by immunostaining. Of the three cell lines, piPS-14 (showing better morphology and differential potentials with larger EBs than other cell lines) was selected for further analyses and culture. (C) The integration of four transgenes into the genome was confirmed. Black and hollow arrows indicate 100 bp and 200 bp size markers, respectively. (D) The piPS-14 cell line could be stably maintained with a normal karyotype (36 + XX). Scale bar = 200 μm in A; 50 μm in B.
Fig 2
Fig 2. Morphological changes of piPSCs in response to culture conditions.
The piPS-14 cells cultured under LIF conditions were transferred to two different media supplemented with LIF + 2i or bFGF. (A) Four days after changing culture media, the cells cultured in bFGF started to change morphologically, becoming flattened. (B) Later, the cells cultured with bFGF showed a primed-like flattened morphology, while those cultured with LIF or LIF + 2i still showed compact dome-shaped morphology. (C) When cultured in the absence of dox, in all cells under the three conditions, the number of colonies gradually decreased within 4 days, and AP-positive colonies were absent after subcultures. Scale bar = 200 μm.
Fig 3
Fig 3. Expression of pluripotent markers in piPSCs.
Expression of endogenous and exogenous pluripotent genes was determined by immunostaining and qPCR. (A) OCT4, SOX2, SSEA1, and SSEA4 were expressed in naïve-like piPSCs cultured with LIF. (B) OCT4, SOX2, and SSEA4 were expressed in primed-like piPSCs cultured with bFGF (C) When treated with 2i, SSEA4 was still expressed in naïve-like piPSCs. (D) Expression of NANOG was not detected under any culture conditions as determined by flow cytometric analysis. (E) Exogenous transgenes were highly expressed when treated with doxycycline in piPSCs, while transgenes were not expressed in the absence of doxycycline. Scale bar = 50 μm.
Fig 4
Fig 4. Expression of pluripotent and MET-related genes as measured by qPCR.
To verify the effects of transgenes on endogenous genes, expression levels of endogenous genes related to pluripotency and MET (pluripotent genes: pOCT4a, pSOX2, pKLF4, pMYC, NANOG, DPPA2 and REX1; epithelial-to-mesenchymal transition (EMT) inducer: TGFB1; epithelial-specific markers: CDH1, EPCAM and OCLN) were determined by qPCR. As reprogramming is processed by exogenous genes, endogenous pluripotent genes (pOCT4a, pSOX2, pKLF4, NANOG, and REX1) and epithelial-specific markers (CDH1, EPCAM, and OCLN) were upregulated. However, pMYC and DPPA2 (known predictors of reprogramming) were not reactivated, while TGFB1 (blocks reprogramming) was not efficiently shut-down by transgene expression. Some genes, such as pSOX2, pKLF4, REX1, and epithelial-specific markers, were preferentially expressed in culture conditions containing bFGF, and pKLF4 and NANOG were downregulated when treated with 2i.
Fig 5
Fig 5. Bisulfite sequencing at promoter regions of OCT4, NANOG, and XIST.
To verify whether epigenetic reprogramming occurred by expression of exogenous genes, DNA methylation patterns at promoter regions of pig OCT4, NANOG, and XIST were evaluated by bisulfite sequencing. (A) OCT core promoter: OCT4 core promoter regions were highly methylated. (B) NANOG promoter: Promoter regions of NANOG were methylated to levels similar to somatic cell control. (C) XIST promoter: X chromosome reactivation did not occur in naïve-like piPSCs. Each circle indicates individual CpG dinucleotides. White and dark circles represent unmethylated and methylated CpGs, respectively. Each row represents one individual clone of amplified PCR products.

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Grant support

This work was supported by the Next-generation BioGreen 21 Program (PJ0113002015), Rural Development Administration, Republic of Korea and also supported by BK21 plus program through the National Research Foundation (NRF), the Ministry of Education of Korea. The funder, LG Life Sciences, provided support in the form of salaries for author [JP], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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