Epigenetic Changes of Lentiviral Transgenes in Porcine Stem Cells Derived from Embryonic Origin

<div><p>Because of the physiological and immunological similarities that exist between pigs and humans, porcine pluripotent cell lines have been identified as important candidates for preliminary studies on human disease as well as a source for generating transgenic animals. Therefore, the establishment and characterization of porcine embryonic stem cells (pESCs), along with the generation of stable transgenic cell lines, is essential. In this study, we attempted to efficiently introduce transgenes into Epiblast stem cell (EpiSC)-like pESCs. Consequently, a pluripotent cell line could be derived from a porcine-hatched blastocyst. Enhanced green fluorescent protein (EGFP) was successfully introduced into the cells via lentiviral vectors under various multiplicities of infection, with pluripotency and differentiation potential unaffected after transfection. However, EGFP expression gradually declined during extended culture. This silencing effect was recovered by <i>in vitro</i> differentiation and treatment with 5-azadeoxycytidine. This phenomenon was related to DNA methylation as determined by bisulfite sequencing. In conclusion, we were able to successfully derive EpiSC-like pESCs and introduce transgenes into these cells using lentiviral vectors. This cell line could potentially be used as a donor cell source for transgenic pigs and may be a useful tool for studies involving EpiSC-like pESCs as well as aid in the understanding of the epigenetic regulation of transgenes.</p></div

Silenced transgenes can be reactivated by treatment with the DNA methylase inhibitor, 5′-aza-2′-deoxycitidine (5-AzadC).

<p>(A) 5-AzadC treatment reactivates the silenced transgene, EGFP. (B) The recovered expression level was measured by flow cytometry. (C) 5-AzadC demethylated the methylated CMV promoter region. Values noted by a–h indicate they are significantly different. Data are the means± S.E.M. (<i>n</i> = 3).</p

Change in EGFP expression during extended culture and <i>in vitro</i> differentiation.

<p>(A) EGFP expression levels of transduced EpiSC-like pESCs declined during extended culture. The decreased expression recovered during <i>in vitro</i> differentiation. (B) To analyze the effects of the epigenetic state on transgene expression, the DNA methylation patterns of the CMV promoter region in the transgene of each sample were evaluated via bisulfite sequencing. Along with decreases in EGFP expression, the DNA methylation levels of the CMV promoter region increased. Although DNA methylation levels increased during extended culture, they were not altered by <i>in vitro</i> differentiation. (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). Values noted by a–h indicate they are significantly different. Data are the mean± S.E.M. (<i>n</i> = 3).</p

Primer sets for bisulfite sequencing.

<p>Primer sets for bisulfite sequencing.</p

Characterization of EGFP-transduced EpiSC-like pESCs.

<p>(A) EGFP-transduced EpiSC-like pESCs have a normal karyotype (36+ XX). (B) The proportion of EGFP-expressing cells increased to 70–80% via selection for the EGFP-expressing part of colonies. (C) Embryoid bodies formed from EGFP-expressing EpiSC-like pESCs and expressed EGFP after aggregation (left panel: bright field, right panel: EGFP; scale bar = 100 µm). (D) EGFP-expressing EpiSC-like pESCs expressed pluripotent genes at the protein level (scale bar = 100 µm; passage number: 13) and (E) could be differentiated into three germ layers (scale bar = 50 µm; passage number: 23).</p

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

<div><p>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 <i>in vitro</i> 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 (<i>DPPA2</i>, <i>CDH1</i>, <i>EPCAM</i>, and <i>OCLN</i>) 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.</p></div

Derivation and characterization of EpiSC-like pESCs.

<p>(A) EpiSC-like pESCs derived from <i>in vitro</i>-produced embryos represented typical morphologies of mouse epiblast stem cells and human embryonic stem cells, and have alkaline phosphatase activity (left panel: no-stained colony; right panel: AP stained colony). (B) EpiSC-like pESCs have a normal karyotype (36+ XX) and (C) expressed genes related to pluripotency, as determined by RT-PCR (NT: non-transfected EpiSC-like pESCs passage 14, T: transfected EpiSC-like pESCs passage 14, W.B.: water blank). (D) Expression of pluripotent markers was detected using immunocytochemistry (passage number: 13). (E) Embryoid bodies were generated in suspension culture and spontaneously differentiated onto culture dishes. (F, G) The differentiated cells expressed differentiation marker genes at the mRNA and protein levels (Ectoderm: <i>CRABP2</i>, neurofilament, Mesoderm: <i>DES</i>, vimentin, Endoderm: <i>AFP</i>, cytokeratin 17; passage number: 17). Scale bars = 100 µm, except for A and G (scale bar = A: 200 µm, G: 50 µm).</p

Morphological changes of piPSCs in response to culture conditions.

<p>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.</p

Expression of pluripotent and MET-related genes as measured by qPCR.

<p>To verify the effects of transgenes on endogenous genes, expression levels of endogenous genes related to pluripotency and MET (pluripotent genes: <i>pOCT4a</i>, <i>pSOX2</i>, <i>pKLF4</i>, <i>pMYC</i>, <i>NANOG</i>, <i>DPPA2</i> and <i>REX1</i>; epithelial-to-mesenchymal transition (EMT) inducer: <i>TGFB1</i>; epithelial-specific markers: <i>CDH1</i>, <i>EPCAM</i> and <i>OCLN</i>) were determined by qPCR. As reprogramming is processed by exogenous genes, endogenous pluripotent genes (<i>pOCT4a</i>, <i>pSOX2</i>, <i>pKLF4</i>, <i>NANOG</i>, and <i>REX1</i>) and epithelial-specific markers (<i>CDH1</i>, <i>EPCAM</i>, and <i>OCLN</i>) were upregulated. However, <i>pMYC</i> and <i>DPPA2</i> (known predictors of reprogramming) were not reactivated, while <i>TGFB1</i> (blocks reprogramming) was not efficiently shut-down by transgene expression. Some genes, such as <i>pSOX2</i>, <i>pKLF4</i>, <i>REX1</i>, and epithelial-specific markers, were preferentially expressed in culture conditions containing bFGF, and <i>pKLF4</i> and <i>NANOG</i> were downregulated when treated with 2i.</p

Characterization of three selected iPSC lines.

<p>(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.</p