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, 8 (1), e52481

Primed Pluripotent Cell Lines Derived From Various Embryonic Origins and Somatic Cells in Pig

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Primed Pluripotent Cell Lines Derived From Various Embryonic Origins and Somatic Cells in Pig

Jin-Kyu Park et al. PLoS One.

Abstract

Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC-like porcine ESC (pESC) lines from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated, and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) into porcine fibroblast cells. In this study, we analyzed characteristics such as marker expression, pluripotency and the X chromosome inactivation status in female of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1-60 and TRA 1-81. Furthermore all of these cell lines showed in vitro differentiation potential, the X chromosome inactivation in female and a normal karyotype. Here we suggest that the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Derivation and gene expression analysis of porcine pluripotent cells.
(A) Early morphology of EpiSC-like primary pESCs derived from porcine blastocysts. (B) Early morphology of EpiSC-like primary piPSCs derived from porcine embryonic fibroblasts. (C) Late morphology of EpiSC-like primary pESCs derived from porcine blastocysts. (D) Late morphology of EpiSC-like primary piPSCs derived from porcine embryonic fibroblasts. (E) AP activity of typical EpiSC-like pESCs. (F) AP activity of typical EpiSC-like piPSCs. (G) All of the pluripotent cells expressed the undifferentiated pluripotent stem cells markers Oct4, Sox2, Nanog, TDGF1 and Rex1 as well as the epiblast stem cell markers bFGF, FGFR1, FGFR2, Activin-A and Nodal. C-kit ligand and C-kit receptor, known growth factor for stem cell renewal, were also expressed in these cell lines. Scale bars = 200 µm.
Figure 2
Figure 2. Expression of pluripotent markers in porcine pluripotent cells by immunocytochemistry analysis.
Expression of pluripotent markers and surface markers Oct4, Sox2, Nanog, SSEA4, TRA 1–60 and TRA 1–81 was observed all six cell lines (A) pESI6, (B) pESP2, (C) pES3X3, (D) pES11, (E) pES12 and (F) piPS-1. Lanes to the left represent hoechst staining. (G) Expression of pluripotent markers Oct4, Sox2 and Nanog was detected in 84.91%, 97.93% and 93.01% of cells from the cell line pESI6, 84.4%, 98.3% and 97.54% in the cell line pESP2, 87.08%, 97.57% and 97.74% in the cell line pES3X3, 99.56%, 94.14% and 99.8% in the cell line pEpiS11, 98.11%, 99.78% and 99.14% in the cell line pES12 and 98.04%, 99.2% and 98.82% in the cell line piPS-1. Scale bars = 100 µm.
Figure 3
Figure 3. Epigenetic characteristics of EpiSC-like pESCs and piPSCs.
(A) Investigation of the XIST gene expression from all cell lines. The result showed the expression of XIST gene in cell line pESI6, pESP2, pES11 and pES12 which are all female. Meanwhile, no expression was identified in male cell lines, pES3X3 and piPS-1. (B) Relative expression levels of XIST gene in individual cell lines clearly demonstrated the XIST gene in female cell lines was highly expressed comparable to male cell lines. Y-axis is expressed as a relative gene expression level in the cell lines. Data are represented as mean ± SEM (n = 5); Bars with different letters (a and b) are significantly different (P<0.05). (C) DNA methylation status of XIST promoter regions in female cell lines pESI6, pESP2, pES11 and pES12 and in male cell line piPS-1. Circles indicated the CpG sites of region analyzed. Open and closed circles mean unmethylation and methylation status.
Figure 4
Figure 4. Global gene expression profile of EpiSC-like pESCs.
Each row represents the expression of a single gene and columns indicate samples; mESCs, pESCs and hESCs. The 3 large groups in the heatmap were identified. Hierarchical clustering result showed the global gene expression pattern of pESCs is similar to hESCs. mm 1, 2 and 3; cell line R1, mm 4, 5 and 6; J1, pig 1, 2 and 3; cell line PES5, hs 1, 2 and 3 : cell line HUES6, hs 4, 5 and 6 : cell line H9.
Figure 5
Figure 5. Differentiation potential and karyotyping of EpiSC-like pESCs and piPSCs.
(A) Representative embryoid bodies derived from EpiSC-like pES cell lines and piPS cell lines through the culture for 5 days by hanging drop method. (B) When EBs cultured continuously onto culture plates, a variety of differentiated cells was observed. We could detect the expression of marker genes AFP (endoderm), DESMIN (mesoderm) and CRABP2 (ectoderm) involving differentiation. (C) Confirmation of the expression of differentiation marker Cytokeratin 17 (endoderm), Desmin (mesoderm) and Neurofilament (ectoderm) from differentiated cells by the immunocytochemistry analysis. Left lane is the cells differentiated from EpiSC-like pESCs and right lane is the cells differentiated from EpiSC-like piPSCs. (D) Both EpiSC-like pESCs and piPSCs have a normal karyotyping. Scale bars = 50 µm.

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Grant support

This work was supported by the Next BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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