DNA repair and replication links to pluripotency and differentiation capacity of pig iPS cells

<div><p>Pigs are proposed to be suitable large animal models for test of the efficacy and safety of induced pluripotent stem cells (iPSCs) for stem cell therapy, but authentic pig ES/iPS cell lines with germline competence are rarely produced. The pathways or signaling underlying the defective competent pig iPSCs remain poorly understood. By improving induction conditions using various small chemicals, we generated pig iPSCs that exhibited high pluripotency and differentiation capacity that can contribute to chimeras. However, their potency was reduced with increasing passages by teratoma formation test, and correlated with declined expression levels of <i>Rex1</i>, an important marker for naïve state. By RNA-sequencing analysis, genes related to WNT signaling were upregulated and MAPK signaling and TGFβ pathways downregulated in pig iPSCs compared to fibroblasts, but they were abnormally expressed during passages. Notably, pathways involving in DNA repair and replication were upregulated at early passage, but downregulated in iPSCs during prolonged passage in cluster with fibroblasts. Our data suggests that reduced DNA repair and replication capacity links to the instability of pig iPSCs. Targeting these pathways may facilitate generation of truly pluripotent pig iPSCs, with implication in translational studies.</p></div

Cell proliferation, epigenetic modification and DNA damage response of pig iPSCs.

<p><b>(A)</b> Reduced cell proliferation of pig iPSCs during prolonged passages. Left upper panel, phase contrast images of representative pig iPSCs and immunofluorescence staining of Ki-67 at early and late passages. Note, these iPSCs appeared as dome-shaped colonies, as they were cultured in the basal medium added with 5% ES quality FBS, so the cells proliferated faster, and could be passaged by single cell after digestion and separation using TrypLE. Right panel at bottom, percentage of Ki-67 positive cells in pig iPSCs induced by OSKM or OSKMN at early and late passages. *, p<0.05. For phase-contrast optics (Ph), scale bar = 100 μm; for immunofluorescence images, scale bar = 10 μm. <b>(B)</b> Epigenetic markers and DNA damage response of PEF and iPSCs at P2 by immunofluorescence staining. Nuclei were stained by Hoechst 33342 (lower panel). PEF at passage 3 as feeder layer served as background control by immunofluorescence. Scale bar = 20 μm. <b>(C)</b> Western blotting analysis of protein levels in PEF, iPSCs induced by OSKM on day 20, iPSCs induced by OSKM at P2 and iPSCs induced by OSKM+Rex1 at P2. H3 and β-tubulin served as loading control. <b>(D)</b> Left panel, representative images showing γH2AX immunofluorescence and foci in PEF and pig iPSCs at early and late passages induced by OSKM or OSKMN. Right panel, percentage of γH2AX-positve cells. *, p<0.05. Scale bar = 10 μm. <b>(E)</b> Left panel, images at higher magnification of γH2AX foci. Right panel, frequency of γH2AX foci in cells fewer than 10 and more than 10, respectively. *, p<0.05, ***, p<0.001. Scale bar = 10 μm. All the experiments, n≥3.</p

Hierarchical clustering and comparative analysis of pathways in pig iPSCs in comparison with PEFs by RNA-sequencing.

<p><b>(A)</b> Hierarchical clustering and heat map of global gene expression patterns in PEF, pig iPSCs at P5, and at P10 by RNA-sequencing. All values are presented as mean of two independent experiments. <b>(B)</b> Scatter plots showing comparison of gene expression profile of PEF, pig iPSCs at P5 and pig iPSCs at P10. Both axes (in log10 scale) represent the gene expression values. Pink and green dots indicate genes with at least two-fold changes. <b>(C)</b> Comparative analysis of expression patterns of key pluripotency genes in PEF, and pig iPSCs at P5 and at P10. All values are presented as mean of two independent experiments. <b>(D)</b> qPCR analysis of expression of selected endogenous pluripotent genes in PEF and pig iPSCs at P5 and at P10. n≥3.</p

Characterization of pig iPSCs <i>in vitro</i>.

<p><b>(A)</b> Teratoma formation test showing differentiation into epithelium (endoderm), muscle (mesoderm) and neural (ectoderm) by H&E histology of pig iPSCs induced from OSKM or OSKMN at passage 3–5. Scale bar = 50 μm. n = 4. <b>(B)</b> Expression of pluripotent markers, Oct4, Sox2, Nanog, SSEA1 or SSEA4 by immunofluorescence microscopy of OSKM or OSKMN-induced pig iPSCs at P10 (representative images were shown). n≥3. <b>(C)</b> Differentiation into three germ layers by embryoid body (EB) formation test of pig iPSCs induced by OSKM or OSKMN (representative images were shown). Endoderm marker, alpha 1-fetoprotein (AFP); mesoderm marker, smooth muscle actin (SMA); ectoderm marker, β-III-tubulin. For phase-contrast optics (Ph) of EB, scale bar = 100 μm, and for immunofluorescence images, scale bar = 50 μm. n≥3. <b>(D-E)</b> qPCR analysis for relative RNA expression of selected endogenous and exogenous pluripotency-associated genes in PEF, pig iPSCs induced by OSKM or OSKMN at passage 5 (P5) and passage 10 (P10). PEF without induction served as negative control and PEF induced by OSKMN on day 2 as positive control of exogenous genes. **, p<0.01. n≥3.</p

Generation of iPSCs from Pig Embryonic Fibroblasts (PEFs) using various factors and small molecules and by extensive clonal selections.

<p><b>(A)</b> Colony identification by alkaline phosphatase (AP) positive staining during iPSC induction on day 18 by various combinations of the transcription factors, OCT4, O; SOX2, S; KLF4, K; c-MYC, M; NANOG, N. n = 3. <b>(B)</b> Small molecule compounds in combinations S-adenosylhomocysteine (SAH), sodium butyrate (NaB) and BIX01294 (BIX) improve the percentage of SSEA-4-positive cells by FACS analysis, n = 3. <b>(C)</b> Schematic outlining the strategy for colonies to be extensively picked and passaged to achieve high quality pig iPSCs. ESC-like colonies were picked at days 14~20 following a standard protocol. High-quality iPSCs were able to subculture by mechanical method, while low-quality iPSCs stoped growing or differentiated quickly before passage 5. However, High-quality iPSCs were difficult to maintain. (<b>D)</b> Phase contrast images showing representative pig iPSCs induced by OSKM or OSKMN at different passages. Scale bar = 100 μm.</p