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Generation of pig induced pluripotent stem cells using an extended pluripotent stem cell culture system.
Stem Cell Res Ther. 2019 06 27; 10(1):193.SC

Abstract

BACKGROUND

Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.

METHODS

iPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.

RESULTS

In this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.

CONCLUSIONS

Our findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.

Authors+Show Affiliations

State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China. College of Biological Sciences, China Agricultural University, Beijing, 100193, China.Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA. Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.Key Laboratory of Animal Gene Editing and Animal Cloning in Yunnan Province, Kunming, 650201, China.State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.College of Biological Sciences, China Agricultural University, Beijing, 100193, China.State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China. College of Biological Sciences, China Agricultural University, Beijing, 100193, China.State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China. College of Biological Sciences, China Agricultural University, Beijing, 100193, China.State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China. College of Biological Sciences, China Agricultural University, Beijing, 100193, China.Key Laboratory of Animal Gene Editing and Animal Cloning in Yunnan Province, Kunming, 650201, China.CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA. Jun2.wu@utsouthwestern.edu. Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA. Jun2.wu@utsouthwestern.edu.Key Laboratory of Animal Gene Editing and Animal Cloning in Yunnan Province, Kunming, 650201, China. hongjiangwei@126.com. College of Veterinary Medicine, Yunnan Agriculture University, Kunming, 650201, China. hongjiangwei@126.com.State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China. hanjy@cau.edu.cn. College of Biological Sciences, China Agricultural University, Beijing, 100193, China. hanjy@cau.edu.cn.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

31248457

Citation

Xu, Junjun, et al. "Generation of Pig Induced Pluripotent Stem Cells Using an Extended Pluripotent Stem Cell Culture System." Stem Cell Research & Therapy, vol. 10, no. 1, 2019, p. 193.
Xu J, Yu L, Guo J, et al. Generation of pig induced pluripotent stem cells using an extended pluripotent stem cell culture system. Stem Cell Res Ther. 2019;10(1):193.
Xu, J., Yu, L., Guo, J., Xiang, J., Zheng, Z., Gao, D., Shi, B., Hao, H., Jiao, D., Zhong, L., Wang, Y., Wu, J., Wei, H., & Han, J. (2019). Generation of pig induced pluripotent stem cells using an extended pluripotent stem cell culture system. Stem Cell Research & Therapy, 10(1), 193. https://doi.org/10.1186/s13287-019-1303-0
Xu J, et al. Generation of Pig Induced Pluripotent Stem Cells Using an Extended Pluripotent Stem Cell Culture System. Stem Cell Res Ther. 2019 06 27;10(1):193. PubMed PMID: 31248457.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Generation of pig induced pluripotent stem cells using an extended pluripotent stem cell culture system. AU - Xu,Junjun, AU - Yu,Leqian, AU - Guo,Jianxiong, AU - Xiang,Jinzhu, AU - Zheng,Zheng, AU - Gao,Dengfeng, AU - Shi,Bingbo, AU - Hao,Haiyang, AU - Jiao,Deling, AU - Zhong,Liang, AU - Wang,Yu, AU - Wu,Jun, AU - Wei,Hongjiang, AU - Han,Jianyong, Y1 - 2019/06/27/ PY - 2018/11/14/received PY - 2019/06/17/accepted PY - 2019/04/26/revised PY - 2019/6/29/entrez PY - 2019/6/30/pubmed PY - 2020/7/1/medline KW - Chimera KW - EPS cells KW - Extended pluripotency KW - Pig iPS cells SP - 193 EP - 193 JF - Stem cell research & therapy JO - Stem Cell Res Ther VL - 10 IS - 1 N2 - BACKGROUND: Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown. METHODS: iPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis. RESULTS: In this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%. CONCLUSIONS: Our findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low. SN - 1757-6512 UR - https://www.unboundmedicine.com/medline/citation/31248457/Generation_of_pig_induced_pluripotent_stem_cells_using_an_extended_pluripotent_stem_cell_culture_system_ L2 - https://stemcellres.biomedcentral.com/articles/10.1186/s13287-019-1303-0 DB - PRIME DP - Unbound Medicine ER -