Abstract
Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4–6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.
Key points
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This protocol describes an optimized culture system for the derivation of porcine expanded potential stem cells (pEPSCs) from preimplantation embryos and reprogrammed somatic cells, including procedures for validation assays and gene targeting approaches.
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This optimized culture system is more robust than previous methods for pEPSC derivation and enables generation of pEPSC lines from preimplantation embryos derived by in vitro fertilization and somatic cell nuclear transfer.
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Data availability
Supporting data of this study can be found in our previous publications4,36. All source data generated or analyzed during this study are included in this published article and its supplementary files. Source data are provided with this paper.
References
Lunney, J. K. et al. Importance of the pig as a human biomedical model. Sci. Transl. Med. 13, eabd5758 (2021).
Hinrichs, A. et al. Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies. Xenotransplantation 28, e12664 (2021).
Reichart, B. et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation? J. Heart Lung Transplant. 39, 751–757 (2020).
Gao, X. et al. Establishment of porcine and human expanded potential stem cells. Nat. Cell Biol. 21, 687–699 (2019).
Guo, G. et al. Naive pluripotent stem cells derived directly from isolated cells of the human inner cell mass. Stem Cell Rep. 6, 437–446 (2016).
Khan, S. A. et al. Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening. Cell Rep. 35, 109233 (2021).
Takashima, Y. et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158, 1254–1269 (2014).
Theunissen, T. W. et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 15, 471–487 (2014).
Yang, J. et al. Establishment of mouse expanded potential stem cells. Nature 550, 393–397 (2017).
Kinoshita, M. et al. Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species. Development https://doi.org/10.1242/dev.199901 (2021).
Zhi, M. et al. Generation and characterization of stable pig pregastrulation epiblast stem cell lines. Cell Res. 32, 383–400 (2022).
Meek, S. et al. Stem cell-derived porcine macrophages as a new platform for studying host-pathogen interactions. BMC Biol. 20, 14 (2022).
Zhao, L. et al. Establishment of bovine expanded potential stem cells. Proc. Natl Acad. Sci. USA 118, e2018505118 (2021).
Brevini, T. A. et al. Culture conditions and signalling networks promoting the establishment of cell lines from parthenogenetic and biparental pig embryos. Stem Cell Rev. Rep. 6, 484–495 (2010).
Ezashi, T., Yuan, Y. & Roberts, R. M. Pluripotent stem cells from domesticated mammals. Annu. Rev. Anim. Biosci. 4, 223–253 (2016).
Haraguchi, S., Kikuchi, K., Nakai, M. & Tokunaga, T. Establishment of self-renewing porcine embryonic stem cell-like cells by signal inhibition. J. Reprod. Dev. 58, 707–716 (2012).
Hou, D. R. et al. Derivation of porcine embryonic stem-like cells from in vitro-produced blastocyst-stage embryos. Sci. Rep. 6, 25838 (2016).
Kues, W. A. et al. Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells. Stem Cells Dev. 22, 124–135 (2013).
Ma, Y., Yu, T., Cai, Y. & Wang, H. Preserving self-renewal of porcine pluripotent stem cells in serum-free 3i culture condition and independent of LIF and b-FGF cytokines. Cell Death Discov. 4, 21 (2018).
Park, J. K. et al. Primed pluripotent cell lines derived from various embryonic origins and somatic cells in pig. PLoS ONE 8, e52481 (2013).
Petkov, S., Hyttel, P. & Niemann, H. The small molecule inhibitors PD0325091 and CHIR99021 reduce expression of pluripotency-related genes in putative porcine induced pluripotent stem cells. Cell Reprogram. 16, 235–240 (2014).
Petkov, S., Glage, S., Nowak-Imialek, M. & Niemann, H. Long-term culture of porcine induced pluripotent stem-like cells under feeder-free conditions in the presence of histone deacetylase inhibitors. Stem Cells Dev. 25, 386–394 (2016).
Vassiliev, I. et al. In vitro and in vivo characterization of putative porcine embryonic stem cells. Cell Reprogram. 12, 223–230 (2010).
Xue, B. et al. Porcine pluripotent stem cells derived from IVF embryos contribute to chimeric development in vivo. PLoS ONE 11, e0151737 (2016).
Huang, S.-M. A. et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009).
Yang, J., Ryan, D. J., Lan, G., Zou, X. & Liu, P. In vitro establishment of expanded-potential stem cells from mouse pre-implantation embryos or embryonic stem cells. Nat. Protoc. 14, 350–378 (2019).
Wilkinson, A. C. et al. Expanded potential stem cell media as a tool to study human developmental hematopoiesis in vitro. Exp. Hematol. 76, 1–12. e15 (2019).
Bauer, B. K. et al. Transcriptional profiling by deep sequencing identifies differences in mRNA transcript abundance in in vivo-derived versus in vitro-cultured porcine blastocyst stage embryos. Biol. Reprod. 83, 791–798 (2010).
Macháty, Z., Day, B. N. & Prather, R. S. Development of early porcine embryos in vitro and in vivo. Biol. Reprod. 59, 451–455 (1998).
Pomar, F. J. et al. Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study. Theriogenology 63, 2254–2268 (2005).
Hao, Y. et al. Apoptosis and in vitro development of preimplantation porcine embryos derived in vitro or by nuclear transfer. Biol. Reprod. 69, 501–507 (2003).
Kim, S. et al. Establishment and characterization of embryonic stem-like cells from porcine somatic cell nuclear transfer blastocysts. Zygote 18, 93–101 (2010).
Siriboon, C. et al. Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning. PLoS ONE 10, e0118165 (2015).
Vassiliev, I. et al. Isolation and in vitro characterization of putative porcine embryonic stem cells from cloned embryos treated with trichostatin A. Cell Reprogram. 13, 205–213 (2011).
Lai, S. et al. Generation of knock-in pigs carrying Oct4-tdTomato reporter through CRISPR/Cas9-mediated genome engineering. PLoS ONE 11, e0146562 (2016).
Gao, X., Ruan, D. & Liu, P. Reprogramming porcine fibroblast to EPSCs. Methods Mol. Biol. 2239, 199–211 (2021).
Marinho, L. S. R., Rissi, V. B., Lindquist, A. G., Seneda, M. M. & Bordignon, V. Acetylation and methylation profiles of H3K27 in porcine embryos cultured in vitro. Zygote 25, 575–582 (2017).
Park, K. E., Magnani, L. & Cabot, R. A. Differential remodeling of mono- and trimethylated H3K27 during porcine embryo development. Mol. Reprod. Dev. 76, 1033–1042 (2009).
Xu, S., Wang, S., Tam, T., Liu, P. & Ruan, D. Derivation of trophoblast stem cells from human expanded potential stem cells. STAR Protoc. 4, 102354 (2023).
Liu, S. et al. Sox2 is the faithful marker for pluripotency in pig: evidence from embryonic studies. Dev. Dyn. 244, 619–627 (2015).
Hölker, M. Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos. Cloning Stem Cells 7, 35–44 (2005).
Nowak-Imialek, M. et al. Oct4-enhanced green fluorescent protein transgenic pigs: A new large animal model for reprogramming studies. Stem Cells Dev. 20, 1563–1575 (2011).
Nowak-Imialek, M. et al. In vitro and in vivo interspecies chimera assay using early pig embryos. Cell Reprogram. 22, 118–133 (2020).
Nowak-Imialek, M. & Niemann, H. in Cell and Molecular Biology and Imaging of Stem Cells (ed. Schatten, H.) Ch. 5, 137–152 (Wiley-Blackwell, 2014).
McMahon, A. P. & Bradley, A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62, 1073–1085 (1990).
Okae, H. et al. Derivation of human trophoblast stem cells. Cell Stem Cell 22, 50–63 e56 (2018).
Ruan, D. et al. Human early syncytiotrophoblasts are highly susceptible to SARS-CoV-2 infection. Cell Rep. Med. 3, 100849 (2022).
Kong, Q. et al. Lineage specification and pluripotency revealed by transcriptome analysis from oocyte to blastocyst in pig. FASEB J. 34, 691–705 (2020).
Acknowledgements
This project is supported by the National Key Research and Development Program of China (nos. 2022YFA1105401, 2022YFA1105400, 2018YFA0902702); Health@InnoHK, Innovation Technology Commission; HKSAR, Hong Kong Research Council (GRF17127219 and GRF17126421, Germany/Hong Kong travel grant G-HKU704/21); National Natural Science Foundation of China/RGC Collaborative Research Scheme (CRS_HKU703); National Natural Science Foundation of China (nos. 81570202 and 32070869); High Level-Hospital Program, Health Commission of Guangdong Province, China (no. HKUSZH201902025). Work in Germany was financially supported by Deutsche Forschungsgemeinschaft within the research network Regenerative Biology to Reconstructive Therapy. We are grateful to the team involved in IVF embryo production and SCNT, A. Lucas-Hahn, P. Hassel, R. Becker and M. Ziegler.
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Contributions
P.L., D.R. and M.N.-I. conceived the project and drafted the protocol. M.N.-I. contributed to the generation of porcine OCT4–eGFP blastocysts and performed the establishment of porcine pEPSCs from preimplantation embryos. M.N.-I. and D.H. performed testing of different pEPSCM conditions and IF staining of pEPSC outgrowths derived from porcine embryos. D.R., X.G. and Y.X. performed the establishment of pEPSCs from PFFs and the generation of pCD31 and pSOX2 reporter cell lines. D.R., X.G. and X.W. contributed to the generation of pTSCs. Y.X. contributed to the teratoma formation, karyotyping, pEPSCs electrotransfection and GGTA1 knockout assays. D.R., S.X. and Y.X. performed RT–qPCR analysis of gene expression levels, IF staining and FACS assays. P.L., H.N., M.N.-I., X.G. and D.R. contributed to the writing and the critical revision of the manuscript. Z.L. performed scRNA sequencing analysis. T.T.K.K.T., S.X. and L.L. provided intellectual input. All authors read and approved the final manuscript.
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A patent application related to the data presented here is pending on behalf of Center for Translational Stem Cell Biology and the University of Hong Kong. The other authors declare no competing interests.
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Nature Protocols thanks Jianyong Hang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Krüger, L. et al. Virus Res. 294, 198295 (2021): https://doi.org/10.1016/j.virusres.2021.198295
Rawat, H. et al. Front. Cell Dev. Biol. 11, 1111684 (2023): https://doi.org/10.3389/fcell.2023.1111684
Key data used in this protocol
Gao, X. et al. Nat. Cell Biol. 21, 687–699 (2019): https://doi.org/10.1038/s41556-019-0333-2
Gao, X. et al. Methods Mol. Biol. 2239, 199–211 (2021): https://doi.org/10.1007/978-1-0716-1084-8_13
Meek, S. et al. BMC Biol. 20, 14 (2022): https://doi.org/10.1186/s12915-021-01217-8
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Unprocessed gels for Supplementary Fig. 2.
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Statistical source data for Fig. 4d.
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Unprocessed gel for Fig. 7f.
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Unprocessed gel for Fig. 9d.
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Ruan, D., Xuan, Y., Tam, T.T.K.K. et al. An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00958-4
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DOI: https://doi.org/10.1038/s41596-024-00958-4
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