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An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells

Nature Protocols (2024)Cite this article

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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

  • 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.

  • 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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Fig. 1: Schematic diagrams describing the steps for the workflow of the protocol.
Fig. 2: The procedure for establishing pEPSC lines from porcine preimplantation embryos.
Fig. 3: The requirements of pEPSCM components in pEPSC derivation and maintenance.
Fig. 4: The development of opEPSCM.
Fig. 5: A schematic diagram of pEPSC derivation from porcine IVF and SCNT preimplantation embryos in opEPSCM.
Fig. 6: Characterization of opEPSCM-cultured pEPSCs derived from porcine preimplantation embryos.
Fig. 7: Reprogramming PFFs to pEPSCs.
Fig. 8: pEPSC’s epigenetic profiles and the developmental potential to trophoblasts.
Fig. 9: Genome editing in pEPSCs cultured in opEPSCM.
Fig. 10: SNL76/7 feeder preparation and single-colony picking and validation.

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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.

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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.

Author information

Author notes

  1. These authors contributed equally: Degong Ruan, Yiyi Xuan.

Authors and Affiliations

  1. Center for Translational Stem Cell Biology, Science Park, Sha Tin, Hong Kong, China

    Degong Ruan, Shao Xu & Pentao Liu

  2. Shenzhen Key Laboratory of Fertility Regulation, the University of Hong Kong-Shenzhen Hospital, Shenzhen, China

    Degong Ruan & Pentao Liu

  3. Stem Cell & Regenerative Medicine Consortium, School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Pok Fu Lam, Hong Kong, China

    Yiyi Xuan, Timothy Theodore Ka Ki Tam, ZhuoXuan Li, Xiao Wang & Pentao Liu

  4. Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute (FLI), Mariensee, Neustadt, Germany

    Doris Herrmann

  5. Hannover Medical School (MHH), Clinic for Gastroenterology, Hepatology and Endocrinology, Hannover, Germany

    Heiner Niemann

  6. CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China

    Liangxue Lai

  7. Department of Physiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China

    Xuefei Gao

  8. German Center for Cardiovascular Research (DZHK), Munich Heart Alliance, Munich, Germany

    Monika Nowak-Imialek

  9. First Department of Medicine, Cardiology, Klinikum rechts der Isar-Technical University of Munich, Munich, Germany

    Monika Nowak-Imialek

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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.

Corresponding authors

Correspondence to Xuefei Gao, Monika Nowak-Imialek or Pentao Liu.

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Competing interests

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

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and Tables 1 and 2.

Supplementary Data

Unprocessed gels for Supplementary Fig. 2.

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Statistical source data for Fig. 2d.

Source Data Fig. 4

Statistical source data for Fig. 4d.

Source Data Fig. 7

Unprocessed gel for Fig. 7f.

Source Data Fig. 9

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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