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

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

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VIEWED BY OTHERS GENERATION AND CHARACTERIZATION OF STABLE PIG PREGASTRULATION EPIBLAST STEM CELL LINES Article Open access 30 November 2021 PIG BLASTOCYST-LIKE STRUCTURE MODELS FROM

EMBRYONIC STEM CELLS Article Open access 02 July 2024 ESTABLISHMENT OF HUMAN EXPANDED POTENTIAL STEM CELL LINES VIA PREIMPLANTATION EMBRYO CULTIVATION AND SOMATIC CELL REPROGRAMMING Article

29 April 2025 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

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pig. _FASEB J._ 34, 691–705 (2020). Article  CAS  PubMed  Google Scholar  Download references 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 * These authors contributed equally: Degong Ruan, Yiyi Xuan. AUTHORS AND AFFILIATIONS * Center for Translational Stem Cell Biology, Science Park,

Sha Tin, Hong Kong, China Degong Ruan, Shao Xu & Pentao Liu * Shenzhen Key Laboratory of Fertility Regulation, the University of Hong Kong-Shenzhen Hospital, Shenzhen, China Degong Ruan 

& Pentao Liu * 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 * Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute (FLI), Mariensee, Neustadt, Germany Doris

Herrmann * Hannover Medical School (MHH), Clinic for Gastroenterology, Hepatology and Endocrinology, Hannover, Germany Heiner Niemann * 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 * Department of

Physiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China Xuefei Gao * German Center for Cardiovascular Research (DZHK), Munich Heart Alliance, Munich,

Germany Monika Nowak-Imialek * First Department of Medicine, Cardiology, Klinikum rechts der Isar-Technical University of Munich, Munich, Germany Monika Nowak-Imialek Authors * Degong Ruan

View author publications You can also search for this author inPubMed Google Scholar * Yiyi Xuan View author publications You can also search for this author inPubMed Google Scholar *

Timothy Theodore Ka Ki Tam View author publications You can also search for this author inPubMed Google Scholar * ZhuoXuan Li View author publications You can also search for this author

inPubMed Google Scholar * Xiao Wang View author publications You can also search for this author inPubMed Google Scholar * Shao Xu View author publications You can also search for this

author inPubMed Google Scholar * Doris Herrmann View author publications You can also search for this author inPubMed Google Scholar * Heiner Niemann View author publications You can also

search for this author inPubMed Google Scholar * Liangxue Lai View author publications You can also search for this author inPubMed Google Scholar * Xuefei Gao View author publications You

can also search for this author inPubMed Google Scholar * Monika Nowak-Imialek View author publications You can also search for this author inPubMed Google Scholar * Pentao Liu View author

publications You can also search for this author inPubMed Google Scholar 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 p_CD31_ and p_SOX2_

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. ETHICS DECLARATIONS 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. PEER REVIEW PEER REVIEW INFORMATION _Nature Protocols_

thanks Jianyong Hang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral

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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.

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governed by the terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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_ 19, 1710–1749 (2024).

https://doi.org/10.1038/s41596-024-00958-4 Download citation * Received: 23 December 2022 * Accepted: 08 December 2023 * Published: 20 March 2024 * Issue Date: June 2024 * DOI:

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