ABSTRACT Because post-mortem human skeletal muscle is not viable, autologous muscle grafts are typically required in tissue reconstruction after muscle loss due to disease or injury.

However, the use of autologous tissue often leads to donor-site morbidity. Here, we show that intraspecies and interspecies chimaeric pig embryos lacking native skeletal muscle can be

produced by deleting the _MYF5_, _MYOD_ and _MYF6_ genes in the embryos via CRISPR, followed by somatic-cell nuclear transfer and the delivery of exogenous cells (porcine blastomeres or

human induced pluripotent stem cells) via blastocyst complementation. The generated intraspecies chimaeras were viable and displayed normal histology, morphology and function. Human:pig

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PORCINE MYOGENESIS IN CLONED WILDTYPE AND _MYF5_/_MYOD_/_MYF6_-NULL PORCINE EMBRYO Article Open access 11 February 2025 REGENERATING HUMAN SKELETAL MUSCLE FORMS AN EMERGING NICHE IN VIVO TO

SUPPORT PAX7 CELLS Article Open access 02 November 2023 SKELETAL MUSCLE DIFFERENTIATION OF HUMAN IPSCS MEETS BIOENGINEERING STRATEGIES: PERSPECTIVES AND CHALLENGES Article Open access 07

April 2022 DATA AVAILABILITY The single-cell RNA-seq data from WT porcine morula and blastocyst embryos have been deposited in the NCBI Sequence Read Archive (SRA) database, under project

accession no. PRJNA509275. All unique materials used are readily available from the authors or from commercial sources (Supplementary Tables 1–3). Gene-edited primary cell lines are

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references ACKNOWLEDGEMENTS This work was supported by a grant from Regenerative Minnesota Medicine (RMM; D.J.G.) and from the US Department of Defense (M.G.G.). We acknowledge NorthStar

Genomics, DeSoto Biosciences, Recombinetics, MOFA Global, the University of Minnesota Genomics Center and the University of Minnesota Imaging Center for their technical assistance. We

acknowledge the Cytogenomics Core at the University of Minnesota for karyotyping services. No NIH or RMM funding was used for the human chimaera portions of this study. We thank J. Hannah

(Weizmann Institute of Science) for helpful discussions throughout the course of the studies. We also thank R. Prather and the National Swine Resource and Research Center at the University

of Missouri for consultation (U42 OD011140). AUTHOR INFORMATION Author notes * These authors contributed equally: Geunho Maeng, Satyabrata Das. AUTHORS AND AFFILIATIONS * Lillehei Heart

Institute, University of Minnesota, Minneapolis, MN, USA Geunho Maeng, Satyabrata Das, Wuming Gong, Bhairab N. Singh, Stefan Kren, Daniel Mickelson, Erik Skie, Ohad Gafni, Cyprian V. Weaver,

 Daniel J. Garry & Mary G. Garry * School of Kinesiology, University of Minnesota, Minneapolis, MN, USA Sarah M. Greising & Jacob R. Sorensen * Stem Cell Institute, University of

Minnesota, Minneapolis, MN, USA Daniel J. Garry * Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN, USA Daniel J. Garry & Mary G. Garry *

NorthStar Genomics, Eagan, MN, USA Daniel J. Garry & Mary G. Garry Authors * Geunho Maeng View author publications You can also search for this author inPubMed Google Scholar *

Satyabrata Das View author publications You can also search for this author inPubMed Google Scholar * Sarah M. Greising View author publications You can also search for this author inPubMed 

Google Scholar * Wuming Gong View author publications You can also search for this author inPubMed Google Scholar * Bhairab N. Singh View author publications You can also search for this

author inPubMed Google Scholar * Stefan Kren View author publications You can also search for this author inPubMed Google Scholar * Daniel Mickelson View author publications You can also

search for this author inPubMed Google Scholar * Erik Skie View author publications You can also search for this author inPubMed Google Scholar * Ohad Gafni View author publications You can

also search for this author inPubMed Google Scholar * Jacob R. Sorensen View author publications You can also search for this author inPubMed Google Scholar * Cyprian V. Weaver View author

publications You can also search for this author inPubMed Google Scholar * Daniel J. Garry View author publications You can also search for this author inPubMed Google Scholar * Mary G.

Garry View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.G.G. and D.J.G. conceived the project. G.M., S.D., D.J.G. and M.G.G. wrote the

manuscript. G.M., S.D., S.K., B.N.S., D.M., S.M.G., J.R.S., E.S., W.G., C.V.W. and O.G. performed experiments and analysed the data. M.G.G. and D.J.G. supervised the project. All authors

commented on and edited the final version of the paper. CORRESPONDING AUTHORS Correspondence to Daniel J. Garry or Mary G. Garry. ETHICS DECLARATIONS COMPETING INTERESTS D.J.G. and M.G.G.

are co-founders of NorthStar Genomics. All other authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Biomedical Engineering_ thanks the anonymous

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THIS ARTICLE Maeng, G., Das, S., Greising, S.M. _et al._ Humanized skeletal muscle in _MYF5_/_MYOD_/_MYF6_-null pig embryos. _Nat Biomed Eng_ 5, 805–814 (2021).

https://doi.org/10.1038/s41551-021-00693-1 Download citation * Received: 13 September 2020 * Accepted: 29 January 2021 * Published: 29 March 2021 * Issue Date: August 2021 * DOI:

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