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Humanized skeletal muscle in MYF5/MYOD/MYF6-null pig embryos


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 chimaeras generated with TP53-null human induced pluripotent stem cells led to higher chimaerism efficiency, with embryos collected at embryonic days 20 and 27 containing humanized muscle, as confirmed by immunohistochemical and molecular analyses. Human:pig chimaeras may facilitate the production of exogenic organs for research and xenotransplantation.

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Fig. 1: Porcine MYF5/MYOD/MYF6-null embryos lack the skeletal muscle lineage and recapitulate the mouse phenotype.
Fig. 2: Complementation of the skeletal muscle lineage by GFP-labelled blastomeres in MYF5/MYOD/MYF6-null embryos.
Fig. 3: Analysis of human:pig (cGFP-labelled TP53 null complemented) embryos.
Fig. 4: Neuronal and germ line cell lineages in human–pig chimaeric embryos are cGFP-negative.

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 13). Gene-edited primary cell lines are available yet limited in number because of supply constraints.


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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




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.

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Competing interests

D.J.G. and M.G.G. are co-founders of NorthStar Genomics. All other authors declare no competing interests.

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Maeng, G., Das, S., Greising, S.M. et al. Humanized skeletal muscle in MYF5/MYOD/MYF6-null pig embryos. Nat Biomed Eng (2021).

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