Generation of allogeneic and xenogeneic functional muscle stem cells for intramuscular transplantation
Ajda Lenardič, … , Christoph Handschin, Ori Bar-Nur
Ajda Lenardič, … , Christoph Handschin, Ori Bar-Nur
Published May 7, 2024
Citation Information: J Clin Invest. 2024;134(12):e166998. https://doi.org/10.1172/JCI166998.
View: Text | PDF
Research Article Muscle biology

Generation of allogeneic and xenogeneic functional muscle stem cells for intramuscular transplantation

Abstract

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected Dmdmdx mouse induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Notably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

Authors

Ajda Lenardič, Seraina A. Domenig, Joel Zvick, Nicola Bundschuh, Monika Tarnowska-Sengül, Regula Furrer, Falko Noé, Christine L. Trautmann, Adhideb Ghosh, Giada Bacchin, Pjeter Gjonlleshaj, Xhem Qabrati, Evi Masschelein, Katrien De Bock, Christoph Handschin, Ori Bar-Nur

×

Figure 2

Exclusive generation of edited iPSC–derived muscle stem cells in intraspecies chimeras.

Options: View larger image (or click on image) Download as PowerPoint
Exclusive generation of edited iPSC–derived muscle stem cells in intrasp...
(A) A schematic overview of the experimental plan. MEFs, mouse embryonic fibroblasts. (B) Representative images of the specified cell lines. Scale bars: 100 μm. LUTs were individually adjusted. (C) PCR products for dystrophin, amplified from the DNA of non-edited (–) and edited (+) Dmdmdx; Pax7-nGFP iPSCs. (D) DNA sequence of the edited dystrophin PCR product lacking a splice donor site. A black asterisk specifies the mdx mutation. (E) Representative images of non-edited and edited Dmdmdx; Pax7-nGFP iPSC–derived myotubes. Scale bars: 100 μm. LUTs were individually adjusted. (F) PCR for dystrophin in cDNA isolated from non-edited and edited Dmdmdx; Pax7-nGFP iPSC–derived myogenic cultures. (G) Sanger sequence of an edited dystrophin band shown in F, revealing successful exon skipping and reframing of dystrophin at the cDNA level. (H) Representative genotyping for the Pax7-nGFP allele in non-chimeric and chimeric pups. (I) A graph showing chimera numbers based on Pax7-nGFP allele genotyping. (J) FACS analysis of Pax7-nGFP expression in the indicated animals and conditions. (K) A graph showing quantification of the percentage of Pax7-nGFP+ cells in muscles derived from chimeras with or without tamoxifen treatment. n = 2–3 animals, data are presented as mean ± SD. Statistical analysis was performed using a Student’s 2-tailed t test. (L) A schematic representation outlining the strategy to determine the percentage of iPSC-derived satellite cells within the overall ITGA7+ (host + donor) satellite cell population of intraspecies chimeras. (M) A graph illustrating the percentage of iPSC-derived satellite cells, identified by the Pax7-nGFP reporter, out of the total ITGA7+ satellite cell population in chimeras. n = 3 animals for the non–tamoxifen-injected control, n = 5 animals for the tamoxifen-treated group. Data are presented as mean ± SD. Statistical analysis was performed using a Student’s 2-tailed t test. (N) PCR for dystrophin using DNA of ITGA7+ satellite cell–derived expanded myoblasts of the specified animals and conditions. Note that all chimera-derived myoblasts showed only an edited dystrophin band.