Domenig, S. A. | bioRxiv Science

bioRxiv2023

Interspecies generation of functional muscle stem cells

Satellite cells, the stem cells of skeletal muscle tissue, hold a prodigious regeneration capacity. However, low satellite cell yield from autologous or donor-derived muscles precludes adoption of satellite cell transplantation for the treatment of muscle diseases including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether sufficient quantity of satellite cells can be produced in allogeneic or xenogeneic animal hosts. First, we report on exclusive satellite cell production in intraspecies mouse chimeras by injection of CRISPR/Cas9-corrected DMD-induced pluripotent stem cells (iPSCs) into blastocysts carrying an ablation system of host Pax7+ satellite cells. Additionally, injection of genetically-corrected DMD-iPSCs into rat blastocysts produced interspecies rat-mouse chimeras harboring mouse muscle stem cells that efficiently restored dystrophin expression in DMD mice. This study thus provides a proof-of-principle for the generation of therapeutically-competent stem cells between divergent species, raising the possibility of procuring human stem cells in large animals for regenerative medicine purposes.

cell biology↗

bioRxiv2021

Differentiation of mouse embryonic stem cells into cells with spermatogonia-like morphology with chemical intervention-dependent increased gene expression of LIM homeobox 1 (Lhx1).

During the development of the male germline, spermatogonial stem cells (SSCs) originate from gonocytes that differentiate from primordial germ cells (PGCs). In the developing and regenerating mouse testis, expression of the gene LIM homeobox 1 (Lhx1) marks the most undifferentiated SSCs. However, an enrichment of Lhx1 expression in spermatogonia-like cells generated in vitro has not been reported so far. Previously, it was shown that a chemical intervention in male mouse embryonic stem (ES) cells in serum culture, including a timed combination of the SIRT1 inhibitor Ex-527, the DNA methyltransferase inhibitor RG-108 and the electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with molecular markers of the PGC to gonocyte differentiation process. Here, we report the in vitro differentiation of male mouse ES cells, cultured under dual chemical inhibition of GSK3{beta} and MEK (termed 2i) with leukemia inhibitory factor (LIF) (termed 2iL) and serum, into cells with spermatogonia-like morphology (CSMs) and population-averaged expression of spermatogonia-specific genes. This was achieved by the removal of 2iL and a specific schedule of 2 partial medium replacements per day with alternating 8-hour and 16-hour intervals over a period of 32 days. Combination of this new cell culture protocol with the previously reported chemical intervention in ES cells changed the population-averaged expression of spermatogonia- and gonocyte-specific genes during the differentiation process and increased the population-averaged gene expression of Lhx1 in the resulting CSMs compared to CSMs without chemical intervention. Furthermore, we detected single CSMs with a strong nuclear LHX1/5 protein signal only in the chemical intervention group. Our results provide the first experimental evidence for the generation of CSMs with an enrichment of Lhx1 expression in vitro. We propose that further investigation of the CSMs generated with this in vitro system may provide new insights into male germline and stem cell development.

cell biology↗