Transcriptome and epigenome diversity and plasticity of muscle stem cells following transplantation

Abstract

20Adult skeletal muscles are maintained during homeostasis and regenerated upon injury by muscle stem 21 cells (MuSCs). A heterogeneity in self-renewal, differentiation and regeneration properties has been 22 reported for MuSCs based on their anatomical location. Although MuSCs derived from extraocular 23 muscles (EOM) have a higher regenerative capacity than those derived from limb muscles, the 24 molecular determinants that govern these differences remain undefined. Here we show that EOM and 25 limb MuSCs have distinct DNA methylation signatures associated with enhancers of location-specific 26 genes, and that the EOM transcriptome is reprogrammed following transplantation into a limb muscle 27 environment. Notably, EOM MuSCs expressed host-site specific positional Hox codes after engraftment 28 and self-renewal within the host muscle. However, about 10% of EOM-specific genes showed 29 engraftment-resistant expression, pointing to cell-intrinsic molecular determinants of the higher 30 engraftment potential of EOM MuSCs. Our results underscore the molecular diversity of distinct MuSC 31 populations and molecularly define their plasticity in response to microenvironmental cues. These 32 findings provide insights into strategies designed to improve the functional capacity of MuSCs in the 33 context of regenerative medicine. 34 35 41 proven to be challenging for muscular dystrophies, as they require delivering enough functional MuSCs 42 to the right muscle groups and ex vivo amplification of healthy MuSCs results in a major decline in their 43 regenerative capacity and stemness properties 16 . 44 45 Most of the knowledge about MuSC biology arises from the study of limb muscles, whereas MuSCs 46 from other muscle groups remain less well characterized. Extraocular muscles (EOMs) are responsible 47 75 76 Here we performed parallel DNA methylation and transcriptome sequencing to characterize the specific 77 identity of adult mouse EOM and limb MuSCs at the molecular level. We used heterotopic 78 transplantation of EOM MuSCs into limb muscles to challenge their fate and assess their plasticity. We 79 4show that their specific identity is mostly niche-driven as they adopt a limb-like molecular signature. 80 Nevertheless, we also identify EOM-specific genes that resist reprogramming by the microenvironment, 81 indicating potential candidates to manipulate in limb-derived MuSCs for improving their regenerative 82 capacity in the context of cellular therapies. 83 84 RESULTS 85 To investigate the molecular differences between EOM and TA MuSCs in vivo, MuSCs from both 86 locations were isolated by FACS (Fluorescence-Activated Cell Sorting) from 10 adult Tg:Pax7-nGFP 17 87 mice and processed for RNA-sequencing and BS-sequencing as in 47,48 (Figure 1A). Unsupervised 88 Principal Component Analysis (PCA) on the transcriptomes revealed clear discrimination between TA 89 and EOM MuSCs samples, indicating location-specific transcriptional identities (Figure 1B). To further 90 investigate these transcriptional signatures...

Hoxa10 mediates positional memory to govern stem cell function in adult skeletal muscle

“…Our DNA methylome analysis also revealed hypermethylation of the Hox-A loci and high expression levels of Hox-A cluster genes in adult limb muscles and their associated satellite cell-derived myoblasts. Although these loci are not uniformly hypermethylated and the DNA hypermethylation patterns do not simply correlate with the expression levels of Hox-A cluster genes, our findings are further supported by the study depicting the hypermethylation status of Hox-A loci in hindlimb-derived satellite cells in mice (24). In addition to DNA methylation, histone modifications may also be involved in regulating Hox-A gene cluster expression in satellite cells (23,34).…”

“…Our ectopic transplantation study shows that MAS-derived satellite cells maintained low Hox status 2 weeks following TA niche exposure. However, Evano et al (24) reported that satellite cells isolated from the extraocular muscle, which is derived from the cranial mesoderm, express Hox-A cluster genes at very low levels, but these genes are up-regulated 3 to 4 weeks after transplantation into CTX preinjected TA muscle. This discrepancy may be explained by the different time periods after transplantation or different cell types: A longer duration of exposure to different niches may gradually influence Hox status, or extraocular muscle-derived satellite cells may adapt to different niches compared to MAS-derived satellite cells.…”

“…This discrepancy may be explained by the different time periods after transplantation or different cell types: A longer duration of exposure to different niches may gradually influence Hox status, or extraocular muscle-derived satellite cells may adapt to different niches compared to MAS-derived satellite cells. We also note that for efficient cell engraftment, we depleted intrinsic satellite cells in TA muscle tissue with -irradiation before transplantation, while Evano et al (24) did not use radiation. Thus, these irradiated and nonirradiated transplantation conditions may differentially affect the regenerative niche and plasticity of engrafted satellite cells in the host muscle tissues.…”

“…Hox-A and Hox-C cluster genes are known to be expressed in satellite cells in adult mice (21)(22)(23)(24). Given that the hypermethylation status at Hox-A cluster locus was observed in limb muscles (Fig.…”

Exercise/Resistance Training and Muscle Stem Cells

“…MuSCs are heterogeneous populations with different cell cycling times [24,25], Pax3 [26], Pax7 [27], Myf5 [28], Mx1 [29], or CD34 expression [30]. In addition, MuSC properties are also influenced by myofiber type [31] or the muscle region in the body [32]. MuSCs in both slow (soleus) and fast-type muscles (plantaris) can supply new myonuclei in loading muscles [33,34], suggesting that the responsiveness of MuSCs to mechanical loading is not dramatically different between slow and fast myofibers.…”