Single-Cell Dissection of Muscle Stem Cell Functional Heterogeneity

Muscle stem cells (MuSCs), also known as satellite cells, play a central role in maintaining muscle homeostasis and are regulated by a highly choreographed cascade of cell-fate changes during muscle regeneration. MuSC self-renewal function progressive declines in advanced aging, resulting in a loss of regenerative capacity and muscle atrophy. This functional decline is caused by both muscle microenvironment alterations and dysregulation of key stem cell-intrinsic regulators, together leading to a restricted self-renewal function, which manifests heterogeneously, of MuSCs in aged tissues. We have previously linked MuSC functional variation with aging to aberrant p38 MAPK pathway regulation, but these alterations have not be connected to changes in the MuSC receptor repertoire.

Here, we used single-cell RNA-sequencing of FACS-sorted MuSCs from the hindlimbs of uninjured and notexin-injured young (3 months-old), aged (20 mo), and geriatric (26 mo) mice to gain an unbiased picture of differences in gene expression within individual cells and populations during the aging process. We optimized a Smart-Seq2 protocol to generate single-cell RNA-sequencing libraries with minimal read bias and high yield after sequencing on a NextSeq instrument. We analyzed the single-cell transcriptomes using Monocle to generate hierarchically organized maps of MuSC sub-population variation and diversification. From these, we observed that a family of non-ligand binding transmembrane proteins exhibited significantly heterogeneous single-cell expression patterns in aged and injured MuSCs.

In injury-response experiments, we confirmed this expression diversification at the protein level within the comprehensive α7-integrin⁺ CD34⁺ MuSC population by FACS immunostaining. We found that a particular member of this protein family is expressed trimodally in injured and aged MuSCs, but unimodally in uninjured MuSCs from healthy young adult mice. In particular, we found that geriatric muscles (26 mo) had increased frequencies of both low and high-expressing subpopulations, indicating that this trifurcated expression pattern might distinguish functional from dysfunctional MuSCs.

To experimentally test the self-renewal function of these MuSC sub-populations, we isolated cells from transgenic Luciferase donor mice by FACS, fractionated them into low/mid/high-expressing groups, and transplanted 50 cells from each group into hindlimb muscles of irradiated NOD/scid recipient mice. We measured donor cell-mediated muscle repair by bioluminescence imaging and immunohistochemistry at one-month post-transplant. We observed that only the “mid” subpopulation can engraft and repair muscles, indicating that the “low” and “high” subpopulations are defective in self-renewal in vivo.

To assess the molecular changes that may explain these functional discrepancies, we performed RNA-sequencing on pools of these three MuSCs subpopulations from young mice. We observed that the receptor-mid population was enriched for a Pax7^high / Myogenin^low expression pattern typical of quiescent MuSCs and the “high” and “low” subsets cells had a Pax7^low / Myf5^high pattern typical of myogenic progenitors.

This approach demonstrates the utility of single-cell RNA-sequencing in the unbiased identification of stem-cell subpopulation functional diversification in aging. Our findings suggest specific MuSC subpopulations that may represent the dysfunctional stem cells in aged muscles, and provide new molecular candidates for enhancing MuSC function in the elderly.