The goal of regenerative medicine is to restore form and function to damaged and aging tissues. Muscle stem cells (MuSCs), also known as satellite cells, are responsible for maintenance and regeneration of skeletal muscle mass. We showed that MuSCs in aged muscles have inherent defects in their quiescence and self-renewal mechanisms, and prematurely senesce and differentiate. Our laboratory has established methods to characterize murine MuSCs by flow cytometry in conjunction with in vivo functional assays and has been able to successfully culture MuSCs ex-vivo in artificial stem cell niches that recapitulate the biophysical environment in which muscle stem cells reside. Here we propose to discern the role of extrinsic factors within or near the niche in modulating MuSC function during aging. Critical to the approach is single- cell analysis, as the behavior of slow proliferating MuSC may be masked by more rapidly proliferating progenitors and the MuSC population, though highly enriched, is heterogeneous and contains multiple subsets based on expression profiles, signaling and functional studies. We will capitalize on two single-cell technologies: (a) a bioengineered culture platform that supports maintenance of MuSC phenotype ex-vivo and long-term single-cell lineage tracking by time-lapse microscopy and (b) single cell mass cytometry, which allows high-throughput analysis of multiple biological cell surface and intracellular markers with single-cell resolution enabling an unbiased approach to the identification of novel subsets within a complex cell population. We will compare MuSCs from young and aged tissues, which have inherently different quiescence and self-renewal capacities.
We aim (1) to identify extrinsic factors that regulate MuSC fate in vitro. A family of cytokines identified in a 660-peptide library of secreted and transmembrane ectodomain proteins will be assayed for their potential to alter MuSC proliferation and phenotype; (2) to characterize specific members of the family of cytokines that regulate MuSC quiescence, asymmetric, or symmetric self- renewal to alter cell fate; (3) to elucidate the MuSC subsets and signaling networks stimulated by this cytokine family. We will employ single-cell mass cytometry to resolve distinct functional subsets of MuSCs, investigate how these subsets differentially activate signaling networks in response to extrinsic quiescence, proliferation, and commitment-inducing stimulation by the cytokine family members, and determine how those differences affect MuSC contributions to regeneration and strength. Together, these studies will provide insights into the role of extrinsic factors in the stem cell microenvironment on stem cell function and suggest novel therapeutic approaches to muscle degenerative diseases and muscle aging.

Public Health Relevance

The ability of the resident muscle stem cell population to maintain and repair skeletal muscle progressively declines in the eldery, severely affecting muscle strength, mobility and quality of life. Here we will capitalize on our extensive experience in the isolation, culture and transplantation of muscle stem cells in mice. Additionally, we will develop novel technologies to test the effects of micro environmental proteins on young and aged muscle stem cell function in regeneration in order to identify novel therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG020961-14
Application #
9414569
Study Section
Skeletal Muscle Biology and Exercise Physiology Study Section (SMEP)
Program Officer
Williams, John
Project Start
2002-05-15
Project End
2020-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
14
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Stanford University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
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