Year 2

Aging is characterized by a decline in skeletal muscle mass and strength, coupled with accumulation of fat tissue, impairing mobility and quality of life in 45% of individuals over 65 years of age. While muscle stem cells (MuSCs) have a remarkable capacity to maintain and repair skeletal muscle throughout adulthood, they decline in regenerative capacity during aging. The goal of this proposal is to elucidate dysregulated functions of MuSCs from human aged tissues and identify pharmacological targets that could enhance the function of dysfunctional subsets or amplify the residual functional subsets. We have adapted the treatment conditions that we uncovered from analyzing MuSCs in a mouse muscle regeneration model. To date, we have performed MuSC characterization using multidimensional single-cell mass cytometry (CyTOF) and identified a set of surface markers CD34- CD29+ α7-integrin+ and transcription factor Pax7 expression, which allow the prospective isolation of the human MuSC population by FACS. We have transplanted the isolated MuSCs from young and aged human biopsies into irradiated TA muscle of NSG mice and optimized the protocol to improve muscle transplantation. We are capitalizing on CyTOF analysis to investigate dynamic cellular and molecular properties of stem and progenitor populations for the elucidation of regulatory networks that underlie cell state transitions in aging and muscle diseases.

We have achieved the milestones of aim 1 and made significant advances toward the remaining aims that will allow us to complete the proposed experiments from the original grant. Up to this year 2 grant period, we have achieved:

• Identification of novel subsets within the human muscle stem/progenitor cell population using multidimensional single-cell mass cytometry (CyTOF);

• Demonstration that prospectively isolated human muscle stem cell (hMuSC) population are fully functional following transplantation into NSG mice;

• Demonstration that transplanted hMuSC home to the stem cell niche and respond to muscle injury thereby reconstituting a stem cell reservoir to meet future needs for regeneration;

• Characterization showing that aged MuSCs decline in number and function;

• Identification of additional MuSC markers that enable resolution of the heterogeneity aged muscle tissue;

• Analysis of MuSC CyTOF data using a new multivariate algorithm, called X-shift together with a force-directed layout visualization to generate unsupervised clusters, in which stem cell fate and functions can be characterized based on differential expression of the developmentally regulated myogenic transcription factors Pax7, Myf5, MyoD and Myogenin.