Grant Award Details
Mass Cytometry to Delineate the Human Muscle Stem Cell Hierarchy and Dysfunction in Aging
To delineate the cellular hierarchy in human skeletal muscle and determine signaling networks that are disregulated in aged muscle stem cells (MuSC, defined as Lin- CD34+ CD20+ a7integrin+). In order to achieve this, the awardee will identify novel subsets of MuSC using multidimensional single-cell mass cytometry and then subsequently evaluate the specific cell populations' expression and properties at different periods of human aging.
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 in human aged tissues and identify pharmacological targets to enhance the function of dysfunctional subsets or amplify the residual functional subsets. During the first 12 months of this grant period we have made significant advances toward the aims that will allow us to address the remaining aspects of the goals of the grant.
We have, for the first time, achieved:
Identification of novel subsets within the human muscle stem/progenitor cell population using multidimensional single-cell mass cytometry (CyTOF);
Prospective isolation of a fully functional human muscle stem cell population;
Demonstration that human muscle stem cells regenerate injured muscle fibers following transplantation into NSG mice;
Verification that engrafted human muscle stem cells home to the stem cell niche and respond to muscle injury, thereby constituting a stem cell reservoir to meet future needs for regeneration;
Observed that the number of human muscle stem cells decrease in aged muscle tissue.
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.
<p>Stem cell therapies are poised to transform the way medicine is delivered to patients with diseases for which currently no treatment exists, such as muscle wasting due to injury, disease, and aging. Muscle stem cells (MuSCs) are indispensable for muscle regeneration and could meet the need to treat muscle wasting alleviating the disease burden and burgeoning healthcare costs in California associated with living longer. While adult MuSCs have potent self-renewal and regenerative potential, the MuSC population becomes increasingly heterogeneous and dysfunctional with aging.</p><p>The approach developed here provides a new tool for identifying and isolating myogenic progenitors in murine and human muscle biopsies via the single-cell mass cytometry (CyTOF) technology. The simultaneous analyses of multiple cell surface markers and intracellular targets in well-defined populations can be performed and therefore provides the unprecedented way to characterize cellular signals that control stem cell functions. We have identified specific markers, validated reagents and methods for CyTOF analysis of muscle stem and progenitor cells in mouse and human skeletal muscle tissue. In particular, we have developed 50 metal-conjugated antibodies that allow us to uncover different populations of muscle cells and identify a potent stem cell population that robustly responses to specific niche signaling factors to enhance muscle repair during injury. In summary, our approach sets the stage for investigating the cellular and molecular defects that characterize aging and muscle diseases and opens the door to the discovery of novel therapeutic targets to improve muscle regeneration.</p>
Grant Application Details
- Mass Cytometry to Delineate the Human Muscle Stem Cell Hierarchy and Dysfunction in Aging
Aging is characterized by a decline in skeletal muscle mass and strength (sarcopenia), coupled with an accumulation of fat tissue, impairing mobility and quality of life in 45% of individuals over 65 years of age. In aged mice, the regeneration defect arises in part from a diminished regenerative capacity of resident muscle stem cells (MuSCs). The goal of this proposal is to characterize muscle stem cell subsets residing in human skeletal muscle tissue and investigate the mechanisms responsible for their regenerative defects in aging. We will capitalize on two cutting edge technologies, single-cell bioengineered stem cell niches, which will allow tracking individual muscle cell fates, and multi-dimensional single-cell mass cytometry, which will allow resolution of the molecular characteristics of skeletal muscle cells from aged humans with detail previously unattainable. We expect that this combined approach will elucidate the functional defects observed in aged MuSC populations and therefore facilitate the development of a therapeutic agent to target muscle stem cells for the treatment muscle weakness in aged humans.
Statement of Benefit to California:
The state of California is the front-runner in stem cell research, having gathered not only private investments, as demonstrated by the numerous biotechnology companies that are developing innovative tools, but also extensive public funds via Prop 71, that allows the state, through CIRM, to sponsor stem cell research in public and private institutions. In order to preserve its leadership position and encourage research on stem cells, the CIRM is calling for research proposals that could lead to significant breakthroughs or the development of technologies useful for studying stem cells in order to improve human health. Aging is characterized by a decline in tissue function and regenerative capacity that leads to degeneration and loss of muscle mass and strength (sarcopenia). Muscle stem cells (MuSCs), also known as satellite cells, are responsible for the maintenance and regeneration of skeletal muscle mass. Several studies have shown a decrease in MuSC function in aged mice. However, the mechanisms responsible for their reduced function are not yet defined. The work we propose here focuses on human muscle and discerning differences among subsets of muscle stem cells. The results will facilitate the development of targets to augment a muscle stem cell based therapy to treat muscle weakness in aged humans.
Source URL: https://www.cirm.ca.gov/our-progress/awards/mass-cytometry-delineate-human-muscle-stem-cell-hierarchy-and-dysfunction-aging