Andrew S. Brack - US grants

DESCRIPTION (provided by applicant): Effective tissue repair relies on an organs ability to control the number and function of stem cells or tissue specific progenitors. Satellite cells (SCs) are the "cell of choice" for adult skeletal muscle repair. Based on the assumption that all somatic cells, including SCs, have a finite capacity;reducing the number of SCs within the pool while keeping intrinsic capacity unchanged will lead to eventual functional exhaustion and impaired muscle repair. Similarly, if intrinsic capacity is impaired, while the number of SCs in the pool is maintained, this will also lead to a detrimental outcome for muscle function. During aging, muscle regeneration capacity is significantly impaired. This coincides with a decline in SC number and function. It remains unresolved whether age related impairment in muscle regeneration is caused by a loss in SC number and/or function. The first part of the proposal focuses on understanding the heterogeneity within the SC pool throughout life, moreover whether subsets of SCs are 'age-resistant. We will use a novel GFP reporter that allows proliferative output to be determined on a cell-by-cell basis in vivo. Slow and fast dividing SCs will be tested for functional diversity. This aim will answer whether aging is associated with a loss of specific functional subpopulations of SCs. The second part of the proposal focuses on studying the importance of the number of SCs available in the pool for effective repair. We will systematically decrease the number of SCs within adult and aged muscle to ask, 1) whether a decline in SC number causes impaired regeneration, 2) whether forced proliferative demand induced by limiting the number of SCs forces premature SC exhaustion. This aim will be achieved using novel genetic strategies that enable SC specific cell ablation. The third part of the proposal focuses on the intrinsic capacity of SCs to effectively repair muscle, in particular the role of P16INK4A as a regulator of SC progenitor proliferation and lineage progression during aging. P16INK4A, an aging biomarker, has been implicated in regenerative impairment of aged tissue. The role of P16INK4A in myogenesis has not been studied previously. Finally by combining SC-ablation technology and P16INK4A loss-of-function approaches, the interaction between SC number and function for effective tissue repair will be interrogated. Understanding the coordination between the number and intrinsic capacity of SCs will be critical for treatment of sarcopenia and other muscle degeneration pathologies. The specific aims of this proposal are: 1) to study heterogeneity in the SC pool as it relates to proliferative output throughout life, 2) to determine if proliferative output is causally related to sarcopenia and 3) to study the effect of genetically manipulating SC function through P16INK4A loss-of-function approaches for efficient muscle repair in young and aged muscle. PUBLIC HEALTH RELEVANCE: Deciphering the coordination between satellite cell number and function during muscle regeneration is critical for harnessing their potential to treat sarcopenia in an increasingly aging population. Furthermore, the biology of adult satellite cells may serve as a paradigm for stem cells in other tissues.

DESCRIPTION (provided by applicant): Adult stem cells are capable of self renewal and differentiation. Although they possess self-renewal properties, this potential is not limitless. This begs the following question: how are stem cells maintained throughout life? The answer lies in the reversible state of quiescence. Adult stem cells are predominantly in a quiescent state interspersed with rare traverses into the cell cycle unlike their downstream descendents that spend more time in cycle. Using cell labeling techniques to monitor cell division history, stem cells that divide less frequently are enriched for self-renewal potential. In muscular dystrophies, muscle stem cells (satellite cells, SCs) undergo continuous bouts of proliferation, which leads to a depletion of the stem cell pool over time. In aged muscle the SC pool is diminished and displays impaired renewal and differentiation. Therefore understanding how 'proliferation-restricted' stem cells may hold the key to optimizing stem cell function during aging and disease. In this grant proposal we will interrogate 1) the functional heterogeneity between slow and faster dividing SCs. 2) How the slow dividing SC population is specified and maintained throughout life through Spry, an inhibitor of growth factor signaling. 3) The consequences of losing slow dividing subset of SCs SC function and muscle homeostasis during aging. Slow dividing SCs will be identified in vivo based on label retaining character (LRC) using a transgenic H2B-GFP approach; cells that undergo fewer divisions retain more label. Satellite cells will be characterized for LRC and non-LRC; both subpopulations will be tested for their functional differences based on in vivo and in vitro assays. We have generated preliminary data that demonstrates Spry1 expression is enriched in LRCs and genetic disruption of Spry1 leads to a loss of LRC in growing muscle and an accelerated decline in the number of SCs during aging. We will extend these studies to delete and overexpress Spry1 specifically in SCs in a temporally inducible manner. We will disrupt Spry1 to alter LRC establishment and maintenance and study the effects on SC function and muscle phenotype. The specific aims of this proposal are: 1) To study the slow division dynamics of satellite cells during developmental and postnatal myogenesis, 2) to identify whether Spry1 is required and sufficient to regulate LRC SCs, and 3) to understand the consequences of losing SC LRC throughout life.

PROJECT SUMMARY The ability of stem cells to survive and maintain function during tissue turnover and stress is a key determinant of long-term regenerative success. Skeletal muscle stem cells lose fitness under contexts of high stress including aging, muscular disease and irradiation (IR). Hence there is a critical need to identify the properties that maintain or enhance SC stress resistance. During the previous funding cycle, we demonstrated that self- renewal potential was restricted to slowly dividing SCs (based on label retention). Our long-term goal is to understand SC heterogeneity and identify the molecular regulators that govern SC potency. We have identified Pax3 as a discrete marker of stress-resistant SCs. These cells are rare in number, express typical satellite cell markers, proliferate slowly, as indicated by label retention analysis, and are capable of self-renewal and differentiation. Under conditions of stress this subset is preserved and becomes the dominant source of stem cells for muscle repair and stem cell replenishment after injury. This is the first demonstration of clonal expansion of a discrete muscle stem cell subset in vivo. While Pax3 has been studied in development, its role in stress resistance of quiescent adult SCs is completely unknown. The overall goal of this proposed study is therefore to understand the functional capacity and regulation of a specific subset of stress-resistant SCs. The first aim will combine lineage tracing (Tmx-inducible Pax3CreERT2.ROSA26-nTNG) and cell ablation (DTAfl/fl) strategies to determine the cellular contribution and requirement of adult Pax3+ SC lineage to muscle repair during normal injury or after increased stress (IR and transplantation), to ask whether Pax3+ SCs fulfill the definition of a reserve stem cell. The second aim will determine the dynamics between pax3 expression (pax3gfp+/- knockin allele) or proliferative output (TetO-H2B-mCHerry) in the progeny of Pax3+ and Pax3- SC lineages (Pax3CreERT2.ROSA26-reporter) during muscle tissue turnover. The third aim will dissect the molecular mechanism underpinning Pax3+ SC function. We will examine the requirement and sufficiency of Pax3 in SC function using loss and gain of function approaches and its regulation through cell survival/apoptosis machinery. Understanding the requirement and regulation of Pax3 in adult SCs will provide new insights into adult SC biology independent of Aim 1 and 2. Successful completion of the proposed aims will provide evidence for a pre-determined subset of Pax3+ SCs, and reveal the clonal dynamics among muscle stem cells that manifests under the selective pressure of stress. Understanding the potency and regulation of stress resistant SCs will lead to important insights for cell therapy, patients undergoing radiation therapy and other physiological stressors including degenerative diseases and aging.

PROJECT SUMMARY The niche is a critical regulator of long-term stem cell function. Our long-term goal is to understand the role the niche plays in regulation of muscle stem cell function. SCs reside in a quiescent state and undergo a series of transitions as they activate and proliferate. We have identified Wnt4 as a tissue resident inhibitory factor that represses activation and migration of quiescent satellite cells (QSCs) via Rho-FAK signaling. This is the first demonstration of a single paracrine acting niche factor coordinates the growth, migration and activation of QSCs. The overall goal of this proposed study is therefore to understand how Wnt4-Rho-FAK signaling axis represses activation and migration in QSCs and its implications for SC function in adult and aged mice. The first aim will combine temporal regulated and cell specific genetic strategies to modulate Wnt4 levels in muscle fibers to determine the role of Wnt4 (in muscle fibers) and Rho-FAK (in QSCs) on activation and migration in QSCs and the response to injury. The second aim will dissect the molecular mechanism that prevents QSC activation and migration via Wnt4. We will examine the role of Wnt4-Rho signaling in the repression of mTORC1 and Hippo signaling in QSCs. Understanding the role of the niche on SC function will lead to important insights for cell therapy, muscle degenerative diseases and aging.

Project Summary During tissue repair, many stem cell populations undergo a dynamic phenotypic change from a quiescent state to an activated state. In muscle stem cells (MuSCs), this dynamic activation process is essential for effective tissue regeneration. Despite the conserved nature of these activation processes, the dynamics of stem cell activation and their contribution to disease states remains largely unknown. We have generated single cell assays that allowed us to study state transitions of adult and aged MuSCs during activation. These results support a conceptual view of the aged stem cell phenotype as a combination of pathological steady-states and deficiencies in cell state dynamics. This provides us with the opportunity to identify factors that rejuvenate MuSC function during aging. In this project we will examine the role of physiological rejuvenation interventions on MuSC heterogeneity and activation state transitions. Understanding how rejuvenation interventions control MuSC activation response is critical for the effective treatment of the ever-expanding aged population.

PROJECT SUMMARY Coordination of cell migration, proliferation and cell fate determination is critical for stem cell function and tissue repair. We have identified that P16Ink4a, the classic pro-senescence and aging regulator, participates in the regulation of healthy adult muscle stem cells. We find a role for P16Ink4a in cell migration and cell fate decisions via the cytoskeleton and contractile machinery. This has not been reported previously. The long-term goal is to understand how P16Ink4a is induced and regulates cytoskeletal function of healthy progenitor cells in non-aged tissues. The first aim will combine temporal regulated and cell specific genetic strategies to determine the role of P16Ink4a in healthy adult muscle stem cells. The second aim will dissect the functional and molecular mechanism that regulates cell mechanics, migration and cell fate via P16Ink4a. The third aim will examine the role of P16Ink4a in cell autonomous regulation of its own microenvironment. Understanding the role of P16Ink4a in progenitor cell function will lead to important insights for aging, senescence, cancer and muscle degenerative diseases.