Biology subjects

Fong, E. H. H.

Publications and source records attributed to Fong, E. H. H..

3 recordsLinked to original sources

bioRxiv2023

Single-cell transcriptomic analysis of skeletal muscle regeneration across mouse lifespan identifies altered stem cell states associated with senescence

Skeletal muscle regeneration is driven by the interaction of myogenic and non-myogenic cells. In aging, regeneration is impaired due to dysfunctions of myogenic and non-myogenic cells, but this is not understood comprehensively. We collected an integrated atlas of 273,923 single-cell transcriptomes from muscles of young, old, and geriatric mice ([~]5, 20, 26 months-old) at six time-points following myotoxin injury. We identified eight cell types, including T and NK cells and macrophage subtypes, that displayed accelerated or delayed response dynamics between ages. Through pseudotime analysis, we observed myogenic cell states and trajectories specific to old and geriatric ages. To explain these age differences, we assessed cellular senescence by scoring experimentally derived and curated gene-lists. This pointed to an elevation of senescent-like subsets specifically within the self-renewing muscle stem cells in aged muscles. This resource provides a holistic portrait of the altered cellular states underlying skeletal muscle regenerative decline across mouse lifespan. EXTENDED SUMMARYSkeletal muscle regeneration relies on the orchestrated interaction of myogenic and non-myogenic cells with spatial and temporal coordination. The regenerative capacity of skeletal muscle declines with aging due to alterations in myogenic stem/progenitor cell states and functions, non-myogenic cell contributions, and systemic changes, all of which accrue with age. A holistic network-level view of the cell-intrinsic and -extrinsic changes influencing muscle stem/progenitor cell contributions to muscle regeneration across lifespan remains poorly resolved. To provide a comprehensive atlas of regenerative muscle cell states across mouse lifespan, we collected a compendium of 273,923 single-cell transcriptomes from hindlimb muscles of young, old, and geriatric (4-7, 20, and 26 months-old, respectively) mice at six closely sampled time-points following myotoxin injury. We identified 29 muscle-resident cell types, eight of which exhibited accelerated or delayed dynamics in their abundances between age groups, including T and NK cells and multiple macrophage subtypes, suggesting that the age-related decline in muscle repair may arise from temporal miscoordination of the inflammatory response. We performed a pseudotime analysis of myogenic cells across the regeneration timespan and found age-specific myogenic stem/progenitor cell trajectories in old and geriatric muscles. Given the critical role that cellular senescence plays in limiting cell contributions in aged tissues, we built a series of tools to bioinformatically identify senescence in these single-cell data and assess their ability to identify senescence within key myogenic stages. By comparing single-cell senescence scores to co-expression of hallmark senescence genes Cdkn2a and Cdkn1a, we found that an experimentally derived gene-list derived from a muscle foreign body response (FBR) fibrosis model accurately (receiver-operator curve AUC = 0.82-0.86) identified senescent-like myogenic cells across mouse ages, injury time-points, and cell-cycle states, in a manner comparable to curated gene-lists. Further, this scoring approach pinpointed transitory senescence subsets within the myogenic stem/progenitor cell trajectory that are related to stalled MuSC self-renewal states across all ages of mice. This new resource of mouse skeletal muscle aging provides a comprehensive portrait of the changing cellular states and interaction network underlying skeletal muscle regeneration across mouse lifespan.

systems biology↗

bioRxiv2022

Eliminating elevated p53 signaling in Lmna-mutant mice fails to rescue skeletal muscle defects or extend survival

Lamins A and C, encoded by the LMNA gene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation. LMNA mutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which many LMNA mutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragile Lmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts of Lmna mutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity in Lmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.

cell biology↗

bioRxiv2020

Long-term high-yield skeletal muscle stem cell expansion through staged perturbation of cytokine signaling in a soft hydrogel culture platform

Muscle stem cells (MuSCs) are an essential stem cell population for skeletal muscle homeostasis and regeneration throughout adulthood. MuSCs are an ideal candidate for cell therapies for chronic and acute muscle injuries and diseases given their inherent ability to self-renew and generate progenitor cells capable of myogenic commitment and fusion. Given their rarity and propensity to lose stem-cell potential in prolonged culture, methods for ex vivo MuSC expansion that achieve clinical-scale stem cell yields represent a critical unmet need in muscle cell-therapeutic development. Here, we tested a microenvironment engineering approach to achieve long-term adult mouse MuSC expansion suitable for clinical demands through the combined optimization of techniques previously reported to achieve small-yield MuSC expansion in short-term cultures. We developed an optimized protocol for high-yield MuSC expansion through the combination of inflammatory cytokine and growth factor co-stimulation, temporally-staged inhibition of the p38/{beta} mitogen activated protein kinase signaling pathway, and modulation of substrate rigidity in long-term hydrogel cultures. We found that, on soft, muscle-mimicking (12 kPa) hydrogel substrates, a mixture of the cytokines TNF-, IL-1, IL-13, and IFN-{gamma} and the growth factor FGF2 stimulated robust exponential proliferation of adult MuSCs from both wildtype and mdx dystrophic mice for up to five weeks of culture that was accompanied by a phenotype shift towards committed myocytes. After observing that the temporal variation in myogenic commitment coincided with an oscillatory activation of p38/{beta} signaling, we tested a late-stage p38/{beta} inhibition strategy and found that blocking p38/{beta} signaling after three weeks, but not earlier, substantially enhanced cell yield, stem-cell phenotypes, and, critically, preserved transplantation potential for up to five weeks of FGF2/cytokine mix culture on soft hydrogels. Notably, this retention of transplant engraftment potency was not observed on traditional plastic substrates. We estimate that this protocol achieves >108-fold yield in Pax7+ stem cells from each starting MuSC, which represents a substantial improvement in stem-cell yield from long-term cultures compared to established methods. HighlightsO_LITNF-/IL-1/IL-13/IFN-{gamma} cytokine cocktail supports prolonged MuSC proliferation ex vivo but induces differentiation. C_LIO_LICytokine cocktail regulates cell signaling with varied prolonged activation signatures. C_LIO_LIEffects of p38/{beta} inhibition on cytokine-induced MuSC expansion are stage-dependent. C_LIO_LISoft hydrogels with late-stage p38/{beta} inhibition expand functional Pax7+ MuSCs long-term. C_LI Short summaryCosgrove and colleagues develop a long-term muscle stem cell expansion protocol by combining a tunable stiffness hydrogel substrate, an inflammatory cytokine cocktail, and targeted inhibition of p38 MAPK signaling. They show that soft, muscle-mimicking hydrogels with delayed p38 inhibition yield robust quantities of Pax7+ functional muscle stem cells.

bioengineering↗