Instructor, Neurology & Neurological Sciences
Honors & Awards
K99 Pathway to Independence, NIA/NIH
T32 Training Grant, NIA/NIH/Buck Institute
Stem cell fate determination and microenvironment dynamics
The necessity of mesenchymal stromal cells, called fibroadipogenic progenitors (FAPs), in skeletal muscle regeneration and maintenance remains unestablished. We report the generation of a PDGFRαCreER knockin mouse model that provides a specific means of labeling and targeting FAPs. Depletion of FAPs using Cre-dependent diphtheria toxin expression results in loss of expansion of muscle stem cells (MuSCs) and CD45+ hematopoietic cells after injury and impaired skeletal muscle regeneration. Furthermore, FAP-depleted mice under homeostatic conditions exhibit muscle atrophy and loss of MuSCs, revealing that FAPs are required for the maintenance of both skeletal muscle and the MuSC pool. We also report that local tamoxifen metabolite delivery to target CreER activity in a single muscle, removing potentially confounding systemic effects of ablating PDGFRα+ cells distantly, also causes muscle atrophy. These data establish a critical role of FAPs in skeletal muscle regeneration and maintenance.
View details for PubMedID 31091443
Skeletal muscle has an extraordinary regenerative capacity due to the activity of tissue-specific muscle stem cells. Consequently, these cells have received the most attention in studies investigating the cellular processes of skeletal muscle regeneration. However, efficient capacity to rebuild this tissue also depends on additional cells in the local milieu, as disrupting their normal contributions often leads to incomplete regeneration. Here, we review these additional cells that contribute to the regenerative process. Understanding the complex interactions between and among these cell populations has the potential to lead to therapies that will help promote normal skeletal muscle regeneration under conditions in which this process is suboptimal.
View details for PubMedID 30016618
Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.
View details for PubMedID 30101152
Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues, such as T lymphocytes and endothelial cells from different anatomical locations. Two distinct technical approaches were used for most organs: one approach, microfluidic droplet-based 3'-end counting, enabled the survey of thousands of cells at relatively low coverage, whereas the other, full-length transcript analysis based on fluorescence-activated cell sorting, enabled the characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.
View details for DOI 10.1038/s41586-018-0590-4
View details for PubMedID 30283141
Coordinated activation of muscle stem cells (known as satellite cells) is critical for postnatal muscle growth and regeneration. The muscle stem cell niche is central for regulating the activation state of satellite cells, but the specific extracellular signals that coordinate this regulation are poorly understood. Here we show that macrophages at sites of muscle injury induce activation of satellite cells via expression of Adamts1. Overexpression of Adamts1 in macrophages in vivo is sufficient to increase satellite cell activation and improve muscle regeneration in young mice. We demonstrate that NOTCH1 is a target of ADAMTS1 metalloproteinase activity, which reduces Notch signaling, leading to increased satellite cell activation. These results identify Adamts1 as a potent extracellular regulator of satellite cell activation and have significant implications for understanding the regulation of satellite cell activity and regeneration after muscle injury.Satellite cells are crucial for growth and regeneration of skeletal muscle. Here the authors show that in response to muscle injury, macrophages secrete Adamts1, which induces satellite cell activation by modulating Notch1 signaling.
View details for PubMedID 28939843
Heterotopic ossification is a debilitating condition that can result from traumatic injury, surgery, or genetic disease. We investigated the cellular origins of heterotopic skeletogenesis in the mouse using lineage tracing and bioassays of heterotopic ossification based on intramuscular transplantation. We identified, characterized, and purified a tissue-resident stem/progenitor cell population that exhibits robust osteogenic potential and represents a major cell-of-origin for heterotopic ossification. These progenitors reside in the interstitium of skeletal muscle and other tissues, and are distinct from the endothelium, which does not exhibit osteogenic activity in response to bone morphogenetic protein 2 (BMP2) stimulation. Intramuscular transplantation, together with clonal analysis in culture, revealed that these progenitors are multipotent, exhibiting the capacity for both BMP-dependent skeletogenic differentiation and spontaneous adipogenic differentiation. Identifying the cells-of-origin responsible for heterotopic ossification provides a potential therapeutic target to treat, mitigate, or prevent this disabling condition.
View details for DOI 10.1002/jbmr.1562
View details for Web of Science ID 000302939400006
View details for PubMedID 22307978
View details for PubMedCentralID PMC3361573
Individuals who have fibrodysplasia ossificans progressiva develop an ectopic skeleton because of genetic dysregulation of bone morphogenetic protein (BMP) signaling in the presence of inflammatory triggers. The identity of progenitor cells that contribute to various stages of BMP-induced heterotopic ossification relevant to fibrodysplasia ossificans progressiva and related disorders is unknown. An understanding of the cellular basis of heterotopic ossification will aid in the development of targeted, cell-specific therapies for the treatment and prevention of heterotopic ossification.We used Cre/loxP lineage tracing methods in the mouse to identify cell lineages that contribute to all stages of heterotopic ossification. Specific cell populations were permanently labeled by crossing lineage-specific Cre mice with the Cre-dependent reporter mice R26R and R26R-EYFP. Two mouse models were used to induce heterotopic ossification: (1) intramuscular injection of BMP2/Matrigel and (2) cardiotoxin-induced skeletal muscle injury in transgenic mice that misexpress BMP4 at the neuromuscular junction. The contribution of labeled cells to fibroproliferative lesions, cartilage, and bone was evaluated histologically by light and fluorescence microscopy. The cell types evaluated as possible progenitors included skeletal muscle stem cells (MyoD-Cre), endothelium and endothelial precursors (Tie2-Cre), and vascular smooth muscle (Smooth Muscle Myosin Heavy Chain-Cre [SMMHC-Cre]).Vascular smooth muscle cells did not contribute to any stage of heterotopic ossification in either mouse model. Despite the osteogenic response of cultured skeletal myoblasts to BMPs, skeletal muscle precursors in vivo contributed minimally to heterotopic ossification (<5%), and this contribution was not increased by cardiotoxin injection, which induces muscle regeneration and mobilizes muscle stem cells. In contrast, cells that expressed the vascular endothelial marker Tie2/Tek at some time in their developmental history contributed robustly to the fibroproliferative, chondrogenic, and osteogenic stages of the evolving heterotopic endochondral anlagen. Importantly, endothelial markers were expressed by cells at all stages of heterotopic ossification. Finally, muscle injury and associated inflammation were sufficient to trigger fibrodysplasia ossificans progressiva-like heterotopic ossification in a setting of chronically stimulated BMP activity.Tie2-expressing progenitor cells, which are endothelial precursors, respond to an inflammatory trigger, differentiate through an endochondral pathway, contribute to every stage of the heterotopic endochondral anlagen, and form heterotopic bone in response to overactive BMP signaling in animal models of fibrodysplasia ossificans progressiva. Thus, the ectopic skeleton is not only supplied by a rich vasculature, but appears to be constructed in part by cells of vascular origin. Further, these data strongly suggest that dysregulation of the BMP signaling pathway and an inflammatory microenvironment are both required for the formation of fibrodysplasia ossificans progressiva-like lesions.
View details for DOI 10.2106/JBJS.H.01177
View details for PubMedID 19255227
The Cre/lox and FLP/FRT recombination systems have been used extensively for both conditional knockout and cell lineage analysis in mice. Here we report a new multifunctional Cre/FLP dual reporter allele (R26(NZG)) that exhibits strong and apparently ubiquitous marker expression in embryos and adults. The reporter construct, which is driven by the CAG promoter, was knocked into the ROSA26 locus providing an open chromatin domain for consistent expression and avoiding site-of-integration effects often observed with transgenic reporters. R26(NZG) directs Cre-dependent nuclear-localized beta-galactosidase (beta-gal) expression, and can be converted into a Cre-dependent EGFP reporter (R26(NG)) by germline excision of the FRT-flanked nlslacZ cassette. Alternatively, germline excision of the floxed PGKNEO cassette in R26(NZG) generates an FLP-dependent EGFP reporter (R26(ZG)) that expresses beta-gal in FLP-nonexpressing cells. Finally, by the simultaneous use of both Cre and FLP deleters, R26(NZG) allows lineage relationships to be interrogated with greater refinement than is possible with single recombinase reporter systems.
View details for DOI 10.1002/dvg.20474
View details for Web of Science ID 000263887700006
View details for PubMedID 19165827
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