Abnormalities in skeletal muscle repair can lead to poor function and complications such as scarring or heterotopic ossification (HO). Here, we use fibrodysplasia ossificans progressiva (FOP), a disease of progressive HO caused by ACVR1R206H (Activin receptor type-1 receptor) mutation, to elucidate how ACVR1 affects skeletal muscle repair. Rare and unique primary FOP human muscle stem cells (Hu-MuSCs) isolated from cadaveric skeletal muscle demonstrated increased ECM marker expression, showed skeletal muscle-specific impaired engraftment and regeneration ability. Human induced pluripotent stem cell (iPSC)-derived muscle stem/progenitor cells (iMPCs) single cell transcriptome analyses from FOP also revealed unusually increased ECM and osteogenic marker expression compared to control iMPCs. These results show that iMPCs can recapitulate many aspects of Hu-MuSCs for detailed in vitro study, that ACVR1 is a key regulator of Hu-MuSC function and skeletal muscle repair; and that ACVR1 activation in iMPCs or Hu-MuSCs may contribute to HO by changing the local tissue environment.
Sequencing data have been deposited in GEO under accession codes GSE151918. All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 4,5,6. The dataset used for the primary Hu-MuSCs can be found here, https://datadryad.org/stash/landing/show?id=doi%3A10.7272%2FQ65X273X.
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All mouse studies were performed using protocols approved by the UCSF Institutional Animal Care and Use Committee. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (AN181101-02A) of the University of California, San Francisco.
Human subjects: Human samples were collected through the University of California - San Francisco Biospecimens and Skeletal Tissues for Rare and Orphan Disease Genetics (BSTROnG) Biobank, using protocols approved by the UCSF Institutional Review Board (10-03053 and 11-06711). All participants provided written consent, which includes consent to collect, use, and publish research data.
© 2021, Barruet et al.
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DNA base lesions, such as incorporation of uracil into DNA or base mismatches, can be mutagenic and toxic to replicating cells. To discover factors in repair of genomic uracil, we performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluorouracil into DNA. We identified known factors, such as uracil DNA N-glycosylase (UNG), and unknown factors, such as the N6-adenosine methyltransferase, METTL3, as required to overcome floxuridine-driven cytotoxicity. Visualized with immunofluorescence, the product of METTL3 activity, N6-methyladenosine, formed nuclear foci in cells treated with floxuridine. The observed N6-methyladenosine was embedded in DNA, called 6mA, and these results were confirmed using an orthogonal approach, liquid chromatography coupled to tandem mass spectrometry. METTL3 and 6mA were required for repair of lesions driven by additional base-damaging agents, including raltitrexed, gemcitabine, and hydroxyurea. Our results establish a role for METTL3 and 6mA in promoting genome stability in mammalian cells, especially in response to base damage.
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