GSE196612
A skeletal-muscle senescence blueprint defines an aged-like inflamed niche that inhibits regeneration over lifetime [ATAC-seq]
A new sorting protocol was used to identify and isolate different senescent cell types from damaged muscles of young and geriatric mice. Analysis revealed conservation of two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time, and aging, while cell identity traits were preserved. Senescent cells create an “aged-like” inflamed niche, mirroring inflammation-associated with aging (inflammaging), that arrests stem cell proliferation and regeneration. Overall design: Muscles were mechanically disaggregated and incubated in Dulbecco's Modified Eagle's medium (DMEM) containing liberase (Roche) and dispase at 37°C with agitation for 1-2 hours. When required, SPiDER-b-gal reagent (Dojindo) was added during the second hour. The supernatant was then filtered and cells were incubated in lysis buffer (BD Pharm Lyse) for 10 min on ice, resuspended in PBS with 2.5% fetal bovine serum (FBS), and counted. BV711-conjugated anti- CD45 (BD), APC-Cy7-conjugated anti F4/80 (Biolegend), PE- conjugated anti-a7-integrin (Ablab), APC-conjugated anti-CD31 (eBioscience) and PE-Cy7-conjugated anti-Sca-1 (Biolegend) antibodies were used to isolate MCs (CD45+ and F4/80+), SCs (a7-integrin+, CD45+, F4/80+ and CD31+) and FAPs (Sca-1+, MCs CD45+, F4/80+, a7-integrin+ and CD31+). PE-Cy7-conjugated anti-CD45 antibody (Biolegend) was used to isolate CD45 positive and negative populations. SPiDER-b-gal (SPiDER) was employed to isolate senescent cells (SPiDER+) from non-senescent cells (SPiDER–) of each cell type. Isolated cells were subjected to ATAC-seq. Pools of 3 animals were used for each sample.