Fig 2: Identification of Oncostatin M as a potent inducer of muscle stem cell engraftment. a Schematic of combined in vitro/in vivo bioluminescence imaging (BLI) screening strategy. b Schematic of deconvolution for positive protein pools. Pools of 22 proteins scoring for engraftment in the initial round of screening were iteratively deconvolved into pools of 4 proteins, and then individual proteins. c Results of multiplexed in vitro/in vivo protein screening. Black, results of in vitro BLI; red, results of in vivo BLI. Ctl: control buffer; SB: SB202190; FGF: recombinant human basic fibroblast growth factor. Symbols below the axis mark pools containing OSM (star), and all other hits (circle; includes FGF, TNFR, and incompletely deconvolved pools). d Representative BLI results following treatment of 800 FACS-sorted MuSC with either FGF or Oncostatin M (OSM; 100 ng/mL) for 6 days in vitro, followed by intramuscular transplantation (3 8-12 week old animals each) and in vivo BLI measurement 6 days (open circles) or 2 weeks (shaded circles) after transplant. e Schematic (left) demonstrating transplantation of 250 counted progeny of CD11b-Sca1-CD31-CD45-CD34+aa7 integrin+ stem cells following long-term 2 week culture with OSM control conditions (FGF). Serial BLI following transplantation of counted cells (right). Each point indicates an individual hindlimb that received a 250 cell transplant, n = 10 per condition. BLI is shown for weeks 1 (white) and 2 (light gray) pre-CTX, and weeks 1 (dark gray) and 10 (red) post-CTX. Double asterisks indicate p < 0.01 in comparison with control condition at the same timepoint

Fig 3: Gating strategy of sorting iHECs by flow cytometry at day 11. The differentiated cells were collected and enriched by incubation with biotin-conjugated anti-CD31 antibody. Then, the iHECs were sorted from the CD31+ enriched cells using the CD31+CD41lowCD45-ckit+CD201high combined markers.

Fig 4: Histone Acetylation Dynamics during Regeneration Revealed by CyTOF(A) Visualization of t-distributed stochastic neighbor embedding (viSNE) plot of uninjured and injured (D6 after notexin injection) muscle. Sca1/CD31/CD11b/ CD45- (Lin-) a7-integrin+ cells were clustered based on PAX7, MYOD1, and MYOG (Myogenin). viSNE plots of combined biological replicates (n = 3) for each time point shown. Color scale shows CD34, PAX7, MYOD1, or MYOG normalized expression. Significance determined using one-way ANOVA with Tukey’s test for multiple comparisons across samples (injured and uninjured), and repeated-measures ANOVA was used for comparisons between Activated or Self-Renewing Stem and Differentiated subsets within biological samples. Subsets outlined are as follows: (a) quiescent PAX7+CD34+MYOD- MuSCs (D0), gray; (b) self-renewing PAX7+CD34-MYOD- MuSCs (D6), black; (c) activated PAX7+MYOD+ MuSCs (D6), blue; and (d) differentiating PAX7-MYOD+MYOG+ progenitors (D6), red. (B) Expression of PAX7, CD34, MYOD1, and MYOG in subsets in (A).(C) IdU incorporation subsets in (A).(D) Histone acetylation normalized for total histone H3 content in subsets outlined in (A).(E) Representative contour plot of CD34 versus MYOG at D0 and D6. Cells are Lin-a7-integrin+. Quiescent (CD34+MYOG-), Activated (CD34LowMYOG-), and Differentiated (CD34LowMYOG+) subsets are indicated.(F) H4K16 and H3K18 acetylation in Quiescent, Activated, and Differentiated subsets. Acetylation normalized for Quiescent MuSCs. n = 4–11 biological replicates. Significance determined using one-way ANOVA with Tukey’s test for multiple comparisons across samples (injured and uninjured), and Student’s paired t test was used for comparisons between within biological samples.(G) Immunostaining of H4K16ac on extensor digitorum muscle fibers. Freshly isolated fibers were compared to those cultured for 72 h. Quiescent MuSCs identified as PAX7+MYOD1-, activated MuSCs as PAX7+MYOD+, and early differentiated progenitors as PAX7-/MYOD+ cells. Scale bar (white): 10 µm. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001). See Figures S1 and S2 and Table S1.

Fig 5: OSM treatment enhances stem cell serial transplantation and inhibits differentiation. a FACS analysis of stem cells reisolated from primary transplant recipients receiving either control (left), FGF (middle), or OSM-treated (right) stem cells. Only the live, CD11b-Sca1-CD31-CD45-CD34+a7 integrin+ stem cell population is shown. b MuSCs (CD11b-Sca1-CD31-CD45-CD34+a7 integrin+GFP+) were reisolated from OSM primary transplant mice as described in a, transplanted equally into 5 limbs (secondary transplant recipients; 50 MuSC/limb; 8–12 week old recipients), and monitored by serial BLI. At day 25 following transplant, cardiotoxin (CTX) was injected to induce myofiber damage and stimulate stem cell expansion and tissue regeneration. The average radiance at each timepoint is indicated, and demonstrates regenerative capacity (BLI signal loss followed by rebound) of serially transplated OSM-treated MuSC. Percentage of engrafted mice was 40% both pre and post-CTX injury (2/5 total mice). Note that untreated cells give no engraftment, and no MuSC are therefore present for reisolation/transplant. c qRT-PCR analysis of Pax7 expression following 6 day culture of FACS-sorted MuSC in the presence of OSM (gray circles) or buffer control (black). Mean±SD is shown, n = 3 replicates. d qRT-PCR analysis of myogenin expression following 6 day culture of FACS-sorted MuSC with the indicated cytokines and growth factors. Expression was normalized to GAPDH expression. Mean±SD is shown, n = 3 replicates. e Timecourse intracellular flow cytometry of primary mouse myoblasts treated for the indicated period of time with OSM, and stained for either phospho-STAT3 (left) or phospho-STAT5 (right). 0 (unstimulated), 15, 30, and 45 min timepoints are shown following cytokine addition. Frequencies are represented as histograms. f ChIP performed using primary myoblasts cultured for 6 days either with or without OSM. Immunoprecipitation was performed with either control IgG or STAT3-specific antibodies, and PCR was performed using primers specific for a predicted STAT3 binding site adjacent to the Pax7 locus