Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential

Abstract

The development of cell therapy for repairing damaged or diseased skeletal muscle has been hindered by the inability to significantly expand immature, transplantable myogenic stem cells (MuSCs) in culture. To overcome this limitation, a deeper understanding of the mechanisms regulating the transition between activated, proliferating MuSCs and differentiation-primed, poorly engrafting progenitors is needed. Here, we show that methyltransferase Setd7 facilitates such transition by regulating the nuclear accumulation of β-catenin in proliferating MuSCs. Genetic or pharmacological inhibition of Setd7 promotes in vitro expansion of MuSCs and increases the yield of primary myogenic cell cultures. Upon transplantation, both mouse and human MuSCs expanded with a Setd7 small-molecule inhibitor are better able to repopulate the satellite cell niche, and treated mouse MuSCs show enhanced therapeutic potential in preclinical models of muscular dystrophy. Thus, Setd7 inhibition may help bypass a key obstacle in the translation of cell therapy for muscle disease.

Keywords: SET domain; WNT; differentiation; methylation; methyltransferase; muscle stem cells; myogenesis; satellite cells; skeletal muscle; β-catenin.

Figure 1. Setd7 is expressed in activated MuSCs and is required for skeletal muscle regeneration

(A) Setd7 expression in myofiber-associated MuSCs fixed immediately after isolation (quiescent, top panel) or after 48 hours ex vivo culture (activated, lower panel). Scale bar = 50µm (B) Percentage of Setd7+ myofiber associated MuSCs (quantification from 5 mice). (C) Setd7 expression in FACS isolated MuSCs following acute damage. (D) Setd7 protein expression in MuSC during in vitro differentiation. (E) Western blot analysis of Setd7 protein expression in whole muscle lysates. (F) Representative images and quantification of quiescent Pax7+ MuSCs from myofibers isolated from control (CON - MyoDi-Cre/Setd7WT) and Setd7 mKO (mKO - MyoDi-Cre/Setd7^FL/FL) mice. (n=3 per group). Scale bar = 50µm (G) Body weights of control and Setd7 mKO mice during development, adulthood and aging (n≥15 per group). (H) Skeletal muscle wet weights at 15 weeks of age. EDL – extensor digitorum longus, TA – tibialis anterior, GAS – gastrocnemius, QUD – quadriceps. (n≥15 per group) (I) Evan’s blue incorporation in TA muscle of NTX damaged control and Setd7 mKO mice (n≥5 per group). (J) TA muscles of control and Setd7 mKO mice following NTX damage. Quantification of myofiber cross sectional area (CSA) (n≥5 per group). Scale bar = 100µm Data represented as mean ± SEM. NS = p > 0.05, *p<0.05, **p<0.01, ***p<0.005

Figure 2. Loss of Setd7 impairs myogenic differentiation and enhances proliferation of MuSCs ex vivo

(A) Allele excision efficiency in FACS sorted MuSCs (CD45/31-, Sca1-, alpha7-integrin/Vcam1+) from MyoDiCre/Setd7^FL/FL mice (mKO) and controls (CON) (n=3 per group). (B) Frequency of Pax7+ and MyoG+ myofiber-associated MuSCs after 72 hours in culture (n=3 per group). (C) Pax7 and MyoG immuno-staining of myofiber-associated MuSCs after 72 hours in culture. Scale bar = 50µm. (D) Quantification of the percentage of cells positive for MyHC, MyoG and Pax7 in cultured primary MuSCs following differentiation for 72 hours (n=3 per group). (E) MyHC, MyoG and Pax7 immuno-staining of cultured primary MuSCs following differentiation for 72 hours. Scale bar = 100µm (F) Quantification of total MyoD+ and percentage of EdU+ myofiber-associated MuSCs after 72 hours in culture. (n=3 per group). (G) MyoD and EdU immunostaining of myofiber-associated MuSCs after 72 hours in culture. Scale bar = 100µm (H) RNA-Seq scatter plot with key myogenic regulatory genes indicated. Each data point represents the mean Log₂(RPKM) from two independent biological replicates. Red and blue indicate upregulated and downregulated genes respectively. (I) Gene Ontology analysis of RNA-Seq data highlighting top 10 associated biological processes. (J) Heat map of key differentially expressed genes associated with MuSC identity, cell cycle and myogenic differentiation. Data represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.005

Figure 3. Inducible deletion of Setd7 in adult MuSCs impairs myogenic commitment ex vivo and skeletal muscle regeneration in vivo

(A) Schematic experimental design for ex vivo experiments. (B) Quantification of total YFP+ and frequency of Pax7+ and MyoG+ myofiber-associated MuSCs from EDL of control and Pax7^CreERT2/YFP/Setd7^FL/− mice cultured for 72 hours (n=3 per group). (G) Pax7, MyoG and YFP immunostaining of myofiber-associated MuSCs from control and Pax7^CreERT2/YFP/Setd7^FL/− mice cultured for 72 hours. Scale bar = 50µm (D) Schematic experimental design for in vivo injury experiments. (E) Quantification of frequency of eMyHC+ myofibers 10 days post injury (n=3 per group). Scale bar = 100µm (F) H+E staining of cross sections from TA muscles of control and Pax7CreERT2/YFP/Setd7^FL/− mice 14 days following NTX damage. Scale bar = 50µm (G) Quantification of myofiber cross sectional area (CSA) from control and Pax7CreERT2/YFP/Setd7^FL/− mice 14 days following NTX damage (n≥5 per group). Data represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.005

Figure 4. Little evidence of Setd7 interacting with MyoD or acting as an H3K4me1 histone methyltransferase in MuSCs

(A) Immunostaining of Setd7 in plated MuSCs highlighting cytoplasmic localization. Scale bar = 50µm. (B) Western blot analysis of Setd7 in Cytoplasmic (cyto), nuclear (nuc) and chromatin (chrom) fractions from plated MuSCs and myotubes. (C) Western blot analysis of H3K4me1 in WT and Sets7KO plated MuSCs. (D) Western blot of H3K4me1, me2, me3 in plated MuSCs and myotubes treated with or without a Setd7 inhibitor (PFI-2). (E+F) H3K4me1 ChIPSeq scatter plot correlating mean peak density in plated MuSCs treated with or without PFI-2 across whole genome (E) or at TSS (+/− 5KB) sites (F). (G) Mean signal of normalized peak density surrounding TSSs for H3K4me1 ChIPSeq for control treated MuSCs (blue line) and PFI-2 treated MuSCs (red line) (H) Heatmap displaying single gene resolution of H3K4me1 ChIP-Seq TSS enrichment data in (G). (I) Comparisons in H3K4me1 enrichment (RPKM) at myoblast (MB) and myotube (MT) enhancer regions in MuSCs treated with or without PFI-2. (J) Setd7 and MyoD co-immunostaining in plated MuSCs highlighting lack of co-localization. Scale bar = 100µm. (K) Western blot analysis of Setd7 and MyoD in cytoplasmic (cyto) and nuclear (nuc) fractions of plated MuSCs. (L) Setd7 IP samples blotted and probed with an anti-MyoD antibody. (M) H3K4me1 genome browser tracks in proximal promoters and gene bodies of MyoD targets (MyoG, Myh1 and Myl1). Control MuSCs (blue line) and PFI-2 treated MuSCs (red line) R values calculated with Pearson’s correlation coefficient.

Figure 5. Setd7 regulates β-catenin nuclear accumulation and transcriptional output via BCL-9 to control myogenic differentiation of MuSCs

(A) Heat map of key β-catenin responsive genes in MuSCs and their differential expression in Control (CON) vs Setd7 mKO cells. (B) β-catenin and Setd7 subcellular localization in C2C12 cells following stimulation with Wnt3a in the presence or absence of PFI-2. (C) Confocal microscopy images of β-catenin localization in C2C12 cells following treatment with Wnt3a in the presence of absence of PFI-2. (D) Quantification of β-catenin localization in C2C12 cells following treatment with Wnt3a in the presence of absence of PFI-2 (n=3). (E) TopFLASH reporter assay of β-catenin transcriptional output (TCF/LEF) by plated MuSCs in response to Wnt3a stimulation and in the presence or absence of PFI-2 (n=3). (F) β-catenin IP samples blotted and probed with anti-Setd7 antibody (top panel) and Setd7 IP samples blotted and probed with anti-β-catenin antibody (bottom panel) in the presence and absence of Wnt3a. (G) β-catenin IP samples blotted and probed with an anti-pan-methyl lysine antibody from cultured wild type (WT) and Setd7 null (KO) MEFs. (H) Frequency of MyoG+ plated MuSCs following 24 treatment with Wnt3a with or without PFI-2. Results from 3 independent experiments (n=3). Scale bar = 100µm. (I) BCL-9 and β-catenin protein-protein interactions evaluated using in-situ proximity ligation assay (PLA). Complexes visualized as red dots. Scale bar = 100µm. Quantification of BCL-9/β-catenin PLA assay. Red dots quantified in cytoplasm and nucleus from at least 800 cells per condition (n=4). *p<0.05, Wnt3a treated and untreated cells in cytoplasm. #p<0.05, Wnt3a treated and untreated cells in nucleus. (J) Schematic summary of interplay between Setd7 and Wnt signaling. Data represented as mean ± SEM. *p<0.05, **p<0.01

Figure 6. Setd7 inhibition enhances MuSC expansion, maintains MuSC potency and improves therapeutic efficacy

(A) Schematic of experimental design. (B) Pax7/MyoD co-immunostaining of plated MuSCs treated with or without PFI-2 for 7 days. Scale bar = 100µm. (C+D) Quantification of expansion (C) and frequency of Pax7/MyoD+ (D) MuSCs after seven days of culture with or with without PFI-2. (n=3). (E) Schematic of β-Actin-GFP transplantation experimental design. (F) GFP+ myofibers following transplantation of MuSCs expanded with or without PFI2. Scale bar = 100µm. (G) Quantification of GFP+ myofibers following transplantation of MuSCs expanded from 10,000 cells in the presence or absence of PFI-2. (n=5. Data from 5 mice each) (H) Quantification of GFP+ myofibers following transplantation of 10,000 MuSCs expanded in the presence or absence of PFI-2. (n=6. Data from 6 mice each) (I) Representative FACS plots and quantification of GFP+ MuSCs recovered from recipient mice transplanted with MuSCs expanded in vitro for 7 days with or without PFI-2. (n=8. Data from 8 mice each) (J) Schematic of GFP-Luc transplantation experimental design. (K) Quantification of bioluminescence over 28 days following transplantation of GFP-Luc labelled MuSCs expanded in vitro with or without PFI-2. (n=8. Data from 8 mice each) (L) Quantification of specific tetanic force production in TA muscle of NSG-MDX mice 28 days following transplantation of GFP-Luc labelled MuSCs expanded in vitro with or without PFI-2. (n=8. Data from 8 mice each) Data represented as mean ± SEM. *p≤0.05, **p<0.01, ***p<0.005.

Figure 7. Setd7 inhibition enhances expansion of transplantable human MuSCs

(A) Schematic of experimental design. hSKM (Human skeletal muscle). (B) Freshly isolated hMuSCs culture-expanded with/without PFI-2 for 7 days (n=3 donor samples). Scale bars = 20µm (main image) and 10µm (inset). (C) Quantification of bioluminescence over 11 days following transplantation of GFP-Luc transduced hMuSCs expanded in vitro with or without PFI-2. (Data from 6 mice (n=6) transplanted with donor cells from three operative samples). Data represented as mean ± SEM. **p≤0.01, ***p<0.005.