ACVR1R206H increases osteogenic/ECM gene expression and impairs myofiber formation in human skeletal muscle stem cells

Abnormalities in skeletal muscle repair 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, and 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 contributes to HO by changing the local tissue environment.


Abstract:
26 Abnormalities in skeletal muscle repair lead to poor function and complications such as scarring 27 or heterotopic ossification (HO). Here, we use fibrodysplasia ossificans progressiva (FOP), a 28 disease of progressive HO caused by ACVR1 R206H (Activin receptor type-1 receptor) mutation, to 29 elucidate how ACVR1 affects skeletal muscle repair. Rare and unique primary FOP human muscle 30 stem cells (Hu-MuSCs) isolated from cadaveric skeletal muscle demonstrated increased ECM 31 marker expression, and showed skeletal muscle-specific impaired engraftment and regeneration 32 ability. Human induced pluripotent stem cell (iPSC)-derived muscle stem/progenitor cells 33 (iMPCs) Single cell transcriptome analyses from FOP also revealed unusually increased ECM and 34 osteogenic marker expression compared to control iMPCs. These results show that iMPCs can 35 recapitulate many aspects of Hu-MuSCs for detailed in vitro study, that ACVR1 is a key regulator 36 of Hu-MuSC function and skeletal muscle repair; and that ACVR1 activation in iMPCs or  MuSCs contributes to HO by changing the local tissue environment. 38 MuSCs, but not when FOP diaphragm Hu-MuSCs were transplanted (Figure 1G, H). No 105 differences in the number of human PAX7 cells were identified (Figure 1G, H). No    expression showed some heterogeneity among the different hiPSC lines but these differences were 126 not statistically significant. Adding a SMAD inhibitor of the BMP pathway (LDN193189) into the 127

3-PAX7 expressing cells can be isolated from myogenic differentiation 137
To test the regenerative properties of PAX7-expressing MuSCs, we used FACS to purify HNK1 -138 CD45 -CD31cells co-expressing CD29, CXCR4, and CD56 markers present on human

4-Isolated iMPCs can regenerate injured mouse muscle and form human fibers 147
To assess iMPC regenerative capacity in vivo, we injected 1,000-10,000 iMPCs derived from 148 control or FOP hiPSCs (Figure 3-Source Data 1) into the tibialis anterior (TA) muscle of whole-149 body irradiated NSG immunocompromised mice (to hinder endogenous satellite cells) previously 150 injured with bupivacaine (Garcia, Tamaki, Xu, & Pomerantz, 2017). The bupivicaine step induces 151 myofiber injury and promotes engraftment of donor SCs. New fibers expressing human 152 DYSTROPHIN and PAX7 cells were found after 5 weeks, showing that iMPCs could engraft and 153 promote muscle regeneration ( Figure 3C). However, the number of human fibers and human 154 PAX7 + cells remained low ( Figure 3D) compared to primary Hu-MuSCs. While some iMPC 155 transplants yielded up to 60 new human fibers, some did not yield any human fibers. By 156 comparison, 2,000 primary non-FOP Hu-MuSCs resulted in an average of 155 human fibers based 157 on prior assessments using the same assay (Garcia et al., 2018). No significant differences between 158 control and FOP iMPCs were identified, though some individual FOP samples showed higher 159 engraftment ( Figure 3D). 160 These results showed that iMPCs can engraft into a muscle injury site in mice, but engraftment 161 efficiency may be lower than primary Hu-MuSCs or be the result of differences in experimental 162 conditions. Also, ACVR1 R206H did not significantly impact muscle fiber regeneration in this assay. 163 164

5-Transcriptional profiling of iMPCs 165
The lower engraftment of iMPCs compared to primary Hu-MuSCs suggested that the FACS-166 purified population was still heterogeneous or that iMPCs do not fully recapitulate adult primary 167 Hu-MuSCs. Single cell RNA sequencing (scRNAseq) from control (1323-2) and FOP (F3-2) 168 iMPCs (the lines were selected based on their lower intra-line variability, Figure 2         Comparing primary FOP Hu-MuSCs from muscles that develop HO (biceps) and non-affected 345 muscle (diaphragm) suggests that the source of the Hu-MuSCs impacts engraftment efficiency. 346 The re-injury model showed that engraftment of Hu-MuSCs from FOP biceps, but not from 347 diaphragm, remained significantly decreased. Thus, it is intriguing to consider that the clinical 348 sparing of the diaphragm from HO in patients with FOP may result from a less impaired or 349 unimpaired muscle repair process in diaphragm satellite cells. Further delineation of muscle-specific Hu-MuSC properties will be revealing to understand this observation. Our finding that 351 primary FOP Hu-MuSCs have lower engraftment ability provides a potential explanation for the 352 poor skeletal muscle repair observed in patients with FOP (Shore, 2012). However, the FOP and 353 control iMPCs showed no major differences in engraftment, possibly due to decreased assay 354 sensitivity from the lower engraftment efficiency of iMPCs in general, because iMPCs may be 355 more immature than primary Hu-MuSCs, or that iMPCs represent a subtype of cells that may be 356 more reflective of non-ossifying skeletal muscle like diaphragm. despite FACS purification. This heterogeneity is an ongoing problem among all published iMPC 367 protocols to date. In our case, this was counter-balanced by our consistent findings across multiple 368 lines and between iMPCs and the availability of primary Hu-MuSCs. Although our primary cell 369 studies were limited by the rarity of FOP (estimated at 1 in 1.4 million people) and even rarer 370 suitable cadaveric samples, the combination of iPSC-derived lineages with the rare primary 371 samples provided multiple avenues for supporting our conclusions. Finally, the ACVR1 R206H 372 mutation increases BMP signaling but also introduces neofunction to Activin A. Our studies are 373 not able to distinguish between these two contributing pathways. Our finding of increased p38 374 activity in the FOP iMPCs was also seen in FOP subject monocyte-derived macrophages (

RT-PCR and Quantitative Analysis 457
Tissues were collected in TRI Reagent (Sigma-Aldrich) to isolate total RNA using the Arcturus TM 458 PicoPure TM RNA kit (Applied Biosystems) as previously described for small samples (Schepers,459 Hsiao         Week 5 Week 10      Figure 1A Psoas 52 F FOP Figure 1A Intercostal 46 F FOP Figure 1B  Biceps Brachii 32 F FOP Figure 1C-H and S1 Biceps Brachii 44 F Control Figure 1C-H and S1 Biceps Brachii 32 F FOP Figure 1C-H and S1 Diaphragm 32 F FOP 840 Week 5 on the myogenic cell subset of the merged hiPSCs samples only to identify sub-clusters 928 corresponding to different myogenic states. To order the cells in pseudotime based on their 929 transcriptional similarity we used Monocle 2.12. Variable genes from Seurat analysis were used 930 as input and clusters were projected onto the minimum spanning tree after ordering. Gene 931 expression patterns were plotted with plot_genes_branched_heatmap, 932 plot_genes_branched_pseudotime, and plot_multiple_branches_pseudotime. The BEAM (branch 933 expression analysis modeling) function was used to score gene significance in a branch-dependent 934 manner. Cells were re-ordered using the orderCells function to set branch A (myocytes) in Figure  935 5F as the "root-state". This allowed us to determine genes that were significantly branch dependent 936 in branch B (mainly Hu-MuSCs) vs branch C (hiPS-derived cells) using the BEAM analysis. 937