Spatial RNA sequencing analysis confirm ACTC1+ myofibers support a transplanted SMPC pool

Spatial transcriptomics analysis of three different experiments (describe below) identify regenerating ACTC1+ myofibers as a supportive cell type for transplanted SMPCs. Data correspond to Figures 6-7 and Extended Data Figures 8-10 in Hicks et al., "Regenerating Human Skeletal Muscle Forms an Emerging Niche In Vivo to Support PAX7 cells". Overall design: Experiment 1: Spatial RNAseq of fetal skeletal muscle tissue. Fetal human week 20 quadriceps, hamstring, and gastrocnemius skeletal muscle were sectioned onto slides and prepared for spatial RNAseq analysis. Morphology markers included ACTC1+ myofibers and DAPI+ACTC1- cells adjacent to myofibers, which would likely represent a large population of PAX7 cells. A third group DAPI+ACTC1- cells away from myofibers was also sequencing which likely represents connective tissues, tenocyotes, or other non-myogenic tissue. This set of tissues was iniatially used as a positive control while optimizing spatial RNA sequencing parameters that would later be used for the engraftment experiments. Experiment 2: Spatial RNAseq of engrafted fetal SMPCs in vivo. Primary fetal SMPCs were FACS-enriched from fetal week 18 skeletal muscle using CD45-CD235a-ERBB3+NGFR+. 500,000 SMPCs were then transplantated into mdx-DBA/2-NSG PAX7-knockout mice. Prior to transplantation, mice were subjected to BaCl2 injury to tibialis anterior muscle, and adminstered tamoxifen to inducibly ablate endogenous Pax7+ cells. Mice were terminated and had their tibialis anteriors sectioned after 2months, 4months, and a final group was subjected to a BaCl2 reinjury at the 2month mark but collected and sectioned at the 4month mark. These tissue samples were then processed for spatial RNAseq accordingly. Experiment 3: Spatial RNAseq of engrafted genetically modified hPSC-derived SMPCs in vivo with and without AP1903 treatment. H9 hPSCs were genetically edited to inducibly ablate ACTC1+ cells by inserting an ACTC1-iCasp9 into the 3' UTR of ACTC1. After isolating a clonal knock-in line, the hPSCs was differentiated to skeletal muscle in vitro, and SMPCs were enriched using flow cytrometry HNK1-ERBB3+NGFR+. The SMPCs were expanded in vitro for 3 days and 1,000,000 SMPCs were then transplantated into mdx-DBA/2-NSG PAX7-knockout mice. and before transplantation into PAX7-Cre mice. Prior to transplantation, mice were subjected to BaCl2 injury to tibialis anterior muscle, and adminstered tamoxifen to inducibly ablate endogenous Pax7+ cells. After transplantation, mice were treated with the AP1903 by IP injections starting at two weeks, and continued every two weeks, throughout the 90 days to ablate ACTC1+ myofibers. Control mice were treated with 0.1% DMSO in saline. After 90 days tissues were sectioned and tissue samples were processed for Spatial RNAseq.