Dissecting cell diversity and connectivity in skeletal muscle for myogenesis

Figures

• 

  Fig. 1. RACs promote myogenic capability of MPCs. a Workflow of the protocol to generate skeletal muscle organoid from seed cells and matrigel via myogenic cultured in differentiation medium (DM). b Images of skeletal organoid under camera during formation after 14 days culture, R: RACs as seed cells cultured alone, MR: MPCs and RACs co-cultured, M: MPCs as seed cells cultured alone, Scale bars, 1 mm. c Images of skeletal organoid under ×40 microscope during formation after 14 days culture, Scale bars, 100 μm. d Representative images of myosin heavy chain (MHC) immunofluorescence staining in skeletal muscle organoid three groups: R, MR, M. e Ultrastructure of skeletal muscle organoid at 2 weeks of culturing. From left to right: Transmitted electron microscopy images of MPCs and RACs co-cultured in 3D culturing environment for 2 weeks (yellow arrowhead: z lines); Transmitted electron microscopy images of RACs cultured in 3D culturing environment for 2 weeks; Transmitted electron microscopy images of MPCs cultured in 3D culturing environment for 2 weeks (yellow arrowhead: z lines)

• 

  Fig. 2. Single-cell RNA-Seq uncovers RACs heterogeneity. a Workflow showing the collection of RACs and SACs by preplate technique and RACs and SACs. separately loaded on C1 high-throughput IFC for single-cell cDNA libraries, then by illumine HiSeq platform acquire single-cell RNA-seq library. b t-distributed stochastic neighbor embedding (t-SNE) plot of RACs and SACs reveals 4 cell clusters. c Violin plots showing the gene expression characteristics of cluster1, cluster2, cluster3, and cluster4 from mice skeletal muscle. d Representative images of tendon cells specific marker (Tnmd) and myo-tendon cells, a new cell type not reported before, markers (Myod1, Tnmd) immunofluorescence staining. e t-SNE plot of cluster1 reveal five subclusters: cluster1–1, cluster1–2, cluster1–3, cluster1–4 and cluster1–5. f Violin plots showing the gene expression characteristics of the 5 sub-clusters

• 

  Fig. 3. Connectivity map reveals ECM and paracrine signals promote muscle organoid formation. a Schematic showing receptor–ligand pairing screen between RACs and MPCs with examples of paracrine. b Heatmap showing the mean number of cell–cell interactions per cell type of RACs with MPCs for selected receptor–ligand pairings. c GO of the top 50 receptor–ligand parings that participate the cell–cell interaction of RACs with MPCs

• 

  Fig. 4. RACs secretome is indispensable during skeletal muscle organoid formation. a. Overview of the trans-well culture system, in which RACs and MPCs are cultured separately in a same well but can’t contact with each other. b Images of five groups of skeletal muscle organoids under ×40 microscope during formation after 14 days: MR (MPCs and RACs co-culture), TM (Trans-well cultured MPCs), TR (Trans-well cultured RACs), M (MPCs cultured alone), R (RACs cultured alone). c Representative images of myosin heavy chain (MHC) immunofluorescence staining slices of skeletal muscle organoid from five groups, Scale bar, 100 μm. d Figure generated after contractive intensity quantified by Moveheat (Methods) according to contraction video, the brighter in the figure the stronger of organoid contraction intensity. e Quantitative statistics of contraction intensity of MR, TM, TR, M, R (n = 3, **p < 0.01, ***p < 0.001). f Images of s1010 treated skeletal muscle organoid group and control group. g Diameter divergence statistic results of s1010 treated skeletal muscle organoid and vehicle control group. h Figure generated after contractive intensity quantified by Moveheat (Methods) according to…