Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors
- Ruhr University Bochum, Medical Faculty, Institute of Anatomy, Department of Anatomy and Molecular Embryology, Germany
- Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Germany
- Department of Neurology with Heimer Institute for Muscle Research, University Hospital Bergmannsheil, Germany
- Max Planck Institute for Molecular Biomedicine, Sequencing Core Facility, Germany
- Luxembourg Centre for Systems Biomedicine, LCSB, Developmental and Cellular Biology, University of Luxembourg, Luxembourg
- Ruhr University Bochum, Medical Faculty, Department of Cellular Physiology, Germany
- Max Planck Institute for Molecular Biomedicine, Flow Cytometry Unit, Germany
- Center for Cellular Nanoanalytics Osnabrück, CellNanOs, University of Osnabrück, Germany
- Max Planck Institute for Molecular Biomedicine, Electron Microscopy Unit, Germany
- Max Planck Institute for Molecular Biomedicine, Department of Tissue Morphogenesis, Germany
- Computational Biology and Systems Biomedicine, Biodonostia Health Research Institute, Spain
- School of Mechanical Engineering, Korea University, Republic of Korea
- R&D Research Center, Next & Bio Inc, Republic of Korea
- School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Republic of Korea
- Ruhr University Bochum, Medical Faculty, Department of Molecular GI Oncology, Germany
- Westphalian Wilhelms University Münster, Medical Faculty, Germany
Figures
Figure 1 with 1 supplement
Skeletal muscle organoid protocol and correlation to fetal development.
(A) Brightfield images of myogenic development stages, with corresponding cytokines/growth factors protocol applications. (B) Graph depicting organoid development in size (day 0, n = 51; day 3, n = …
Figure 1—figure supplement 1
Lineage representation and organoid culture progression at early stages of differentiation protocol.
(A) qPCR analysis depicts relative expression of pluripotent (OCT4, SOX2, NANOG), mesodermal/posterior somitic (BRACHYURY, MESOGENIN, TBX6, HES7), anterior somitic (PAX3, UNCX, TBX18, MEOX2), …
Figure 2 with 1 supplement
Neural lineage development during skeletal muscle organoid progression.
(A) Stepwise brightfield images depicting delamination/migration of progenitor population during organoid culture progression and corresponding myofiber formation. (B) Gene ontology enrichment …
Figure 2—figure supplement 1
Myogenic versus neural fate during organoid development.
(A) Heatmap of limb myogenic progenitor markers emphasizes upregulation through organoid protocol development. (B) Differential expression comparison highlights upregulated (n = 5919) and …
Figure 3 with 3 supplements
Skeletal muscle organoid characterization at single-cell resolution.
(A) t-SNE visualization of color-coded clustering (n = 4323 cells) at 12 wk post differentiation highlights the predominant presence of skeletal muscle lineage, represented by clusters corresponding …
Figure 3—figure supplement 1
Single-cell RNA-seq expression profiling and lineage representation in organoid culture at week 12.
(A) Gene signatures of t-SNE-described clusters based on relative expression levels of the 50 most significant markers for each of the seven clusters. (B) t-SNE plot of myogenic and fibroadipogenic …
Figure 3—figure supplement 2
Skeletal muscle organoid culture maturation and identity.
(A) Differential expression comparison highlights upregulated (n = 2856) and downregulated (n = 2369) genes between 16 and 8 wk organoids (Padj<0.001). (B, C) Gene ontology (GO) enrichment analysis …
Figure 3—figure supplement 3
Functional properties of organoid-derived skeletal muscle myofibers.
(A) Second harmonic generation imaging (SHG) from areas with dense fiber network reveals myofibril formation with distinct sarcomeres (z plane of stack right). (B) Electron microscopy depicts …
Figure 4 with 7 supplements
Myogenic progenitor identity and comparison to progenitors derived from fetal and adult muscle tissue.
(A) t-SNE plot visualization of color-coded clustering indicates myogenic progenitor subcluster with distinct molecular signatures: ‘dormant’ PAX7high/CHODLhigh/FBN1high, ‘activated’ CD44high/CD98+/M…
Figure 4—figure supplement 1
Subclustering of myogenic progenitors and NOTCH signaling.
(A) Dot plot showing expression of representative genes related to satellite cell identity across the seven main clusters. (B) Dot plot showing expression of representative genes related to NOTCH …
Figure 4—figure supplement 2
Pseudotime ordering of myogenic progenitor revealing distinct states and cell fate decisions.
(A) Gene signatures of t-SNE described clusters based on relative expression levels of the 50 most significant markers for each of the three clusters. (B) Expression of selected genes along …
Figure 4—figure supplement 3
Organoid-derived myogenic progenitors and correlation to fetal muscle progenitors.
(A) Gating strategy applied for FACS sorting CD29+/CXCR4+ cells from 15 to 16 wk skeletal muscle organoids. (B, C) Re-plating CD29+/CXCR4+ cells and culturing for 14 d highlights fetal myogenic …
Figure 4—figure supplement 4
Organoid-derived myogenic progenitors and correlation to adult human satellite cells.
(A) Violin plots highlighting developmental status for organoid-derived myogenic progenitors and satellite cells through relative expression of key signature markers ZFP36, CD44, FBN1, NEB, and SPRY1…
Figure 4—figure supplement 5
Characterization of cell–cell communication network for all clusters at week 12 of human skeletal muscle organoid development.
(A) Circle plot illustrates the aggregated cell–cell communication network for all clusters at week 12 of human skeletal muscle organoids development. Circle sizes are proportional to the number of …
Figure 4—figure supplement 6
Reproducibility of organoid culture at early and mature stages.
(A) Pseudotemporal ordering of organoids from days 2, 7, and 11 based on qPCR expression profiling of selected 32 genes indicates robust homogeneous development of organoids during WNT activation …
Figure 4—figure supplement 7
Comparison between 2D in vitro myogenic differentiation protocols and in vivo staging.
Violin plots highlighting expression of key myogenesis markers: Comparison between myogenic progenitors derived in 2D differentiation protocols (HX protocol, Xi et al., 2017; JC protocol, Chal et …
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Additional files
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Supplementary file 1
Tables of qPCR primer pairs.
(a) qPCR primer pairs applied to detect relative expression of key markers during skeletal muscle organoid development. (b) qPCR primer pairs applied for diffusion map analysis of early skeletal muscle organoid development.
- https://cdn.elifesciences.org/articles/87081/elife-87081-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/87081/elife-87081-mdarchecklist1-v1.docx
