iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease modeling
- Wellstone Muscular Dystrophy Program, Department of Neurology, University of Massachusetts Chan Medical School, United States
- Li Weibo Institute for Rare Disease Research, University of Massachusetts Chan Medical School, United States
- Transgenic Animal Modeling Core, University of Massachusetts Chan Medical School, United States
- Genea Biocells, United States
- Conrad Prebys Center for Chemical Genomics, Sanford Burnham Prebys Medical Discovery Institute, United States
- Center for Genetic Muscle Disorders, Kennedy Krieger Institute, United States
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, United States
Figures
Figure 1 with 2 supplements
Isolation and characterization of iMyoblasts.
(A) Schematic of a three-stage transgene-free iPSC induction, iMyoblast reserve cell isolation, and iMyotube differentiation protocol. Images of S2 cells and iMyoblasts immunostained with MYOD1 …
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Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Transgene-free myogenic induction of FSHD1 and Ctrl iPSC and ESC and FKRP LGMDR9 and LGMDR7 iPSCs.
(A) Three-stage transgene-free myogenic induction (Caron et al., 2016). (B) Normalized qPCR assays of PAX3, PAX7, MYOD1, and MYH8 RNAs during myogenic induction of FSHD1 (pink) and Ctrl (teal) ESCs, …
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Reserve cell isolation of induced tertiary Myoblasts.
(A) Ctrl (17UM) and FSHD1 (17AM) tertiary iMyoblast cell lines isolated from iMyotube cultures using reserve cell selection and cultured for three passages in bMyoblast growth medium followed by …
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Figure 1—figure supplement 2—source data 1
Source data for Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig1-figsupp2-data1-v1.xlsx
Figure 2 with 1 supplement
iMyoblasts have a distinct gene expression signature compared to S1, S2, and muscle biopsy cells.
(A) Single-cell transcriptome sequencing (scRNA-Seq) was performed on S1, S2, iMyoblasts and bMyoblasts from three FSHD1 and three healthy (Ctrl) donors. A total of 24,991 cells satisfied criteria …
Figure 2—figure supplement 1
Morphology of different cell classes used in scRNA-Seq.
Phase images of S1, S2, iMyoblast (iMB), and bMyoblast (bMB) of cohort 17. Scale bar=100 µm.
Figure 3
scRNA-seq transcriptome signatures of iMyoblasts from FSHD1 and Ctrl subjects compared to S1, S2A, S2B, bMyo, and bMes myogenic cell classes.
Dot plot of manually curated genes in cells from six different subjects for each of the six cell classes (bottom row). Purple to orange colors define the low to high average expression of each gene …
Figure 4 with 2 supplements
iMyoblasts upregulated muscle genes during ex vivo differentiation in response to specialized differentiation media.
(A) Normalized qPCR assays of muscle RNAs CKM, MYH1, and MYH8 in cultures of bMyoblasts and iMyoblasts from FSHD1, Ctrl, and LGMDR7 iPSCs during their differentiation for 6 days (D) in N2 serum-free …
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Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Effects of serum-free media and iPSC reprogramming on muscle and DUX4 target gene expression.
(A) Normalized qPCR assays of the expression of muscle and DUX4 target genes during the myotube differentiation of FSHD1 and Ctrl bMyoblasts and iMyoblasts at Days 0, 2, 4, and 6, comparing Opti-MEM …
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig4-figsupp1-data1-v1.xlsx
Figure 4—figure supplement 2
iMyotubes and bMyotubes respond differently to specialized differentiation media.
Phase images of Ctrl and FSHD1 (A) iMyotubes and (B) bMyotubes after differentiation in designated specialized differentiation media cocktails described in text. Scale bar=250µm.
Figure 5 with 1 supplement
DUX4 and DUX4 target gene expression by iMyotubes, bMyotubes, and S3 iMyocytes in response to differentiation media.
(A) Normalized qPCR assays of DUX4 target genes MBD3L2, TRIM43, LEUTX, and ZSCAN4 in cultures of bMyoblasts and iMyoblasts of family cohorts 17 and 15 during differentiation for 6 days (D) in N2 …
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Figure 5—source data 1
Source data for Figure 5.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Losmapimod treatment decreases DUX4 target gene expression in FSHD1 iMyotubes.
(A) Schematic of Losmapimod treatment protocols for Pre-drug administration at the initiation of iMyotube differentiation and Post-drug administration after 4 days of iMyotube differentiation in N2 …
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Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 2 supplements
Epigenetic regulation of DUX4 and MYOD1 during iPSC reprogramming and iMyocyte and iMyotube differentiation.
(A) Normalized qPCR assays of DUX4 target genes MBD3L2, ZSCAN4, and TRIM43 during myogenic induction of FSHD1 and Ctrl ESC or iPSC. (B) Bisulfite sequencing of the DUX4 4qA locus in bMyoblasts from …
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Figure 6—source data 1
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
DUX4 expression by FSHD1 and Ctrl iPSCs and iMyoblasts and bMyoblasts.
(A) Normalized qPCR assays of DUX4 RNA expressed by Ctrl (17UM) and FSHD (17AM) in proliferating iMyoblasts and iMyotubes after 7 days in serum-free Opti-MEM differentiation medium. (B) Normalized …
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Figure 6—figure supplement 1—source data 1
Source data for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
CpG methylation of the 4qA allele of FSHD1 and Ctrl ESCs.
Bisulfite sequencing of the DUX4 4qA alleles of Genea Biocells' three Ctrl and three FSHD1 ESCs. (A) Bisulfite sequencing showing CpG methylated (red) and unmethylated (blue) CpG sites in DNA …
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Figure 6—figure supplement 2—source data 1
Source data for Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig6-figsupp2-data1-v1.xlsx
Figure 7 with 1 supplement
In vivo differentiation of FSHD1 and Ctrl iMyoblasts in muscle xenografts.
(A) Schematic of muscle xenograft protocol. (B) Representative cryosections of 2 and 4 weeks Ctrl bMyoblasts (17Ubic) and Ctrl and FSHD1 iMyoblasts (17UM and 17AM) xenoengrafted TA muscles were …
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Figure 7—source data 1
Source data for Figure 7.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
bMyoblasts and iMyoblasts efficiently xenoengraft into irradiated and injured TA muscle of NSG mice.
Cryosections of TA muscles were xenoengrafted with Ctrl (17Ubic) and FSHD (17Abic) bMyo and Ctrl (17UM) and FSHD (17AM) iMyoblasts. Xenografted TAs recovered 2 weeks (top panel) or 4 weeks (bottom …
Figure 8
Embryonic/adult MYH isoform switching during maturation of iMyoblast and bMyoblast muscle xenografts.
NanoString digital assays of the expression of MYH isoforms in cultures of FSHD1 (17Abic) and Ctrl (17Ubic) bMyotubes and FSHD1 (17AM) and Ctrl (17UM) iMyotubes at Day 6 differentiation in Opti-MEM …
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Figure 8—source data 1
Source data for Figure 8.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig8-data1-v1.xlsx
Figure 9
iMyoblast modeling LGMDR7 and FKRP dystroglycanopathies.
(A) Representative images from LGMDR7 muscle xenograft cryosections immunostained with human-specific antibodies (left) lamin A/C (red) and collagen VI (green) or (right) lamin A/C and spectrin β1 …
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Figure 9—source data 1
Source data for Figure 9.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig9-data1-v1.xlsx
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Figure 9—source data 2
Source data for Figure 9F.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig9-data2-v1.pptx
Figure 10
iMyoblasts have regenerative potential after secondary muscle injury.
(A) Representative images from 17UM and WWS muscle xenograft cryosections immunostained with PAX3 and stained with Hoechst. The percentage of PAX3+ nuclei is shown on the right. N=4, 17UM engrafted …
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Figure 10—source data 1
Source data for Figure 10.
- https://cdn.elifesciences.org/articles/70341/elife-70341-fig10-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | NOD.Cg-PrkdcscidIL2rγtmiWjl/SzJ | Jackson Lab | Stock No: 005557 | |
| Cell line(Homo sapiens) | 15Abic biopsy myoblast | Homma et al., 2012; Jones et al., 2012 | ||
| Cell line(H. sapiens) | 15Vbic biopsy myoblast | Homma et al., 2012; Jones et al., 2012 | ||
| Cell line(H. sapiens) | 17Abic biopsy myoblast | Jones et al., 2012 | ||
| Cell line(H. sapiens) | 17Ubic biopsy myoblast | Jones et al., 2012 | ||
| Cell line(H. sapiens) | 30Abic biopsy myoblast | Jones et al., 2012 | ||
| Cell line(H. sapiens) | 30Wbic biopsy myoblast | University of Massachusetts Medical School https://www.umassmed.edu/wellstone/ | ||
| Cell line(H. sapiens) | 15AM iPSCs | This paper | 15AM iPSCs were reprogrammed from 15Abic biopsy CD56+ myoblast at UMMS | |
| Cell line(H. sapiens) | 15VM iPSCs | This paper | 15VM iPSCs were reprogrammed from 15Vbic biopsy CD56+ myoblast at UMMS | |
| Cell line(H. sapiens) | 17AM iPSCs | This paper | 17AM iPSCs were reprogrammed from 17Abic biopsy CD56+ myoblast at UMMS | |
| Cell line(H. sapiens) | 17AF iPSCs | This paper | 17UM iPSCs were reprogrammed from 17Abic biopsy CD56- fibroblast at UMMS | |
| Cell line(H. sapiens) | 17UM iPSCs | This paper | 17UM iPSCs were reprogrammed from 17Ubic biopsy CD56+ myoblast at UMMS | |
| Cell line(H. sapiens) | 30AM iPSCs | This paper | 30AM iPSCs were reprogrammed from 30Abic biopsy CD56+ myoblast at UMMS | |
| Cell line(H. sapiens) | 30WM iPSCs | This paper | 30WM iPSCs were reprogrammed from 30Wbic biopsy CD56+ myoblast at UMMS | |
| Cell line(H. sapiens) | 54574/75 iPSCs | This paper | 54574/75 iPSCs were reprogrammed from skin fibroblast at UMMS | |
| Cell line(H. sapiens) | 54585 iPSCs | This paper | 54585 iPSCs were reprogrammed from skin fibroblast at UMMS | |
| Cell line(H. sapiens) | LGMDR7 iPSCs | Formerly LGMD2G (Iyer et al., 2019) | ||
| Cell line(H. sapiens) | LGMDR9 FP iPSCs | This paper | FP iPSCs were reprogrammed from skin fibroblast at Sanford Burnham Prebys Medical Discovery Institute | |
| Cell line(H. sapiens) | WWS iPSCs | This paper | WWS iPSCs were reprogrammed from skin fibroblast at Sanford Burnham Prebys Medical Discovery Institute | |
| Cell line(H. sapiens) | 15AM iMyoblasts | This paper | 15AM iMyoblasts were made from 15AM iPSCs | |
| Cell line(H. sapiens) | 15VM iMyoblasts | This paper | 15VM iMyoblasts were made from 15VM iPSCs | |
| Cell line(H. sapiens) | 17AM iMyoblasts | This paper | 17AM iMyoblasts were made from 17AM iPSCs | |
| Cell line(H. sapiens) | 17AF iMyoblasts | This paper | 17AF iMyoblasts were made from 17AF iPSCs | |
| Cell line(H. sapiens) | 17UM iMyoblasts | This paper | 17UM iMyoblasts were made from 17UM iPSCs | |
| Cell line(H. sapiens) | 30AM iMyoblasts | This paper | 30AM iMyoblasts were made from 30AM iPSCs | |
| Cell line(H. sapiens) | 30WM iMyoblasts | This paper | 30WM iMyoblasts were made from 30WM iPSCs | |
| Cell line(H. sapiens) | 54574/75 iMyoblasts | This paper | 54574/75 iMyoblasts were made from 30WM iPSCs | |
| Cell line(H. sapiens) | 54585 iMyoblasts | This paper | 54585 iMyoblasts were made from 54585 iPSCs | |
| Cell line(H. sapiens) | LGMDR7 iMyoblasts | This paper | LGMDR7 iMyoblasts were made from LGMDR7 iPSCs | |
| Cell line(H. sapiens) | LGMDR9 FP iMyoblast | This paper | LGMDR9 FP iMyoblasts were made from LGMDR9 iPSCs | |
| Cell line(H. sapiens) | WWS iMyoblasts | This paper | WWS iMyoblasts were made from WWS iPSCs | |
| Antibody | MyoD1 (Clone: 5.8A) (Mouse Monoclonal) | Dako | Cat #: M3512 | IF (1:50) |
| Antibody | MF20 (Mouse Monoclonal) | DSHB | Cat #: AB_2147781 | IF (1:100) |
| Antibody | MEF2C (Rabbit polyclonal) | Sigma-Aldrich | Cat #: HPA005533 | IF (1:100) |
| Antibody | Collagen Type VI (Mouse Monoclonal) | Sigma-Aldrich | Cat #: MAB1944 | IF (1:250) |
| Antibody | Lamin A/C (Mouse Monoclonal) | Thermo Fisher Scientific | Cat #: MA3-1000 | IF (1:100) |
| Antibody | Laminin β1 (clone 4E10) | MilliporeSigma | Cat #: MAB1921P | IF (1:100) |
| Antibody | Myosin heavy chain, neonatal | Leica Biosystems | Cat #: NCL-MHCn | IF (1:100) |
| Antibody | PAX3 | Abcam | Cat #: Ab180754 | IF (1:50) |
| Antibody | Spectrin β1 (Mouse Monoclonal) | Leica Biosystems | Cat #: NCL-SPEC1 | IF (1:50) |
| Antibody | APC Mouse Anti-Human CD56 (Mouse Monoclonal) | BD Biosciences | Cat #: 555518 | Flow (100 μl per million cells) |
| Antibody | PE anti-human CD82 (Mouse Monoclonal) | BioLegend | Cat #: 342103 | Flow (3 μl per million cells) |
| Antibody | FITC anti-human CD318 (Mouse Monoclonal) | BioLegend | Cat #: 324004 | Flow (5 μl per million cells) |
| Antibody | APC anti-human ERBB3 (Mouse Monoclonal) | BioLegend | Cat #: 324708 | Flow (5 μl per million cells) |
| Antibody | FITC anti-human NGFR (Mouse Monoclonal) | BioLegend | Cat #: 345104 | Flow (5 μl per million cells) |
| Antibody | PE anti-human CD18 (Mouse Monoclonal) | BioLegend | Cat #: 373407 | Flow (5 μl per million cells) |
| Sequence-based reagent | RT-qPCR primers | This paper | Supplementary file 4 | |
| Commercial assay or kit | StemMACS iPS-Brew XF, human | Miltenyi Biotec | Cat #: 130-104-368 | |
| Commercial assay or kit | Skeletal Muscle Differentiation Kit | Amsbio | Amsbio Skeletal Muscle Differentiation Kit | SKM01, SKM02, and SKM03 were used for human iPSCs skeletal muscle differentiation |
| Commercial assay or kit | RNeasy Plus Mini Kit | QIAGEN | Cat #: 74136 | |
| Commercial assay or kit | Mouse on Mouse (M.O.M.) Basic Kit | Vectorlab | Cat #: BMK-2202 | |
| Commercial assay or kit | SuperScript III First-Strand Synthesis System | Invitrogen | Cat #: 18080051 | |
| Commercial assay or kit | Emerson lab custom muscle NanoString panel | NanoString Technologies | NanoString Technologies developed the muscle NanoString panel based a gene list provided by Emerson lab | |
| Commercial assay or kit | Chromium Single Cell 3' GEM, Library &Gel Bead Kit v2, 4 rxns | 10× Genomics | Cat #: 120267 | |
| Commercial assay or kit | Chromium Chip A Single Cell Kit, 16 rxns | 10× Genomics | Cat #: 1000009 | |
| Commercial assay or kit | Chromium i7 Multiplex Kit, 96 rxns | 10× Genomics | Cat #: 120262 | |
| Chemical compound, drug | ROCK Inhibitor Y-27632 | STEMCELL Technologies | Cat #: 72307 | |
| Chemical compound, drug | SB431542 | SelleckChem | Cat #: S1067 | |
| Chemical compound, drug | CHIR99021 | STEMCELL Technologies | Cat #: 72052 | |
| Chemical compound, drug | Prednisolone | Sigma-Aldrich | Cat #: P6004 | |
| Chemical compound, drug | DAPT | SelleckChem | Cat #: S2215 | |
| Chemical compound, drug | Dexamethasone | SelleckChem | Cat #: S1322 | |
| Chemical compound, drug | Forskolin | SelleckChem | Cat #: S2449 | |
| Software, algorithm | GraphPad Prism | GraphPad Prism, RRID:SCR_002798 | https://www.graphpad.com/ | |
| Software, algorithm | nSolver 4.0 Analysis Software | nSolver Analysis Software, RRID:SCR_003420 | http://www.nanostring.com/products/nSolver | |
| Software, algorithm | Bisulfite Sequencing DNA Methylation Analysis | BISMA, RRID:SCR_000688 | http://services.ibc.uni-stuttgart.de/BDPC/BISMA/ | |
| Software, algorithm | Cell Ranger | Cell Ranger, RRID:SCR_017344 | https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger | Version 3.1.0 |
| Software, algorithm | STAR 2.5.1b | Implemented in Cell Ranger version 3.1.0 | https://github.com/alexdobin/STAR (Dobin, 2022) | Version 2.5.1b |
| Software, algorithm | Python | Python Programming Language, RRID:SCR_008394 | http://www.python.org/ | Version 2.7.9 |
| Software, algorithm | Opossum 0.2 | Oikkonen and Lise, 2017 | https://github.com/BSGOxford/Opossum (BSG Oxford, 2022) | Version 0.2 |
| Software, algorithm | Platypus | Platypus, RRID:SCR_005389 | https://www.rdm.ox.ac.uk/research/lunter-group/lunter-group/all-platypus-and-stampy-versions | Version 0.8.1 |
| Software, algorithm | Demuxlet | Kang et al., 2018 | https://github.com/statgen/demuxlet (The Center for Statistical Genetics at the University of Michigan School of Public Health, 2021) | Version 08/03/2018 |
| Software, algorithm | Seurat | SEURAT, RRID:SCR_007322 | http://seurat.r-forge.r-project.org/ | Version 3.1.4 |
| Software, algorithm | R Project for Statistical Computing | R Project for Statistical Computing, RRID:SCR_001905 | http://www.r-project.org/ | Version 3.6.2 |
| Software, algorithm | edgeR | edgeR, RRID:SCR_012802 | http://bioconductor.org/packages/edgeR/ | Version 3.30.3 |
Additional files
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Supplementary file 1
Differential expression for pairwise cell type comparisons from edgeR analysis.
For each of the 15 pairwise comparisons (shown in separate tabs), genes with differential expression (in either up or down directions) were ranked by p-value if the P-value < 1.0E-06 and |log2(FC)| > 1. The table includes columns for p-values, log2(FC), log2(CPM), QL F-test (F), and false discovery rate (FDR) from edgeR, and several columns for gene annotations.
- https://cdn.elifesciences.org/articles/70341/elife-70341-supp1-v1.xlsb
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Supplementary file 2
Top 20 Up and Down GO terms (BP, CC, MF) and KEGG pathways for pairwise cell type comparisons.
For each of the 15 pairwise comparisons (shown in separate tabs), the goana and kegga functions in edgeR were used to rank the top 20 Gene Ontology (GO) terms from each of the biological process (BP), cellular component (CC) and molecular function (MF) branches of GO, and the top 20 KEGG pathways, based on overrepresentation among the DE genes. Terms comprising N ( < 500) genes are sorted by P.Up and P.Down. For each term, the top DE Genes.Up and Genes.Down (ordered by P Value) are listed, up to a maximum of 30 genes.
- https://cdn.elifesciences.org/articles/70341/elife-70341-supp2-v1.xlsx
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Supplementary file 3
Pseudobulk counts for 36 samples based on scRNA-Seq data.
Raw single-cell counts were summed for each of the 6 cell types or subclusters of interest (S1, S2A, S2B, iMyoblast, bMyo, bMes) in each of the six donors (15 A, 15 V, 17 A, 17 U, 30 A, 30 W).
- https://cdn.elifesciences.org/articles/70341/elife-70341-supp3-v1.xlsx
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Supplementary file 4
Primer sequences.
Table lists all primers for qPCR and bisulfite sequencing.
- https://cdn.elifesciences.org/articles/70341/elife-70341-supp4-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70341/elife-70341-transrepform1-v1.pdf
