Journal: Cell Reports Methods
Article Title: African pygmy mouse iPSCs as a model for in vitro embryogenesis, interspecies chimerism, and blastocyst complementation
doi: 10.1016/j.crmeth.2025.101293
Figure Lengend Snippet: Ablation of mouse PAX7 + cells promotes APM satellite cell formation in mouse-APM chimeras (A) Schematic of experimental design to preferentially obtain APM satellite cells in mouse-APM chimeras through elimination of host mouse PAX7 + cells during postnatal growth. (B) Representative photos of mouse-APM chimeras generated from Pax7 Cre/ERT2 ; Rosa26 LSL-DTA blastocysts. Scale bars, 1 cm. (C) Representative bright-field and fluorescence overlay images of tibialis anterior (TA) muscles from the indicated animals. Scale bars, 5 mm. (D) Immunostaining images of TA muscle cross-sections for the indicated markers in the specified chimeras. Note the presence of PAX7 + /mCHERRY + cells with and without tamoxifen treatment. Scale bars, 100 μm. (E) Representative FACS plots displaying the percentage of mCHERRY + satellite cells within the ITGA7 + satellite cell population of a tamoxifen-treated vs. a B6 chimera. (F) Graph showing quantification of the representative FACS plots shown in (E) for a larger group of chimeras. Data are shown as mean ± SD. N = 3 tamoxifen-treated chimeras and N = 8 B6 chimeras. ∗∗ p ≤ 0.01. (G) Uniform manifold approximation and projection (UMAP) based on scRNA-seq of all cell populations in tamoxifen-treated mouse-APM chimera skeletal muscles, colored by the indicated cell type. SCs, satellite cells; FAPs, fibro-adipogenic progenitors. (H) Dot plot showing individual gene expression in mouse-APM cell populations used to annotate the UMAP shown in (G). (I) UMAP of all cells from skeletal muscles of a mouse-APM chimera, colored by detection of the H2B-mCherry transcript. (J) Representative bright-field and fluorescence overlay images of FACS-purified ITGA7 + /mCHERRY + and ITGA7 + /mCHERRY − myoblasts. Scale bars, 100 μm. (K) Representative immunostaining images for the skeletal muscle differentiation marker ACTN2 in ITGA7 + /mCHERRY + myoblast-derived myotubes. Scale bars, 100 μm.
Article Snippet: For satellite cell FACS-purification, cells were stained with conjugated anti-mouse LY-6A/E (SCA1) (1:100, Thermo Fisher, 11-5981-82), anti-mouse ITGA7 (1:100, R&D Systems, FAB3518S), anti-mouse CD45 (1:100, Biogend, 103121), anti-mouse PECAM1/CD31 (1:100, BioLegend, 102414), and DAPI (1:1000, Sigma-Aldrich MBD0015-1ML).
Techniques: Generated, Fluorescence, Muscles, Immunostaining, Gene Expression, Purification, Marker, Derivative Assay
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