Human iPSC-Derived Muscle Cells as a New Model for Investigation of EDMD1 Pathogenesis

Figure 1. Morphology and pluripotency markers expression analysis of iPSC lines. (A) Images show the morphology of properly formed iPSC colonies in transmitted light (10x objective). The scale bar is 100 μm. (B) IF staining of iPSCs shows proper localization and equal distribution of pluripotency markers Oct4 (AF488) in nucleus and SSEA4 (TRITC) in cell surface of representative iPSC colonies. Control negative staining of HeLa cells is shown in Supplementary Figure S1. The scale bar is 500 μm. (C) qPCR analysis shows relative gene expression levels of iPSC pluripotency markers, normalized to the previously published E1M40 1.7 clone [25] and to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level. Error bars present standard deviations, n = 4 (technical replicates).

Figure 2. Validation of the iPSC lines—genetics, purity, and differentiation ability. (A) G-banding analysis shows proper 46, XY karyotypes. (B) DNA sequencing chromatogram of fragment of amplified EMD gene confirmed wild-type sequence in control lines, cytidine deletion c.153del (CCCCC_AG) in E1M40 1.4 clone, and guanine duplication c.451dup (GGGGAAC) in both E1M19 lines. The upper sequence shows the reference wild-type EMD, while the bottom sequence shows the results of DNA sequencing. Red square indicates the mutation position. (C) PCR verification shows the loss of each reprogramming vector (KOS, c-MYC, KLF4, SeV) in iPSC lines, together with positive control GAPDH. (D) PCR analysis shows lack of mycoplasma contamination in each iPSC clone. (E) IF staining shows germ layers markers. Each iPSC clone was differentiated independently into three germ layers on coverslips, followed by cells staining for adequate markers: Sox17 (Cy5) for endoderm, Brachyury (Cy5) for mesoderm, and Otx2 (Cy5) for ectoderm. Control negative staining of undifferentiated iPSCs is shown in Supplementary Figure S3. The scale bar is 25 μm.

Figure 3. iPSCs differentiation into subsequent developmental stages of muscle cells. (A) Graphical representation of the reprogramming of fibroblasts from skin biopsies and differentiation process to subsequent developmental stages of muscle cells. (B) Images of SCs (day 6–7), myoblasts (day 6–7), and myotubes (up to 5 days in horse serum (HS)); transmission light, 10x magnification, scale bar 300 µm; with multinucleated myotubes, 40x magnification, scale bar 50 µm. Green arrows point to nuclei within an exemplary multinucleated myotube. (C) qPCR analyses of relative transcript levels of muscle early markers (Pax3, Pax7, Myf5, MyoD) in SCs (day 5–6) and myoblasts (day 6–7). Boxes depict 25th to 75th percentiles of relative gene expression with mean value marked with horizontal line, whiskers illustrate minimal and maximal values, asterisks indicate statistical significance (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns—not significant). Samples were normalized to GAPDH and HPRT and referred to C1M35 donor cell lines; to SCs for Pax3 and Pax7; and to myoblasts for Myf5 and MyoD, n ≥ 12 (≥4 biological replicates, 3 technical replicates).

Figure 4. Characterization of myoblasts and myotubes obtained by iPSCs differentiation in vitro. (A) Graph shows the minimal duration of myoblasts culture that is necessary for ability to form myotubes in subsequent differentiation medium. Each point indicates time spent by myoblasts in myoblast medium before performing successful differentiation to myotubes. The mean is marked with a horizontal line, error bars show standard deviations, and asterisks indicate statistical significance (ns—not significant, * p ≤ 0.05, ** p ≤ 0.01); n ≥ 3. (B) IF images show myotubes derived from patients’ cells, triple-stained for chromatin (DAPI), lamin B1 (AF488), and titin (DyLight650). Titin staining shows sarcomere structure in myotubes (white arrowheads), while yellow arrows point to nuclei within an exemplary multinucleated myotube. The scale bar is 20 μm. (C) WB analysis of emerin level and size in myotubes. Full-length emerin was present in control cells, a lower level of its truncated form was detected in E1M19 clones, while emerin was absent in E1M40 and E1M51 myotubes. IF images of myoblasts (D) and myotubes (E) triple-stained for chromatin (DAPI), emerin (AF488), and muscle markers: MyoD…