Tissue-specific promoter-based reporter system for monitoring cell differentiation from iPSCs to cardiomyocytes

Figures

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  Figure 1. Myoblast characteristics. (A) Flow cytometry detected approximately 83% of CD56+ myoblasts cells in the isolated skeletal muscle population. (B) Isotype control (IgG1-PC5). (C) Immunofluorescence image of myoblasts stained with anti-desmin antibody (green) with nuclear dye DAPI (blue); (D) Immunofluorescence image of myoblasts stained with anti-α-MHC (myosin heavy chain) antibody (green) with nuclear dye DAPI (blue) (E) Multinuclear tube formation test confirmed the ability of cells to differentiate in vitro, scale bars = 50 µm.

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  Figure 2. Morphology and expression of iPSC markers. (A) iPSCs derived from human myoblasts were expanded and characterized. Expression of undifferentiated cell markers was confirmed by immunostaining with anti-c-myc, anti-TRA, anti-Sox2 and anti-SSEA antibodies. Nuclear dye = DAPI (blue). Scale bar = 250 µm. (B) The in vitro spontaneous differentiation of iPSCs to embryoid bodies (EBs) in suspension in vitro culture was followed by monolayer culture. Cells of ectoderm, endoderm and mesoderm lineages were confirmed by immunostaining with neural class III, β-tubulin (TUJ-1), α-fetoprotein (AFP) and smooth muscle actin (SMA). Nuclear dye = DAPI (blue). Scale bar = 250 µm.

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  Figure 3. Morphology and marker expression in TNNT2/ACTC reporter-modified iPSC line. (A) The phase contrast image showed that modified TNNT2/ACTC iPSCs preserved their typical cell colony morphology; the GFP signal indicated the transduction efficiency. Scale bar = 250 µm. (B) TNNT2/ACTC reporter-modified iPSC line expressed classical undifferentiated cell markers: c-myc, TRA-60, Sox2 and SSEA; nuclear dye = DAPI (blue). Scale bar = 250 µm. (C) TNNT2/ACTC reporter-modified iPSC line spontaneously differentiated in vitro via embryoid bodies (EBs) and expressed ectoderm (neural class III β-tubulin- TUJ-1), endoderm (α-fetoprotein - AFP) and mesoderm (smooth muscle actin - SMA) markers; nuclear dye = DAPI (blue). Scale bar = 250 µm.

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  Figure 4. Determination of TNNT2 copy number in TNNT2/ACTC reporter-modified iPSCs. (A) Representative images of I-FISH signals with complementary probe generated from TNNT2 vector in the cells at passages 34, 37, 40, 43 and 46. As a negative control, reporter unmodified cells were used. Images were taken at 100x magnification. (B) Graphic presentation of I-FISH signals in 50 nuclei for cells at passages 34, 37, 40, 43, and 46. Unmodified cells were used as negative control. Values are given as the mean ± SD, ***p < 0.001. (C) TNNT2 insert copy in modified iPSCs at cell passages 34, 37, 40, 43 and 46, and reporter-unmodified cells were evaluated by ddPCR; values are given as the mean ± SD for ddPCR triplicates, ***p < 0.001. (D) The reporter-modified iPSC line was expanded, and the average insert copy number in the cell population was estimated in cell passages 34, 37, 40, 43 and 46 compared to reporter-unmodified cells by qPCR relative to Albumin; values are given as the mean ± SD for qPCR triplicates; *p < 0.05, ***p < 0.001.

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  Figure 5. Differentiation of TNNT2/ACTC reporter-modified iPSCs at passages 34, 37, 40, 43, and 46 and reporter-unmodified cells towards human cardiomyocytes. (A) iPSCs were differentiated using a PSC protocol. Fluorescence images were taken at the beginning of differentiation (day 0), showing positive GFP signal generated by constitutively expressed mCMV promoter in the TNNT2-luc-T2A-Puro-CMV-GFP plasmid. Images taken at day 4 show emerging mCherry signal (passages 34–46) that became clearly visible at day 10 and increased to the end of experiment (day 14). Higher mCherry and GFP expression levels were found in cell samples at passages 34–40 than in cells at passages 43–36. Unmodified iPSCs were used as a negative control. Scale bar = 250 µm. (B) The level of firefly luminescence measured at day 0 and 4, 10 and 14 days after initiation of differentiation in TNNT2/ACTC reporter-modified cells at passages 34, 37, 40, 43, and 46. A significantly elevated luminescence signal was detected in cells at passage 34 vs. cells at passages 37, 40, 43 and 46 at day 10 and at day 14. Unmodified cells were used as a control. Measurements were performed in triplicate for each cell passage number; values…

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  Figure 6. TNNT2 and ACTC promoter reporter system verification in contracting human cardiomyocytes. (A) Reporter unmodified iPSCs were differentiated into contracting cardiomyocytes (14 d) and were then transduced with TNNT2-luc-T2A-Puro-CMV-GFP and hACTC-mcherry-WPRE-EF1-Neo lentiviruses. The figure shows corresponding images of phase-contrast, mCherry reporting actin (ACTC) expression and GFP signals generated from the constitutive mCMV promoter of the TNNT2-luc-T2A-Puro-CMV-GFP plasmid. Scale bar = 250 µm. (B) Firefly luminescence signal intensity reporting Troponin 2 (TNNT2) expression in TNNT2/ACTC-transduced cardiomyocytes measured at 72 h post transduction vs. unmodified cardiomyocytes. Values are given as the mean ± SD for the triplicates, **p < 0.01. (C) The immunofluorescence staining confirmed the presence of cardiomyocyte markers: ACTC, TNNT2 and cMHC, in reporter unmodified and TNNT2/ACTC reporter-modified cells. Scale bar = 50 µm.