Fig. 6

1 The process of generating cardiomyocytes from iPSCs lacking the dystrophin gene. The figure consists of two parts. In Part A, a diagram depicts the dystrophin gene and the specific mutations found in the cell lines. The UC1015.6 line has a CRISPR-induced mutation that leads to the production of a truncated dystrophin protein without the N terminus. The DMD19 and DMD16 lines are derived from patients and possess nonsense mutations. In Part B, immunostaining is performed on day 30 iPSC-derived cardiomyocytes (iPSC-CMs) to visualize the presence of cTnT (cardiac troponin T) and dystrophin. The UC lines are stained using the MANEX1A antibody, which detects the N terminus of dystrophin. On the other hand, the DMD19 and DMD16 lines are stained using the ab15277 antibody, which recognizes the C terminus of dystrophin. Nuclei are marked in blue using DAPI. The scale bar represents a length of 100 µm. 2 The observed deficiencies in cell size, nuclear size, and sarcomere density of DMD iPSC-CMs (induced pluripotent stem cell-derived cardiomyocytes) on the 30th day of the differentiation process. The figure includes images of immunostaining for cTnT (cardiac troponin T) and DAPI (4',6-diamidino-2-phenylindole) staining for nuclei, comparing UC3.4 and UC1015.6 iPSC-CMs (A), DMD19 iso and DMD19 iPSC-CMs (B), and DMD16 iso and DMD16 iPSC-CMs (C). The scale bar in the images represents a length of 50 µm. Additionally, the figure presents the cell area measurements for UC3.4 and UC1015.6 iPSC-CMs (Figure D), DMD19 iso and DMD19 iPSC-CMs (Figure E), and DMD16 iso and DMD16 iPSC-CMs (F). The nuclear size measurements are provided for UC3.4 and UC1015.6 iPSC-CMs (Figure G), DMD19 iso and DMD19 iPSC-CMs (Figure H), and DMD16 iso and DMD16 iPSC-CMs (I). Furthermore, the figure displays the sarcomere density, quantified by the cTnT signal relative to the cell area, for UC3.4 and UC1015.6 iPSC-CMs (J), DMD19 iso and DMD19 iPSC-CMs (K), and DMD16 iso and DMD16 iPSC-CMs (L). The data were collected from three differentiation experiments, involving a total of 97 to 205 cells. 3 The effect of TRF2 overexpression on rescuing telomere attrition. The shelterin complex, consisting of six subunits, includes TRF1 and TRF2, which directly bind to telomere sequences. To investigate the impact of TRF2, cardiomyocytes were differentiated from iPSCs and transduced with either an empty retroviral vector without an open reading frame (ev) or TRF2 on day 10. Assays were conducted on day 30 of differentiation. Southern blot analysis was performed on telomere restriction fragments of iPSC-derived cardiomyocytes from UC, DMD19, and DMD16. The signal distribution of telomere lengths from the Southern blots is represented in arbitrary units (AU) for UC iPSC-CMs, DMD19 iPSC-CMs, and DMD16 iPSC-CMs. 4 The impact of TRF2 on the DNA damage response and cell survival. The experiment involved transducing cells with either ev (control) or TRF2 on day 10 and assessing them on day 30. The figure presents several Western blot analyses and survival percentages for different cell types. (A) TRF2 levels were analyzed using Western blot, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. TRF2 signal was normalized to the GAPDH signal and measured in arbitrary units (AU). The expected sizes for TRF2 and GAPDH are 65 kDa and 35 kDa, respectively. (B) Western blot analysis of P53 levels normalized to the GAPDH signal in AU. The expected size for P53 is 50 kDa. (C) Western blot analysis of gH2AX levels normalized to the GAPDH signal in AU. The expected size for gH2AX is 17 kDa. (D) Western blot analysis of CHK2 phosphorylated at threonine 68 (phosphor-CHK2) and total CHK2. The signals were normalized to the GAPDH signal in AU. The expected size for phosphor-CHK2 is 62 kDa. (E), (F), and (G) show the percentage of cells that survived on day 40 compared to day 30 of differentiation for UC iPSC-CMs, DMD19 iPSC-CMs, and DMD16 iPSC-CMs, respectively. Survival percentages were determined based on three to five differentiation experiments, with cell numbers ranging from 375 to 12,036 on day 30. 5 The effects of TRF2 on various cellular characteristics, including cell size, nuclear size, and sarcomere density. The experiment involved transducing cells with either the control vector (ev) or TRF2 on day 10 and assessing them on day 30. The images (A), (B), and (C) show immunostaining for cTnT and DAPI staining for nuclei in UC iPSC-CMs, DMD19 iPSC-CMs, and DMD16 iPSC-CMs, respectively. The scale bar represents a length of 20 µm. The area of cells in (D), (E), and (F) represents UC iPSC-CMs, DMD19 iPSC-CMs, and DMD16 iPSC-CMs, respectively. The nuclear size in (G), (H), and (I) corresponds to UC iPSC-CMs, DMD19 iPSC-CMs, and DMD16 iPSC-CMs, respectively. The sarcomere density, indicated by the cTnT signal over the cell area, is depicted in (J), (K), and (L) for UC iPSC-CMs, DMD19 iPSC-CMs, and DMD16 iPSC-CMs, respectively. The cells were evaluated based on three differentiation experiments, with a total of 90 to 230 cells analyzed. Reprinted from [91] with permission from the PNAS