Functionally Competent DNA Damage-free iPS-Derived Cardiomyocytes for Myocardial Repair

Experimental and preclinical evidence demonstrate that cardiac cell transplantation can moderately improve the LV contractile performance of failing hearts.¹ The quality of delivered cells determines the efficiency of remuscularization and improvement of host cardiac function. Cardiac cell transplantation also requires millions of stem cells or their differentiated derivatives. The commonly used strategies for generating large number of cells involves cell propagation under conditions of accelerated growth, which leads to “culture stress”. The lethal form of culture stress is DNA damage. Intracellular repair mechanisms are typically activated in response to such stress to correct damaged DNA. However, rapid propagation and insufficient time allowed for repair processes could lead to accumulation of damaged DNA and eventually reflecting in phenotype changes. These DNA-damaged (Dd) cells are not suitable for cell transplantation and have to be removed from cell preparations. We found that the transcription factor p53 activation by a small molecule in induced pluripotent stem cells (iPSC) selectively induces apoptosis in Dd cells while sparing DNA damage-free (DdF) cells. We tested whether transplanting DdF-iPSCs derived cardiomyocytes (CM) can engraft into an ischemic mouse heart.

The transcription factor p53 is activated in response to a wide range of cellular stresses including DNA damage. Depending on the stimuli, through transactivation of appropriate target genes, p53 can activate cell cycle arrest, cellular senescence or apoptosis in Dd cells.^{2, 3} We activated p53 in iPSCs using 4 μM Nutlin-3a, a MDM2 inhibitor, for 24 hrs. (Fig. 1A). Then the Nutlin-3a and the dead cells were washed off from the culture. The surviving DdF cells were cultured normally and differentiated into cardiomyocytes. As a response to DNA damage, γH2A.X protein accumulates at regions of DNA strand breaks, allowing for the recognition of DNA damage. The localization of γH2A.X at the sites of DNA damage was evident in the Ctrl-iPSCs and Ctrl-CMs, indicating that prolonged culture induces DNA damage in these cells. However, the fraction of γH2A.X cells were significantly reduced in DdF-iPSCs and DdF-CMs (Fig. 1B, C). Fluorescent micrographs of DdF-CMs are shown in figure 1D. To determine the engraftment efficiency of DdF-CMs, animal study was performed as outlined in figure 1A. Animal experimental protocol was approved by IACUC, UAB and performed in accordance with the NIH publication No 85–23. After coronary artery ligation in SCID mice, 9×10⁵ iPSC-CMs were injected into the border zone of LV. Following transplantation, cell engraftment was monitored by luciferase BLI assay at weeks 1 & 4. At week-1, no significant difference in cell engraftment was observed between Ctrl-CM and DdF-CM transplanted groups. However, at week-4 there was a significant difference between the two groups (Fig. 1E). At week-4, the hearts were perfused, harvested and fixed in 4% paraformaldehyde. Cardiac sections of 10 μm thickness were made and transplanted iPSC-CM were identified by hcTnT immunolabeling (Fig. 1F). Cells that expressed hcTnT were counted in every tenth serial section of the whole heart from base to apex, and the total was multiplied by 10 to obtain the number of engrafted iPSC-CMs per heart. The engraftment rate was calculated by dividing the total number of engrafted iPSC-CMs by the number of injected cells and expressed as a percentage. Engraftment of iPSC-CMs were found in both treatment groups, however, a significantly higher engraftment rate was found in mouse hearts that received DdF-CMs (Fig. 1F, lower right). Importantly, the confocal images showed that engrafted DdF-CMs displayed striated muscle phenotype (Fig. 1F, lower left), a CM maturation phenotype.

Figure 1. Enhanced engraftment of DNA damage-free iPS derived cardiomyocytes in ischemic mouse hearts.

A, Schematics of DNA damage-free cell selection by p53 stabilization and in vivo experiment design. DdF-iPSCs and Ctrl-iPSCs were differentiated into cardiomyocytes and transplanted into the border zone of myocardial infarcted mice. B, C, γH2A.X in nuclei of iPSCs (B) and CMs (C) stained by DAPI. F-actin in iPSCs were labeled by phalloidin (B, scale bar = 40 μm). Cardiac troponin T (hcTnT) in CMs are shown as red (C, scale bar = 50 μm). Respective magnified area under the white box are shown in the left panels (B, scale bar = 5 μm; C, 10 μm). Fraction of cells positive for γH2A.X are shown in the right panels (n = 3). Data are mean ± s.d. *P < 0.05. D, Confocal micrograph of DdF-CM immunolabled for human specific cardiac troponin T (hcTnT, red) and alpha-sarcomeric actin (αSA, green), and nuclei counter stained with DAPI (blue) at sixty days after differentiation. Higher magnification of the area under the box in left panel (Scale bar = 40 μm) is shown in the middle panel (Scale bar = 10 μm). Higher magnification of the area under the box in middle panel is shown in the right panel (Scale bar = 2.5 μm). E, In vivo bioluminescence imaging of engrafted cardiomyocytes at week one (upper panels) and week four (lower panels) in Sham, MI, Ctrl-CM and DdF-CM groups. Cardiomyocyte engraftment as measured by total photon counts is shown in the graph (right panel). n = 5 to 7 mice in each group at week one, n = 5 to 6 mice in each group at week four. Data are mean ± s.d. *P < 0.05 vs. MI, ^#P < 0.05 vs. Ctrl-CM. F, Immunolabeling of human specific cardiac troponin T (hcTnT; red, all panels), cardiac troponin T (cTnT; green, right upper panel), cell membrane (WGA; white, left upper panel) and nuclei counter stained with DAPI (blue) in DdF-CM transplanted mice heart section (left upper panel, scale = 500 μm). Left lower panel, engrafted DdF-CMs displaying cardiac striations (scale = 20 μm). Right lower panel, engraftment rate expressed as the fraction of human specific cardiac troponin T positive cardiomyocytes in mice that received Ctrl-CMs and DdF-CMs. Total number of hcTnT positive cells were counted from every ten 10 μm sections from base to apex then divided by total number of cells transplanted. n = 5 in each group. Data are mean ± s.d. Significance (P < 0.05) was determined via the Student’s t-test for comparisons between two groups and via two-way ANOVA with Tukey’s multiple comparisons test for panel E.

DNA damaged senescent cells do not undergo cell death. Instead, they remain within the tissue, gaining altered functions, and changing the tissue microenvironments which can promote aging phenotypes of other cell.³ The DNA intercalating agent Doxorubicin, an effective and frequently used chemotherapeutic drug, induces cardiomyopathy in patients through its DNA damage property.⁴ Aerobic-respiration-mediated oxidative DNA damage regulates the proliferation of pre-existing cardiomyocytes in neonatal and adult mice hearts.⁵ All these studies iterate the impact of DNA damage on the heart and in heart diseases. Our study shows that in vitro cell propagation induces DNA damage and transplanting them into mice models supported a mere engraftment. Previously, we have demonstrated that hypoxia precondition improves mesenchymal stem cell transplantation in non-human primates without increasing arrhythmogenic complications.¹ Here, we show that transplanting DdF-CMs results in a significantly increased engraftment rate. To our knowledge, this is the first study to show that DdF-iPSCs can be selected by p53 activation in iPSC cultures and DdF-CMs have enhanced cardiac engraftment potential. Importantly, this robust approach to select functionally competent, intact-DNA cells from a heterogeneous population can be easily adopted in clinical setups. In clinics, before transplanting cells to regenerate the injured myocardium p53 can be activated in the cells and DdF cells can be isolated and transplanted. These DdF cells would better repopulate the ischemic myocardium and improve the performance of a failing heart.