Doxorubicin (Adriamycin) is an anthracycline chemotherapy agent effective in treating a wide range of malignancies with a well–established dose–response cardiotoxic side effect that can lead to heart failure. At present, it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). Here we demonstrate that patient–specific human induced pluripotent stem cell–derived cardiomyocytes (hiPSC–CMs) can recapitulate individual patients’ predilection to DIC at the single cell level. hiPSC–CMs derived from breast cancer patients who suffered clinical DIC are consistently more sensitive to doxorubicin toxicity, demonstrating decreased cell viability, mitochondrial and metabolic function, calcium handling, and antioxidant pathway activity, along with increased reactive oxygen species (ROS) production compared to hiPSC–CMs from patients who did not experience DIC. Together, our data indicate that hiPSC–CMs are a suitable platform for identifying and verifying the genetic basis and molecular mechanisms of DIC.

Background Despite the promise shown by stem cells for restoration of cardiac function following myocardial infarction (MI), the poor survival of transplanted cells has been a major issue. Hypoxia inducible factor-1 (HIF-1) is a transcription factor that mediates adaptive responses to ischemia. Here we hypothesize that co-delivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF-1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs. Methods and Results Following MI, CPCs were co-delivered intramyocardially into adult NOD/SCID mice with either saline, MC-GFP, or MC-HIF1 versus MC-HIF1 alone (N=10/group). Bioluminescence imaging (BLI) demonstrated better survival when CPCs were co-delivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs + MC-HIF1 had the highest ejection fraction 6 weeks post-MI (57.1±2.6%) followed by MC-HIF1 alone (48.5±2.6%), with no significant protection for CPCs + MC-GFP (44.8±3.3%) compared to saline control (38.7±3.2%, P<0.05). In vitro mechanistic studies confirmed that cardiac endothelial cells (ECs) produced exosomes which were actively internalized by recipient CPCs. Exosomes purified from ECs overexpressing HIF-1 had higher contents of miR-126 and miR-210. These microRNAs activated pro-survival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes. Conclusions In summary, HIF-1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.

Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and was associated with perturbed transforming growth factor beta (TGFβ) signaling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGFβ signaling. TBX20 regulates the expression of TGFβ signaling modifiers including a known genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGFβ signaling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.

We have demonstrated that acute HIF-1α stabilization using either a pharmacological or genetic approach protected the heart against acute IRI by promoting aerobic glycolysis, decreasing mitochondrial oxidative stress, activating HKII, and inhibiting MPTP opening.

Background Human induced pluripotent stem cells (iPSCs) are attractive candidates for therapeutic use, with the potential to replace deficient cells and to improve functional recovery in injury or disease settings. Here we test the hypothesis that human iPSC-derived cardiomyocytes (iPSC-CMs) can secrete cytokines as a molecular basis to attenuate adverse cardiac remodeling after myocardial infarction (MI). Methods and Results Human iPSCs were generated from skin fibroblasts and differentiated in vitro using a small molecule based protocol. Troponin+ iPSC-CMs were confirmed by immunohistochemistry, quantitative PCR, fluorescence activated cell sorting (FACS), and electrophysiological measurements. Afterwards, 2×106 iPSC-CMs derived from a cell line transduced with a vector expressing firefly luciferase and GFP were transplanted into adult NOD/SCID mice with acute left anterior descending (LAD) ligation. Control animals received PBS injection. Bioluminescence imaging (BLI) showed limited engraftment upon transplantation into ischemic myocardium. However, magnetic resonance imaging (MRI) of animals transplanted with iPSC-CMs showed significant functional improvement and attenuated cardiac remodeling when compared to PBS-treated control animals at day 35 (Ejection fraction: 24.5±1.3 vs. 14.5±1.5%; P<0.05). To understand the underlying molecular mechanism, microfluidic single cell profiling of harvested iPSC-CMs, laser capture microdissection (LCM) of host myocardium, and in vitro ischemia stimulation were used to demonstrate that the iPSC-CMs could release significant levels of pro-angiogenic and anti-apoptotic factors in the ischemic microenvironment. Conclusions Transplantation of human iPSC-CMs into an acute mouse MI model can improve left ventricular function and attenuate cardiac remodeling. Because of limited engraftment, most of the effects are possibly explained by paracrine activity of these cells.

Patient-specific pluripotent stem cells (PSCs) can be generated via nuclear reprogramming by transcription factors (i.e., induced pluripotent stem cells, iPSCs) or by somatic cell nuclear transfer (SCNT). However, abnormalities and preclinical application of differentiated cells generated by different reprogramming mechanisms have yet to be evaluated. Here we investigated the molecular and functional features, and drug response of cardiomyocytes (PSC-CMs) and endothelial cells (PSC-ECs) derived from genetically relevant sets of human iPSCs, SCNT-derived embryonic stem cells (nt-ESCs), as well as in vitro fertilization embryo-derived ESCs (IVF-ESCs). We found that differentiated cells derived from isogenic iPSCs and nt-ESCs showed comparable lineage gene expression, cellular heterogeneity, physiological properties, and metabolic functions. Genome-wide transcriptome and DNA methylome analysis indicated that iPSC derivatives (iPSC-CMs and iPSC-ECs) were more similar to isogenic nt-ESC counterparts than those derived from IVF-ESCs. Although iPSCs and nt-ESCs shared the same nuclear DNA and yet carried different sources of mitochondrial DNA, CMs derived from iPSC and nt-ESCs could both recapitulate doxorubicin-induced cardiotoxicity and exhibited insignificant differences on reactive oxygen species generation in response to stress condition. We conclude that molecular and functional characteristics of differentiated cells from human PSCs are primarily attributed to the genetic compositions rather than the reprogramming mechanisms (SCNT vs. iPSCs). Therefore, human iPSCs can replace nt-ESCs as alternatives for generating patient-specific differentiated cells for disease modeling and preclinical drug testing.

Background Both human embryonic stem cell-derived cardiomyocytes (ESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can serve as unlimited cell sources for cardiac regenerative therapy. However, the functional equivalency between human ESC-CMs and iPSC-CMs for cardiac regenerative therapy has not been demonstrated. Here we performed a head-to-head comparison of ESC-CMs and iPSC-CMs in their ability to restore cardiac function in a rat myocardial infarction (MI) model as well as their exosomal secretome. Methods and Results Human ESCs and iPSCs were differentiated into cardiomyocytes using small molecule inhibitors. Fluorescence-activated cell sorting (FACS) analysis confirmed ~85% and ~83% of CMs differentiated from ESCs and iPSCs, respectively, were positive for cardiac troponin T. At a single-cell level, both cell types displayed similar calcium handling and electrophysiological properties, with gene expression comparable to the human fetal heart marked by striated sarcomeres. Sub-acute transplantation of ESC-CMs and iPSC-CMs into nude rats post-MI improved cardiac function, which was associated with increased expression of angiogenic genes in vitro following hypoxia. Profiling of exosomal microRNAs (miRs) and long non-coding RNAs (lncRNAs) revealed that both groups contain an identical repertoire of miRs and lncRNAs, including some that are known to be cardioprotective. Conclusions We demonstrate for the first time that both ESC-CMs and iPSC-CMs can facilitate comparable cardiac repair. This is advantageous because unlike allogeneic ESC-CMs used in therapy, autologous iPSC-CMs could potentially avoid immune rejection when used for cardiac cell transplantation in the future.

Objectives To evaluate whether radiation exposure from cardiac computed tomographic angiography is associated with DNA damage and whether damage leads to programmed cell death and activation of genes involved in apoptosis and DNA repair. Background Exposure to radiation from medical imaging has become a public health concern, but whether it causes significant cell damage remains unclear. Methods We conducted a prospective cohort study in 67 patients undergoing cardiac computed tomographic angiography (CTA) between January 2012 and December 2013 in two US medical centers. Median blood radiation exposure was estimated using phantom dosimetry. Biomarkers of DNA damage and apoptosis were measured by flow cytometry, whole genome sequencing, and single cell polymerase chain reaction. Results The median DLP was 1535.3 mGy·cm (969.7 – 2674.0 mGy·cm). The median radiation dose to the blood was 29.8 milliSieverts (18.8 – 48.8 mSv). Median DNA damage increased 3.39% (1.29 – 8.04%, P<0.0001) post-radiation. Median apoptosis increased 3.1-fold (1.4 – 5.1-fold, P<0.0001) post-radiation. Whole genome sequencing revealed changes in the expression of 39 transcription factors involved in the regulation of apoptosis, cell cycle, and DNA repair. Genes involved in mediating apoptosis and DNA repair were significantly changed post-radiation, including DDB2 [1.9-fold (1.5 – 3.0-fold), P<0.001], XRCC4 [3.0-fold (1.1 – 5.4-fold), P=0.005], and BAX [1.6-fold (0.9 – 2.6-fold), P<0.001]. Exposure to radiation was associated with DNA damage [OR: 1.8 (1.2 – 2.6), P=0.003]. DNA damage was associated with apoptosis [OR: 1.9 (1.2 – 5.1), P<0.0001] and gene activation [OR: 2.8 (1.2 – 6.2), P=0.002]. Conclusions Patients exposed to radiation from cardiac CTA had evidence of DNA damage, which was associated with programmed cell death and activation of genes involved in apoptosis and DNA repair.