Human embryonic stem (hES) cells are pluripotent stem cells capable of self-renewal and differentiation into virtually all cell types. Thus, they hold tremendous potential as cell sources for regenerative therapies. The concurrent development of accurate, sensitive, and noninvasive technologies capable of monitoring hES cells engraftment in vivo can greatly expedite basic research prior to future clinical translation. In this study, hES cells were stably transduced with a lentiviral vector carrying a novel double-fusion reporter gene that consists of firefly luciferase and enhanced green fluorescence protein. Reporter gene expression had no adverse effects on cell viability, proliferation, or differentiation to endothelial cells (human embryonic stem cell-derived endothelial cells [hESC-ECs]). To compare the two popular imaging modalities, hES cells and hESC-ECs were then colabeled with superparamagnetic iron oxide particles before transplantation into murine hind limbs. Longitudinal magnetic resonance (MR) imaging showed persistent MR signals in both cell populations that lasted up to 4 weeks. By contrast, bioluminescence imaging indicated divergent signal patterns for hES cells and hESC-ECs. In particular, hESC-ECs showed significant bioluminescence signals at day 2, which decreased progressively over the following 4 weeks, whereas bioluminescence signals from undifferentiated hES cells increased dramatically during the same period. Post-mortem histology and immunohistochemistry confirmed teratoma formation after injection of undifferentiated hES cells but not hESC-ECs. From these data taken together, we concluded that reporter gene is a better marker for monitoring cell viability, whereas iron particle labeling is a better marker for high-resolution detection of cell location by MR. Furthermore, transplantation of predifferentiated rather than undifferentiated hES cells would be more suited for avoiding teratoma formation. Disclosure of potential conflicts of interest is found at the end of this article.

Rationale:: Cardiac myocytes derived from pluripotent stem cells have demonstrated the potential to mitigate damage of the infarcted myocardium and improve left ventricular ejection fraction. However, the mechanism underlying the functional benefit is unclear. Objective:: To evaluate whether the transplantation of cardiac-lineage differentiated derivatives enhance myocardial viability and restore left ventricular ejection fraction more effectively than undifferentiated pluripotent stem cells after a myocardial injury. Herein, we utilize novel multimodality evaluation of human embryonic stem cells (hESCs), hESC-derived cardiac myocytes (hCMs), human induced pluripotent stem cells (iPSCs), and iPSC-derived cardiac myocytes (iCMs) in a murine myocardial injury model. Methods and Results:: Permanent ligation of the left anterior descending coronary artery was induced in immunosuppressed mice. Intramyocardial injection was performed with (1) hESCs (n=9), (2) iPSCs (n=8), (3) hCMs (n=9), (4) iCMs (n=14), and (5) PBS control (n=10). Left ventricular ejection fraction and myocardial viability, measured by cardiac magnetic resonance imaging and manganese-enhanced magnetic resonance imaging, respectively, was significantly improved in hCM- and iCM-treated mice compared with pluripotent stem cell- or control-treated mice. Bioluminescence imaging revealed limited cell engraftment in all treated groups, suggesting that the cell secretions may underlie the repair mechanism. To determine the paracrine effects of the transplanted cells, cytokines from supernatants from all groups were assessed in vitro. Gene expression and immunohistochemistry analyses of the murine myocardium demonstrated significant upregulation of the promigratory, proangiogenic, and antiapoptotic targets in groups treated with cardiac lineage cells compared with pluripotent stem cell and control groups. Conclusions:: This study demonstrates that the cardiac phenotype of hCMs and iCMs salvages the injured myocardium effectively than undifferentiated stem cells through their differential paracrine effects.

Cardiovascular disease (CVD) is the leading cause of death in modern society. The adult heart innately lacks the capacity to repair and regenerate the damaged myocardium from ischemic injury. Limited understanding of cardiac tissue repair process hampers the development of effective therapeutic solutions to treat CVD such as ischemic cardiomyopathy. In recent years, rapid emergence of induced pluripotent stem cells (iPSC) and iPSC-derived cardiomyocytes presents a valuable opportunity to replenish the functional cells to the heart. The therapeutic effects of iPSC-derived cells have been investigated in many preclinical studies. However, the underlying mechanisms of iPSC-derived cell therapy are still unclear, and limited engraftment of iPSC-derived cardiomyocytes is well known. One facet of their mechanism is the paracrine effect of the transplanted cells. Microvesicles such as exosomes secreted from the iPSC-derived cardiomyocytes exert protective effects by transferring the endogenous molecules to salvage the injured neighboring cells by regulating apoptosis, inflammation, fibrosis, and angiogenesis. In this review, we will focus on the current advances in the exosomes from iPSC derivatives and discuss their therapeutic potential in the treatment of CVD.

Background: Induced pluripotent stem cells and their differentiated cardiomyocytes ( iCM s) have tremendous potential as patient‐specific therapy for ischemic cardiomyopathy following myocardial infarctions, but difficulties in viable transplantation limit clinical translation. Exosomes secreted from iCM s (iCM‐Ex) can be robustly collected in vitro and injected in lieu of live iCM s as a cell‐free therapy for myocardial infarction. Methods and Results: iCM ‐Ex were precipitated from iCM supernatant and characterized by protein marker expression, nanoparticle tracking analysis, and functionalized nanogold transmission electron microscopy. iCM ‐Ex were then used in in vitro and in vivo models of ischemic injuries. Cardiac function in vivo was evaluated by left ventricular ejection fraction and myocardial viability measurements by magnetic resonance imaging. Cardioprotective mechanisms were studied by JC ‐1 (tetraethylbenzimidazolylcarbocyanine iodide) assay, immunohistochemistry, quantitative real‐time polymerase chain reaction, transmission electron microscopy, and immunoblotting. iCM ‐Ex measured ≈140 nm and expressed CD 63 and CD 9. iCM and iCM ‐Ex micro RNA profiles had significant overlap, indicating that exosomal content was reflective of the parent cell. Mice treated with iCM ‐Ex demonstrated significant cardiac improvement post–myocardial infarction, with significantly reduced apoptosis and fibrosis. In vitro iCM apoptosis was significantly reduced by hypoxia and exosome biogenesis inhibition and restored by treatment with iCM ‐Ex or rapamycin. Autophagosome production and autophagy flux was upregulated in iCM ‐Ex groups in vivo and in vitro. Conclusions: iCM‐Ex improve post–myocardial infarction cardiac function by regulating autophagy in hypoxic cardiomyoytes, enabling a cell‐free, patient‐specific therapy for ischemic cardiomyopathy.

Cardiovascular disease is the leading cause of death worldwide today and numerous studies are demonstrating that exosomes improve cardiac function after myocardial injury through various mechanisms. Exosome therapy has great potential as an effective precision medicine biologic by targeting the underlying disease process. However, this innovative approach may face some challenges, such as zeta potential, standardization of exosome collection, biopharmaceutical regulation, and more importantly, specific clinical application of exosome therapy. These issues will be addressed by broadly summarizing the biological plausibility; delivery, dosing, and pharmacokinetics; and reproducibility and manufacturing with a focus on microRNA as molecular cargo.