Nelson, Timothy J

Rationale : Induced pluripotent stem cells (iPS) allow derivation of pluripotent progenitors from somatic sources. Originally, iPS were induced by a stemness-related gene set that included the c-MYC oncogene. Objective : Here, we determined from embryo to adult the cardiogenic proficiency of iPS programmed without c-MYC, a cardiogenicity-associated transcription factor. Methods and Results : Transgenic expression of 3 human stemness factors SOX2, OCT4, and KLF4 here reset murine fibroblasts to the pluripotent ground state. Transduction without c-MYC reversed cellular ultrastructure into a primitive archetype and induced stem cell markers generating 3-germ layers, all qualifiers of acquired pluripotency. Three-factor induced iPS (3F-iPS) clones reproducibly demonstrated cardiac differentiation properties characterized by vigorous beating activity of embryoid bodies and robust expression of cardiac Mef2c, α-actinin, connexin43, MLC2a, and troponin I. In vitro isolated iPS-derived cardiomyocytes demonstrated functional excitation-contraction coupling. Chimerism with 3F-iPS derived by morula-stage diploid aggregation was sustained during prenatal heart organogenesis and contributed in vivo to normal cardiac structure and overall performance in adult tumor-free offspring. Conclusions : Thus, 3F-iPS bioengineered without c-MYC achieve highest stringency criteria for bona fide cardiogenesis enabling reprogrammed fibroblasts to yield de novo heart tissue compatible with native counterpart throughout embryological development and into adulthood.

Background— Nuclear reprogramming provides an emerging strategy to produce embryo-independent pluripotent stem cells from somatic tissue. Induced pluripotent stem cells (iPS) demonstrate aptitude for de novo cardiac differentiation, yet their potential for heart disease therapy has not been tested. Methods and Results— In this study, fibroblasts transduced with human stemness factors OCT3/4, SOX2, KLF4, and c-MYC converted into an embryonic stem cell–like phenotype and demonstrated the ability to spontaneously assimilate into preimplantation host morula via diploid aggregation, unique to bona fide pluripotent cells. In utero, iPS-derived chimera executed differentiation programs to construct normal heart parenchyma patterning. Within infarcted hearts in the adult, intramyocardial delivery of iPS yielded progeny that properly engrafted without disrupting cytoarchitecture in immunocompetent recipients. In contrast to parental nonreparative fibroblasts, iPS treatment restored postischemic contractile performance, ventricular wall thickness, and electric stability while achieving in situ regeneration of cardiac, smooth muscle, and endothelial tissue. Conclusions— Fibroblasts reprogrammed by human stemness factors thus acquire the potential to repair acute myocardial infarction, establishing iPS in the treatment of heart disease.

Abstract Stem cells are an emerging strategy for treatment of myocardial infarction, limited however to postinjury intervention. Preventive stem cell-based therapy to augment stress tolerance has yet to be considered for lifelong protection. Here, pluripotent stem cells were microsurgically introduced at the blastocyst stage of murine embryo development to ensure stochastic integration and sustained organ contribution. Engineered chimera displayed excess in body weight due to increased fat deposits, but were otherwise devoid of obesity-related morbidity. Remarkably, and in sharp contrast to susceptible nonchimeric offspring, chimera was resistant to myocardial infarction induced by permanent coronary occlusion. Infarcted nonchimeric adult hearts demonstrated progressive deterioration in ejection fraction, while age-matched 12–14-months-old chimera recovered from equivalent ischemic insult to regain within one-month preocclusion contractile performance. Electrical remodeling and ventricular enlargement with fibrosis, prominent in failing nonchimera, were averted in the chimeric cohort characterized by an increased stem cell load in adipose tissue and upregulated markers of biogenesis Ki67, c-Kit, and stem cell antigen-1 in the myocardium. Favorable outcome in infarcted chimera translated into an overall benefit in workload capacity and survival. Thus, prenatal stem cell transplant yields a cardioprotective phenotype in adulthood, expanding cell-based indications beyond traditional postinjury applications to include pre-emptive therapy. Disclosure of potential conflicts of interest is found at the end of this article.

KCNJ11 -encoded Kir6.2 assembles with ATP-binding cassette sulphonylurea receptors to generate ATP-sensitive K + (K ATP ) channel complexes. Expressed in tissues with dynamic metabolic flux, these evolutionarily conserved yet structurally and functionally unique heteromultimers serve as high-fidelity rheostats that adjust membrane potential-dependent cell functions to match energetic demand. Genetic defects in channel subunits disrupt the cellular homeostatic response to environmental stress, compromising organ tolerance in the adult. As maladaptation characterizes malignant K ATP channelopathies, establishment of platforms to examine progression of K ATP channel-dependent adaptive behaviour is warranted. Chimeras provide a powerful tool to assay the contribution of genetic variance to stress intolerance during prenatal or post-natal development. Here, KCNJ11 K ATP channel gene knockout↔wild-type chimeras were engineered through diploid aggregation. Integration of wild-type embryonic stem cells into zona pellucida-denuded morula derived from knockout embryos achieved varying degrees of incorporation of stress-tolerant tissue within the K ATP channel-deficient background. Despite the stress-vulnerable phenotype of the knockout, ex vivo derived mosaic blastocysts tolerated intrauterine transfer and implantation, followed by full-term embryonic development in pseudopregnant surrogates to produce live chimeric offspring. The development of adult chimerism from the knockout↔wild-type mosaic embryo offers thereby a new paradigm to probe the ecogenetic control of the K ATP channel-dependent stress response.

Abstract Pathogenic causes underlying nonischemic cardiomyopathies are increasingly being resolved, yet repair therapies for these commonly heritable forms of heart failure are lacking. A case in point is human dilated cardiomyopathy 10 (CMD10; Online Mendelian Inheritance in Man #608569), a progressive organ dysfunction syndrome refractory to conventional therapies and linked to mutations in cardiac ATP-sensitive K+ (KATP) channel subunits. Embryonic stem cell therapy demonstrates benefit in ischemic heart disease, but the reparative capacity of this allogeneic regenerative cell source has not been tested in inherited cardiomyopathy. Here, in a Kir6.2-knockout model lacking functional KATP channels, we recapitulated under the imposed stress of pressure overload the gene-environment substrate of CMD10. Salient features of the human malignant heart failure phenotype were reproduced, including compromised contractility, ventricular dilatation, and poor survival. Embryonic stem cells were delivered through the epicardial route into the left ventricular wall of cardiomyopathic stressed Kir6.2-null mutants. At 1 month of therapy, transplantation of 200,000 cells per heart achieved teratoma-free reversal of systolic dysfunction and electrical synchronization and halted maladaptive remodeling, thereby preventing end-stage organ failure. Tracked using the lacZ reporter transgene, stem cells engrafted into host heart. Beyond formation of cardiac tissue positive for Kir6.2, transplantation induced cell cycle activation and halved fibrotic zones, normalizing sarcomeric and gap junction organization within remuscularized hearts. Improved systemic function induced by stem cell therapy translated into increased stamina, absence of anasarca, and benefit to overall survivorship. Embryonic stem cells thus achieve functional repair in nonischemic genetic cardiomyopathy, expanding indications to the therapy of heritable heart failure. Disclosure of potential conflicts of interest is found at the end of this article.

Abstract Pluripotent stem cells demonstrate an inherent propensity for unrestricted multi-lineage differentiation. Translation into regenerative applications requires identification and isolation of tissue-specified progenitor cells. From a comprehensive pool of 11,272 quality-filtered genes, profiling embryonic stem cells at discrete stages of cardiopoiesis revealed 736 transcripts encoding membrane-associated proteins, where 306 were specifically upregulated with cardiogenic differentiation. Bioinformatic dissection of exposed surface biomarkers prioritized the chemokine receptor cluster as the most significantly over-represented gene receptor family during pre cardiac induction, with CXCR4 uniquely associated with mesendoderm formation. CXCR4+ progenitors were sorted from the embryonic stem cell pool into mesoderm-restricted progeny according to co-expression with the early mesoderm marker Flk-1. In contrast to CXCR4−/Flk-1− cells, the CXCR4+/Flk-1+ subpopulation demonstrated overexpressed cardiac lineage transcription factors (Mef2C, Myocardin, Nkx2.5), whereas pluripotent genes (Oct4, Fgf4, Sox2) as well as neuroectoderm (Sox1) and endoderm alpha-fetoprotein markers were all depleted. In fact, the CXCR4+/Flk-1+ biomarker combination identified embryonic stem cell progeny significantly enriched with Mesp-1, GATA-4, and Tbx5, indicative of pre cardiac mesoderm and the primary heart field. Although the CXCR4+/Flk-1+ transcriptome shared 97% identity with the CXCR4−/Flk-1− counterpart, the 818 divergent gene set represented predominantly cardiovascular developmental functions and formed a primitive cardiac network. Differentiation of CXCR4+/Flk-1+ progenitors yielded nuclear translocation of myocardial transcription factors and robust sarcomerogenesis with nascent cardiac tissue demonstrating beating activity and calcium transients. Thus, the CXCR4/Flk-1 biomarker pair predicts the emergence of cardiogenic specification within a pluripotent stem cell pool, enabling targeted selection of cardiopoietic lineage. Disclosure of potential conflicts of interest is found at the end of this article.