Citations

The advent of induced pluripotent stem cell (iPSC) technology creates new opportunities for transplant-based therapeutic strategies. The potential for clinical translation is currently hindered by the risk of dysregulated cell growth. Pluripotent stem cells reprogrammed by three-factor (Sox2, Klf, and Oct4) and four-factor (Sox2, Klf, Oct4, and c-Myc) strategies result in the capacity for teratogenic growth from residual pluripotent progeny upon in vivo transplantation. However, these pluripotent stem cells also have a stage-specific hypersensitivity to DNA-damaging agents that may allow separation of lineage-specific therapeutic subpopulation of cells. We aimed to demonstrate the selective effect of DNA topoisomerase II inhibitor, etoposide, in eliminating pluripotent cells in the early cardiac progenitor population thus decreasing the effect of teratoma formation. Immunodeficient murine hearts were infarcted and received implantation of a therapeutic dose of cardiac progenitors derived from partially differentiated iPSCs. Etoposide-treated cell implantation reduced mass formation in the intracardiac and extracardiac chest cavity compared with the same dose of iPSC-derived cardiac progenitors in the control untreated group. In vivo bioluminescence imaging confirmed the localization and engraftment of transplanted cells in the myocardium postinjection in both groups. Comparatively, the equivalent cell population without etoposide treatment demonstrated a greater incidence and size of teratoma formation. Hence, pretreatment with genotoxic etoposide significantly lowered the threat of teratogenicity by purging the contaminating pluripotent cells, establishing an adjunctive therapy to further harness the clinical value of iPSC-derived cardiac regeneration.

Mammalian heart development is built on highly conserved molecular mechanisms with polygenetic perturbations resulting in a spectrum of congenital heart diseases (CHD). However, knowledge of cardiogenic ontogeny that regulates proper cardiogenesis remains largely based on candidate-gene approaches. Mapping the dynamic transcriptional landscape of cardiogenesis from a genomic perspective is essential to integrate the knowledge of heart development into translational applications that accelerate disease discovery efforts toward mechanistic-based treatment strategies. Herein, we designed a time-course transcriptome analysis to investigate the genome-wide dynamic expression landscape of innate murine cardiogenesis ranging from embryonic stem cells to adult cardiac structures. This comprehensive analysis generated temporal and spatial expression profiles, revealed stage-specific gene functions, and mapped the dynamic transcriptome of cardiogenesis to curated pathways. Reconciling known genetic underpinnings of CHD, we deconstructed a disease-centric dynamic interactome encoded within this cardiogenic atlas to identify stage-specific developmental disturbances clustered on regulation of epithelial-to-mesenchymal transition (EMT), BMP signaling, NF-AT signaling, TGFb-dependent EMT, and Notch signaling. Collectively, this cardiogenic transcriptional landscape defines the time-dependent expression of cardiac ontogeny and prioritizes regulatory networks at the interface between health and disease.

Abstract The value of induced pluripotent stem cells (iPSCs) within regenerative medicine is contingent on predictable and consistent iPSC differentiation. However, residual influence of the somatic origin or reprogramming technique may variegate differentiation propensity and confound comparative genotype/phenotype analyses. The objective of this study was to define quality control measures to select iPSC clones that minimize the influence of somatic origin on differentiation propensity independent of the reprogramming strategy. More than 60 murine iPSC lines were derived from different fibroblast origins (embryonic, cardiac, and tail tip) via lentiviral integration and doxycycline-induced transgene expression. Despite apparent equivalency according to established iPSC histologic and cytomorphologic criteria, clustering of clonal variability in pluripotency-related gene expression identified transcriptional outliers that highlighted cell lines with unpredictable cardiogenic propensity. Following selection according to a standardized gene expression profile calibrated by embryonic stem cells, the influence of somatic origin on iPSC methylation and transcriptional patterns was negated. Furthermore, doxycycline-induced iPSCs consistently demonstrated earlier differentiation than lentiviral-reprogrammed lines using contractile cardiac tissue as a measure of functional differentiation. Moreover, delayed cardiac differentiation was predominately associated with upregulation in pluripotency-related gene expression upon differentiation. Starting from a standardized pool of iPSCs, relative expression levels of two pluripotency genes, Oct4 and Zfp42, statistically correlated with enhanced cardiogenicity independent of somatic origin or reprogramming strategy (R2 = 0.85). These studies demonstrate that predictable iPSC differentiation is independent of somatic origin with standardized gene expression selection criteria, while the residual impact of reprogramming strategy greatly influences predictable output of tissue-specification required for comparative genotype/phenotype analyses. Stem Cells 2014;32:2350–2359

Regenerative medicine offers a curative approach to treating heart disease through multiple emerging therapeutic concepts. Decellularized organ scaffolds are being optimized to guide and spatially organize stem cell differentiation in efforts to rebuild functional tissues. Additionally, pluripotent stem cells offer a transformative cell source to differentiate into the full spectrum of cellular building blocks. Adult cardiac tissues have been used as extracellular scaffolds as a proof of principle; however, matching the developmental stages of embryonic scaffold with primitive cardiac progenitors may be used to optimize the differentiation and maturation of bioengineered cardiac tissues. Our novel approach uses embryo-derived decellularized hearts as scaffolds to promote embryonic stem cell differentiation. Further, we determined that agitation with 0.25% sodium dodecyl sulfate (SDS) solution was the most effective protocol to maintain matrix integrity while eliminating endogenous cells. The scaffolds were successfully reseeded with different cellular sources derived from pluripotent stem cells to achieve beating cardiac tissues characterized by endothelial, cardiac, and smooth muscle markers. Therefore, embedding stem cells within a tissue-specific environment matched to the developmental stage of the progenitors may offer a practical solution for stem-cell-derived applications such as disease modeling, pharmaceutical safety testing, and screening of novel therapeutic targets.

Contraction regulates heart development via a complex mechanotransduction process controlled by various mechanical forces. Here, we exploit zebrafish embryos as an in vivo animal model to discern the contribution from different mechanical forces and identify the underlying mechanotransductive signaling pathways of cardiogenesis. We treated 2 days postfertilization zebrafish embryos with Blebbistatin, a myosin II inhibitor, to stop cardiac contraction, which induces a response termed cessation of contraction-induced cardiomyocyte (CM) enlargement (CCE). Accompanying the CCE, lateral fusion of myofibrils was attenuated within CMs. The CCE can be blunted by loss of blood in tail-docked zebrafish but not in cloche mutant fish, suggesting that transmural pressure rather than shear stress is accountable for the chamber enlargement. By screening a panel of small molecule inhibitors, our data suggested essential functions of phosphoinositide 3-kinase signaling and protein synthesis in CCE, which are independent of the sarcomere integrity. In summary, we defined a unique CCE response in genetically tractable zebrafish embryos. A panel of assays was established to verify the contribution from extrinsic forces and interrogate underlying signaling pathways.

Background Contractile discordance exacerbates cardiac dysfunction, aggravating heart failure outcome. Dissecting the genesis of mechanical dyssynchrony would enable an early diagnosis before advanced disease. Methods and Results High‐resolution speckle‐tracking echocardiography was applied in a knockout murine surrogate of adult‐onset human cardiomyopathy caused by mutations in cardioprotective ATP‐sensitive K + (K ATP ) channels. Preceding the established criteria of cardiac dyssynchrony, multiparametric speckle‐based strain resolved nascent erosion of dysfunctional regions within cardiomyopathic ventricles of the K ATP channel–null mutant exposed to hemodynamic stress. Not observed in wild‐type counterparts, intraventricular disparity in wall motion, validated by the degree, direction, and delay of myocardial speckle patterns, unmasked the disease substrate from asymptomatic to overt heart failure. Mechanical dyssynchrony preceded widening of the QRS complex and exercise intolerance and progressed into global myocardial discoordination and decompensated cardiac pump function, precipitating a low output syndrome. Conclusions The present study, with the use of high‐resolution imaging, prospectively resolved the origin and extent of intraventricular motion disparity in a K ATP channel–knockout model of dilated cardiomyopathy. Mechanical dyssynchrony established as an early marker of cardiomyopathic disease offers novel insight into the pathodynamics of dyssynchronous heart failure.

Background— Cardiac development is a complex process resulting in an integrated, multilineage tissue with developmental corruption in early embryogenesis leading to congenital heart disease. Interrogation of individual genes has provided the backbone for cardiac developmental biology, yet a comprehensive transcriptome derived from natural cardiogenesis is required to gauge innate developmental milestones. Methods and Results— Stage-specific cardiac structures were dissected from 8 distinctive mouse embryonic time points to produce genome-wide expressome analysis across cardiogenesis. With reference to this native cardiogenic expression roadmap, divergent induced pluripotent stem cell–derived cardiac expression profiles were mapped from procardiogenic 3-factor (SOX2, OCT4, KLF4) and less-cardiogenic 4-factor (plus c-MYC) reprogrammed cells. Expression of cardiac-related genes from 3-factor–induced pluripotent stem cell differentiated in vitro at days 5 and 11 and recapitulated expression profiles of natural embryos at days E7.5-E8.5 and E14.5-E18.5, respectively. By contrast, 4-factor–induced pluripotent stem cells demonstrated incomplete cardiogenic gene expression profiles beginning at day 5 of differentiation. Differential gene expression within the pluripotent state revealed 23 distinguishing candidate genes among pluripotent cell lines with divergent cardiogenic potentials. A confirmed panel of 12 genes, differentially expressed between high and low cardiogenic lines, was transformed into a predictive score sufficient to discriminate individual induced pluripotent stem cell lines according to relative cardiogenic potential. Conclusions— Transcriptome analysis attuned to natural embryonic cardiogenesis provides a robust platform to probe coordinated cardiac specification and maturation from bioengineered stem cell–based model systems. A panel of developmental-related genes allowed differential prognosis of cardiogenic competency, thus prioritizing cell lines according to natural blueprint to streamline functional applications.

Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.

Abstract Defining the functional status of host-associated microbial ecosystems has proven challenging owing to the vast number of predicted genes within the microbiome and relatively poor understanding of community dynamics and community–host interaction. Metabolomic approaches, in which a large number of small molecule metabolites can be defined in a biological sample, offer a promising avenue to ‘fingerprint’ microbiota functional status. Here, we examined the effects of the human gut microbiota on the fecal and urinary metabolome of a humanized (HUM) mouse using an optimized ultra performance liquid chromatography–mass spectrometry-based method. Differences between HUM and conventional mouse urine and fecal metabolomic profiles support host-specific aspects of the microbiota’s metabolomic contribution, consistent with distinct microbial compositions. Comparison of microbiota composition and metabolome of mice humanized with different human donors revealed that the vast majority of metabolomic features observed in donor samples are produced in the corresponding HUM mice, and individual-specific features suggest ‘personalized’ aspects of functionality can be reconstituted in mice. Feeding the mice a defined, custom diet resulted in modification of the metabolite signatures, illustrating that host diet provides an avenue for altering gut microbiota functionality, which in turn can be monitored via metabolomics. Using a defined model microbiota consisting of one or two species, we show that simplified communities can drive major changes in the host metabolomic profile. Our results demonstrate that metabolomics constitutes a powerful avenue for functional characterization of the intestinal microbiota and its interaction with the host.

Abstract Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.