Transcriptional atlas of cardiogenesis maps congenital heart disease interactome.

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.

Selection via pluripotency-related transcriptional screen minimizes the influence of somatic origin on iPSC differentiation propensity.

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 (R(2) = 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.

Cessation of contraction induces cardiomyocyte remodeling during zebrafish cardiogenesis.

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.

Natural cardiogenesis-based template predicts cardiogenic potential of induced pluripotent stem cell lines.

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.

New and TALENted genome engineering toolbox.

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.

Apoptotic susceptibility to DNA damage of pluripotent stem cells facilitates pharmacologic purging of teratoma risk.

Pluripotent stem cells have been the focus of bioengineering efforts designed to generate regenerative products, yet harnessing therapeutic capacity while minimizing risk of dysregulated growth remains a challenge. The risk of residual undifferentiated stem cells within a differentiated progenitor population requires a targeted approach to eliminate contaminating cells prior to delivery. In this study we aimed to validate a toxicity strategy that could selectively purge pluripotent stem cells in response to DNA damage and avoid risk of uncontrolled cell growth upon transplantation. Compared with somatic cell types, embryonic stem cells and induced pluripotent stem cells displayed hypersensitivity to apoptotic induction by genotoxic agents. Notably, hypersensitivity in pluripotent stem cells was stage-specific and consistently lost upon in vitro differentiation, with the mean half-maximal inhibitory concentration increasing nearly 2 orders of magnitude with tissue specification. Quantitative polymerase chain reaction and Western blotting demonstrated that the innate response was mediated through upregulation of the BH3-only protein Puma in both natural and induced pluripotent stem cells. Pretreatment with genotoxic etoposide purged hypersensitive pluripotent stem cells to yield a progenitor population refractory to teratoma formation upon transplantation. Collectively, this study exploits a hypersensitive apoptotic response to DNA damage within pluripotent stem cells to decrease risk of dysregulated growth and augment the safety profile of transplant-ready, bioengineered progenitor cells.

Reprogrammed keratinocytes from elderly type 2 diabetes patients suppress senescence genes to acquire induced pluripotency.

Nuclear reprogramming enables patient-specific derivation of induced pluripotent stem (iPS) cells from adult tissue. Yet, iPS generation from patients with type 2 diabetes (T2D) has not been demonstrated. Here, we report reproducible iPS derivation of epidermal keratinocytes (HK) from elderly T2D patients. Transduced with human OCT4, SOX2, KLF4 and c-MYC stemness factors under serum-free and feeder-free conditions, reprogrammed cells underwent dedifferentiation with mitochondrial restructuring, induction of endogenous pluripotency genes - including NANOG, LIN28, and TERT, and down-regulation of cytoskeletal, MHC class I- and apoptosis-related genes. Notably, derived iPS clones acquired a rejuvenated state, characterized by elongated telomeres and suppressed senescence-related p15INK4b/p16INK4a gene expression and oxidative stress signaling. Stepwise guidance with lineage-specifying factors, including Indolactam V and GLP-1, redifferentiated HK-derived iPS clones into insulin-producing islet-like progeny. Thus, in elderly T2D patients, reprogramming of keratinocytes ensures a senescence-privileged status yielding iPS cells proficient for regenerative applications.

Induced pluripotent stem cells from GMP-grade hematopoietic progenitor cells and mononuclear myeloid cells.

INTRODUCTION: The induced pluripotent stem cell (iPSC) technology allows generation of patient-specific pluripotent stem cells, thereby providing a novel cell-therapy platform for severe degenerative diseases. One of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming. METHODS: We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and peripheral blood mononuclear cells (PBMCs) and tested the pluripotency of derived iPS clones. RESULTS: Ectopic expression of OCT4, SOX2, KLF4, and c-MYC in HPCs and PBMCs resulted in rapid iPSC derivation. Long-term time-lapse imaging revealed efficient iPSC growth under serum- and feeder-free conditions with frequent mitotic events. HPC- and PBMC-derived iPS cells expressed pluripotency-associated markers, including SSEA-4, TRA-1-60, and NANOG. The global gene-expression profiles demonstrated the induction of endogenous pluripotent genes, such as LIN28, TERT, DPPA4, and PODXL, in derived iPSCs. iPSC clones from blood and other cell sources showed similar ultrastructural morphologies and genome-wide gene-expression profiles. On spontaneous and guided differentiation, HPC- and PBMC-derived iPSCs were differentiated into cells of three germ layers, including insulin-producing cells through endodermal lineage, verifying the pluripotency of the blood-derived iPSC clones. CONCLUSIONS: Because the use of blood cells allows minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming, mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation, especially from diabetes patients complicated by slow-healing wounds.