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Background Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature primary cardiomyocytes, especially the ability to use fatty acids (FA) as an energy source, containing high mitochondrial mass, presenting binucleation and increased DNA content per nuclei (polyploidism), and synchronized electrical conduction. This immaturity represents a bottleneck to their application in (1) disease modelling—as most cardiac (genetic) diseases have a middle-age onset—and (2) clinically relevant models, where integration and functional coupling are key. So far, several methods have been reported to enhance iPSC-CM maturation; however, these protocols are laborious, costly, and not easily scalable. Therefore, we developed a simple, low-cost, and rapid protocol to promote cardiomyocyte maturation using two small molecule activators of the peroxisome proliferator-activated receptor β/δ and gamma coactivator 1-alpha (PPAR/PGC-1α) pathway: asiatic acid (AA) and GW501516 (GW). Methods and Results Monolayers of iPSC-CMs were incubated with AA or GW every other day for ten days resulting in increased expression of FA metabolism-related genes and markers for mitochondrial activity. AA-treated iPSC-CMs responsiveness to the mitochondrial respiratory chain inhibitors increased and exhibited higher flexibility in substrate utilization. Additionally, structural maturity improved after treatment as demonstrated by an increase in mRNA expression of sarcomeric-related genes and higher nuclear polyploidy in AA-treated samples. Furthermore, treatment led to increased ion channel gene expression and protein levels. Conclusions Collectively, we developed a fast, easy, and economical method to induce iPSC-CMs maturation via PPAR/PGC-1α activation. Treatment with AA or GW led to increased metabolic, structural, functional, and electrophysiological maturation, evaluated using a multiparametric quality assessment.

The development of new cardioprotective approaches using in vivo models of ischemic heart disease remains challenging as differences in cardiac physiology, phenotype, and disease progression between humans and animals influence model validity and prognostic value. Furthermore, economical and ethical considerations have to be taken into account, especially when using large animal models with relevance for conducting preclinical studies. The development of human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has opened new opportunities for in vitro studies on cardioprotective compounds. However, the immature cellular phenotype of iPSC-CMs remains a roadblock for disease modeling. Here, we show that metabolic maturation renders the susceptibility of iPSC-CMs to hypoxia further toward a clinically representative phenotype. iPSC-CMs cultured in a conventional medium did not show significant cell death after exposure to hypoxia. In contrast, metabolically matured (MM) iPSC-CMs showed inhibited mitochondrial respiration after exposure to hypoxia and increased cell death upon increased durations of hypoxia. Furthermore, we confirmed the applicability of MM iPSC-CMs for in vitro studies of hypoxic damage by validating the known cardioprotective effect of necroptosis inhibitor necrostatin-1. Our results provide important steps to improving and developing valid and predictive human in vitro models of ischemic heart disease.

Most cardiomyocytes (CMs) in the adult mammalian heart are either binucleated or contain a single polyploid nucleus. Recent studies have shown that polyploidy in CMs plays an important role as an adaptive response to physiological demands and environmental stress and correlates with poor cardiac regenerative ability after injury. However, knowledge about the functional properties of polyploid CMs is limited. In this study, we generated tetraploid pluripotent stem cells (PSCs) by fusion of murine embryonic stem cells (ESCs) and somatic cells isolated from bone marrow or spleen and performed a comparative analysis of the electrophysiological properties of tetraploid fusion-derived PSCs and diploid ESC-derived CMs. Fusion-derived PSCs exhibited characteristics of genuine ESCs and contained a near-tetraploid genome. Ploidy features and marker expression were also retained during the differentiation of fusion-derived cells. Fusion-derived PSCs gave rise to CMs, which were similar to their diploid ESC counterparts in terms of their expression of typical cardiospecific markers, sarcomeric organization, action potential parameters, response to pharmacologic stimulation with various drugs, and expression of functional ion channels. These results suggest that the state of ploidy does not significantly affect the structural and electrophysiological properties of murine PSC-derived CMs. These results extend our knowledge of the functional properties of polyploid CMs and contribute to a better understanding of their biological role in the adult heart.

Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) represent an attractive resource for cardiac regeneration. However, survival and functional integration of transplanted iPS-CM is poor and remains a major challenge for the development of effective therapies. We hypothesized that paracrine effects of co-transplanted mesenchymal stromal cells (MSCs) augment the retention and therapeutic efficacy of iPS-CM in a mouse model of myocardial infarction (MI). To test this, either iPS-CM, MSC, or both cell types were transplanted into the cryoinfarction border zone of syngeneic mice immediately after injury. Bioluminescence imaging (BLI) of iPS-CM did not confirm enhanced retention by co-application of MSC during the 28-day follow-up period. However, histological analyses of hearts 28 days after cell transplantation showed that MSC increased the fraction of animals with detectable iPS-CM by 2-fold. Cardiac MRI analyses showed that from day 14 after transplantation on, the animals that have received cells had a significantly higher left ventricular ejection fraction (LVEF) compared to the placebo group. There was no statistically significant difference in LVEF between animals transplanted only with iPS-CM or only with MSC. However, combined iPS-CM and MSC transplantation resulted in higher LVEF compared to transplantation of single-cell populations during the whole observation period. Histological analyses revealed that MSC increased the capillarization in the myocardium when transplanted alone or with iPS-CM and decreased the infarct scar area only when transplanted in combination with iPS-CM. These results indicate that co-transplantation of iPS-CM and MSC improves cardiac regeneration after cardiac damage, demonstrating the potential of combining multiple cell types for increasing the efficacy of future cardiac cell therapies.

The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine offers promising perspectives for the treatment of major depressive disorder. Although ketamine demonstrates rapid and long-lasting effects, even in treatment-resistant patients, to date, the underlying mode of action remains elusive. Thus, the aim of our study was to investigate the molecular mechanism of ketamine at clinically relevant concentrations by establishing an in vitro model based on human induced pluripotent stem cells (iPSCs)-derived neural progenitor cells (NPCs). Notably, ketamine increased the proliferation of NPCs independent of the NMDA receptor, while transcriptome analysis revealed significant upregulation of insulin-like growth factor 2 (IGF2) and p11, a member of the S100 EF-hand protein family, which are both implicated in the pathophysiology of depression, 24 h after ketamine treatment. Ketamine (1 &micro, M) was able to increase cyclic adenosine monophosphate (cAMP) signaling in NPCs within 15 min and cell proliferation, while ketamine-induced IGF2 expression was reduced after PKA inhibition with cAMPS-Rp. Furthermore, 24 h post-administration of ketamine (15 mg/kg) in vivo confirmed phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the subgranular zone (SGZ) of the hippocampus in C57BL/6 mice. In conclusion, ketamine promotes the proliferation of NPCs presumably by involving cAMP-IGF2 signaling.

The possibility to generate cardiomyocytes (CMs) from disease-specific induced pluripotent stem cells (iPSCs) is a powerful tool for the investigation of various cardiac diseases in vitro. The pathological course of various cardiac conditions, causatively heterogeneous, often converges into disturbed cellular Ca(2+) cycling. The gigantic Ca(2+) channel of the intracellular Ca(2+) store of CMs, the ryanodine receptor type 2 (RyR2), controls Ca(2+) release and therefore plays a crucial role in Ca(2+) cycling of CMs. In the present protocol we describe ways to measure and analyze global as well as local cellular Ca(2+) release events in CMs derived from a patient carrying a CPVT-causing RyR2 mutation.

International audience; Chronic granulomatous disease (CGD) is an inherited orphan disorder caused by mutations in one of the five genes encoding reduced nicotinamide-adenine-dinucleotide-phosphate oxidase subunits, which subsequently lead to impairment in the production of microbicidal reactive oxygen species (ROS). In order to offer several cell line models of CGD and therefore support research on pathophysiology and new therapeutic approaches, we optimized protocols to differentiate induced pluripotent stem cells (iPSCs) from wild-type, X(0)-, AR22(0)- and AR47(0)-CGD patient's fibroblasts into neutrophils and into macrophages. Aberrant genetic clones were discarded after chromosome karyotyping and array-comparative genomic hybridization analysis. All remaining iPSC lines showed human embryonic stem cell-like morphology, expressed all tested pluripotency markers and formed embryoid bodies that contained cells originating from all three primary germ layers. Furthermore, each CGD patient-specific iPSC line retained the gp91 (phox) , p47 (phox) , and p22 (phox) mutations found in the corresponding patient's neutrophils. The average production of CD34(+) progenitors was of 1.5×10(6) cells after 10 days of differentiation of 10×10(6) iPSCs. They were terminally differentiated into about 3×10(5) neutrophils or into 3×10(7) macrophages. Based on morphological, phenotypical, and functional criteria both phagocyte types were mature and indistinguishable from the native human neutrophils and macrophages. However, neutrophils and macrophages derived from X(0)-, AR22(0)-, and AR47(0)-CGD patient-specific iPSC lines lacked ROS production and the corresponding mutated proteins. To simplify the phagocytes' production upon request, progenitors can be cryopreserved. In conclusion, we describe a reproducible, simple, and efficient way to generate neutrophils and macrophages from iPSCs and provide a new cellular model for the AR22(0)-CGD genetic form that has not been described before.