Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes.

Noonan Syndrome (NS), an autosomal dominant RASopathy disorder, is caused by germ-line mutations that affect the canonical RAS-MAPK pathway. >95% of NS patients with an S257L mutation in Raf1 exhibit cardiac hypertrophy (CH) at a very young age. However, the molecular and developmental mechanisms that elicit CH in these patients remain unknown. Hence, we aimed at modeling CH by differentiating human NS Raf1 induced-pluripotent stem cells (iPSCs) towards the cardiomyocyte fate. We generated iPSCs from an NS pediatric patient with the S257L/+ mutation in the Raf1 gene and corrected the mutation using CRISPR-Cas9 double nickase system. Differentiation of the mutant NS line led to a significantly increased number of cardiomyocytes (CMs) as measured by FACS sorting at day 20, as compared to isogenic corrected cells (5.71 million ± 0.38 of cTNT+ cells vs 3.77±0.54, n=6, p<0.01). Differentiation of mutant iPSCs into mesodermal cells was normal; however, differentiation into cardiac progenitor cells was enhanced by the mutation, as demonstrated by the increased number of NKX2.5+ cells in the mutant culture at this stage of development. In addition, differentiated S257L/+ CMs had a significantly increased cell surface area compared to the control CMs (3,331 μm 2 ± 325 vs 1,638 μm 2 ± 97, n=6, p<0.01). To uncover the aberrant signaling pathways underlying these phenotypes, we generated RAF1 S257L/+ knockin and RAF1 null (KO) Hela cell lines using CRISPR-Cas9n technology. Interestingly, we found that ERK1/2 activity was enhanced in KO cells, both at baseline and in response to growth factor (GF) stimulation. At the opposite, ERK1/2 activity was reduced in mutant cells, suggesting that the RAF1 S257L/+ mutant actually suppresses ERK1/2 activation. Finally, we found that AKT activation was upregulated in S257L/+ cells under GF stimulation, due to decreased ERK1/2-dependent p70-S6 kinase activity and subsequent increase in mTORC2 activity (n=6). Similar experiments are underway in S257L/+, corrected and KO RAF1 iPSC-derived CMs. In conclusion, we propose that the S257L/+ mutation modulates development of CH in NS RAF1 patients through a reduction in ERK1/2 activity and overactivation of the AKT/mTOR signaling pathway, leading to both an increased number and size of CMs.

Human embryonic stem (hES) cells are already transforming our vision of regenerative medicine and cell replacement therapies because of their unique ability to maintain themselves indefinitely and to form all cell types in the body. hES cell lines with genomes that are predisposed to disease can be used to develop cellular models of human pathologies. The generation of disease‐specific and/or genotypically diverse human stem cell lines that can differentiate into many cell types will have great value for understanding the biology of the cell types affected in disease and analysing disease mechanisms, screening for drug candidates that can slow or prevent disease‐related degeneration as well as toxicological testing and understanding the variation between patients in their responses to therapeutics. In addition, to prepare for cell‐based therapies, new technologies for the generation of stem cell lines that are histocompatible with or specific to individual patients will provide a strategy to overcome the challenges of immune rejection. Several different methods have been developed to make hES cells; to date, these all require the use of human female egg cells as a starting point. Recently, several laboratories have reported that adult cells from human skin and other sources can be induced to revert back to earlier stages of development and exhibit stem cell‐like properties. The methods for ‘reprogramming’ have been developing rapidly but generally involve putting multiple genes into skin cells and then exposing them to specific chemical environments tailored to hES cell growth. While these cells appear to have a developmental potential that is similar to that of hES cells, they are not derived from human embryos. To distinguish these reprogrammed cells from the embryo‐sourced hES cells, they are termed induced pluripotent stem (iPS) cells. The iPS technology is the source of much optimism; »The iPS technology is the source of much …

Background: Cardiomyocytes (CMs) lost during ischemic cardiac injury can't be replaced due to their limited proliferative capacity. Our laboratory has shown that overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 in combination (4F) promotes cell division in 10-20% of post-mitotic cardiomyocytes (CMs). Recently, we found that 4F overexpression transiently decreased CM contractility during the mitotic phase. Temporal bulk and single-cell RNAseq data from 4F-transduced human iPSC-derived CMs (hiPS-CMs) or P7 neonatal mouse CMs (NMCM) indicated a transient downregulation of contractile genes during the mitotic phase; among the most differentially regulated genes was Ras-Related Associated with Diabetes (RRAD), an endogenous inhibitor of the L-type calcium channel (LTCC). Objective: RRAD inhibition of LTCC primes CMs to respond to cell cycle stimulation through unloading CMs from their commitment to contractile function. Methods and Results: We used viral approaches to overexpress and knockdown (KD) RRAD in hiPS-CMs and NMCM. Functionally, RRAD overexpression abolished intracellular calcium transients’ amplitude while RRAD KD significantly increased calcium transients’ amplitude in response to electric stimulation in CMs. RRAD overexpression in hiPS-CMs or P7 NMCM augments the cell cycle induction following 4F transduction, while RRAD KD resulted in a decline in the cell cycle induction as indicated by cell cycle markers; EDU and PHH3 (PHH3(%): 4F 26.0± 1, 4F+RRAD 41.15± 6.7, 4F+KD 15.92± 2.6, P<0.05; EDU(%): 4F 29.9± 3.9, 4F+RRAD 61.04± 2.6, 4F+KD 16.8± 2.8; n=6, P<0.05). Nitrendipine, an LTCC blocker, and Mavacamten, which directly suppress cardiomyocyte contractility, reversed the inhibitory effect of RRAD KD on cell cycle induction. More interestingly, RRAD overexpression enhanced the cell cycle induction in presence of only 2 factors (CDK4 and CCND (2F)), which are known to induce S phase (PHH3(%): 2F 5.1± 0.4, 2F+RRAD 10.4± 6.7, 2F+KD 0.4± 0.1, EDU (%); 2F 38.6±15, 2F +RRAD 44.7± 18, 2F+KD 5.6± 2; n=6, P<0.05). Conclusions: These data suggest that RRAD modulation of contractile function modulates the response of CMs to cell cycle induction and can reduce the number of factors needed to induce cell cycle in CMs.

Three-dimensional co-culturing of stem cell-derived cardiomyocytes and cardiac fibroblasts reveals a role for both cell types in Marfan-related cardiomyopathy

Event Abstract Back to Event Modeling microglial differentiation and function in vitro using induced pluripotent stem cells Florent Ginhoux1* 1 Agency for Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), Singapore Microglia, the resident macrophages of the central nervous system (CNS), play important roles in both CNS homeostasis and inflammation. Dysregulated microglial activity is involved in the pathogenesis of neurodegenerative disorders and neuroinflammatory diseases, rendering modulation of microglial responses an attractive therapeutic target. However, a lack of relevant microglial cell model systems has so far precluded progress toward a comprehensive characterization of microglial activation states and the mechanisms by which these states may be modulated to decelerate neurodegeneration. We have previously shown that microglia arise very early during development from primitive yolk sac (YS) macrophages. Using this concept of microglial origin, we develop methods to derive YS primitive like macrophages from murine embryonic stem cells (ESC) and human induced pluripotent stem cells (iPSC), as in vitro ESC/ iPSC differentiation towards hematopoiesis was shown to recapitulate YS primitive hematopoiesis in vivo. Using our “in house” xeno/serum-free differentiation protocol, we are able to generate primitive-like macrophages that are genetically and phenotypically similar to in vivo YS macrophages and microglia progenitors. We have also been establishing methods for the co-culture of iPSC-derived primitive macrophages with neurons derived from the same mouse iPSC batch and show that iPSC-derived macrophages efficiently differentiate into microglial cells compared to monocytes or bone marrow derived macrophages. Such co-culture model will provide insights into the mechanisms of microglia differentiation, and the interactions of microglia and neurons in both physiological and pathological neuroinflammatory conditions. Furthermore, such co-culture model has the potential to serve as an in vitro drug screening system for the development of novel therapies against brain diseases. Taken together, iPSC-derived primitive macrophages could be a useful microglial source for biological, pathophysiological, and therapeutic studies. Keywords: Induced Pluripotent Stem Cells, Microglia, microglial activation, microglial differentiation, primitive macrophages Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Symposium 10: Microglia: Immune Cells or Brain Cells? Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Ginhoux F (2016). Modeling microglial differentiation and function in vitro using induced pluripotent stem cells. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00044 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jul 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Florent Ginhoux, Agency for Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), Singapore, Singapore, florent_ginhoux@immunol.a-star.edu.sg Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Florent Ginhoux Google Florent Ginhoux Google Scholar Florent Ginhoux PubMed Florent Ginhoux Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The goal of "Recent Developments" is to provide a concise but comprehensive overview of new advances in cardiovascular research, which we hope will keep our readers abreast of recent scientific discoveries and facilitate discussion, interpretation, and integration of the findings.This will enable readers who are not experts in a particular field to grasp the significance and effect of work performed in other fields.It is our hope and expectation that these "Recent Development" articles will help readers to gain a broader awareness and a deeper understanding of the status of research across the vast landscape of cardiovascular research-The Editors.

Cardiovascular disorders and associated morbidities remain the leading cause of premature death worldwide. Since the regeneration of diseased hearts is very limited and the insufficient supply of donor organs persists, hopes rely on new therapies for heart repair. Reviving the proliferation of endogenous cardiomyocytes (CMs) or the administration of adult stem cells to the heart was of limited curative success to date. Thus, the administration of in vitro generated CMs is under investigation to replenish loss of functional heart muscle tissue. This requires a sustainable source of CMs. Induced pluripotent stem cells (iPSC) have raised hopes for developing autologous cell therapies. To serve for heart repair, efficient and safe iPSC differentiation protocols for CMs production are required. iPSC differentiation into CMs and even functional subtypes was indeed achieved in recent years, either by the ectopic expression of cardiac transcription factors or the supplementation of chemical pathway modulators. An alternative approach aims at the direct transdifferentiation of fibroblasts, which are present in the interstitial tissue of many organs, into functional lineage-specific cell types. As a result the formation of induced cardiomyocyte-like cells (iCMs) by the ectopic expression of specific transcription factors combinations has been demonstrated in vitro and in vivo. This is an important proof-of-concept that the intermediate state of iPSC induction is dispensable. However, most of the early experiments were conducted in mice and translation to more relevant large animal models and subsequently to the clinic are challenging. Progress, drawbacks, and perspectives in this field will be discussed.

Among various cardiac safety concerns, proarrhythmia risks, including QT prolongation leading to Torsade de Pointes, is one of major cause for drugs being withdrawn (~45% 1975-2007). Preclinical study requires the evaluation of proarrhythmia using in silico, in vitro, and/or animal models. Considering that the primary consumers of prescription drugs are elderly patients, applications of "aging-in-a-dish" models would be appropriate for screening proarrhythmia risks. However, acquiring such models, including cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs), presents extensive challenges. We proposed the hypothesis that CMs differentiated from iPSCs derived from Hutchinson-Gilford progeria syndrome (HGPS, progeria) patients, an ultra-rare premature aging syndrome, can mimic the phenotypes of aging CMs. Our objective, therefore, was to examine this hypothesis by analyzing the response of 11 reference compounds utilized by the Food and Drug Administration (FDA)'s Comprehensive in vitro Proarrhythmia Assay (CiPA) using progeria and control CMs. As a sensitive surrogate marker of modulating cardiac excitation-contraction coupling, we evaluated drug-induced changes in calcium transient (CaT). We observed that the 80% CaT peak duration in the progeria CMs (0.98 ± 0.04 s) was significantly longer than that of control CMs (0.70 ± 0.05 s). Furthermore, when the progeria CMs were subjected to four doses of 11 compounds from low-, intermediate-, and high-risk categories, they demonstrated greater arrhythmia susceptibility than control cells, as shown through six-parameter CaT profile analyses. We also employed the regression analysis established by CiPA to classify the 11 reference compounds and compared proarrhythmia susceptibilities between the progeria and control CMs. This analysis revealed a greater proarrhythmia susceptibility in the progeria CMs compared to the control CMs. Interestingly, in both CMs, the compounds categorized as low risk did not exceed the safety risk threshold of 0.8. In conclusion, our study demonstrates increased proarrhythmia sensitivity in progeria CMs when tested with reference compounds. Future studies are needed to analyze underlying mechanisms and further validate our findings using a larger array of reference compounds.

Actions of photoreleased cADP-ribose (cADPR), a novel regulator of calcium-induced calcium release (CICR) from ryanodine-sensitive stores, were investigated in cardiac myocytes. Photoreleased cADPR caused an increase in the magnitude of whole-cell calcium transients studied in mammalian cardiac ventricular myocytes (both guinea-pig and rat) using confocal microscopy). Approx. 15 s was required following photorelease of cADPR for the development of its maximal effect. Photoreleased cADPR also increased the frequency of calcium 'sparks', which are thought to be elementary events which make up the whole-cell calcium transient, and were studied in rat myocytes, but had little or no effect on spark characteristics (amplitude, rise time, decay time and distance to half amplitude). The potentiating effects of photoreleased cADPR on both whole-cell transients and the frequency of calcium sparks were prevented by cytosolic application of the antagonist 8-amino-cADPR (5 microM). These experiments, therefore, provide the first evidence in any cell type for an effect of cADPR on calcium sparks, and are the first to show the actions of photoreleased cADPR on whole-cell calcium transients in mammalian cells. The observations are consistent with the effects of cADPR in enhancing the calcium sensitivity of CICR from the sarcoplasmic reticulum in cardiac ventricular myocytes, leading to an increase in the probability of occurrence of calcium sparks and to an increase in whole-cell calcium transients. The slow time-course for development of the full effect on whole-cell calcium transients might be taken to indicate that the influence of cADPR on CICR may involve complex molecular interactions rather than a simple direct action of cADPR on the ryanodine-receptor channels.

Genetic Correction of Huntington's Disease Phenotypes in Induced Pluripotent Stem Cells

To bridge the gap between 2D cell culture and tissue, various 3D cell culture approaches have been developed for the investigation of cardiac myocytes (CM) and fibroblasts (CF), but limitations still exist. The goal of this study was to develop a scaffold-free cardiac culture that could easily and inexpensively create many uniform cardiac microtissues that mimic the cellular distribution and functional behavior of CM and CF in tissue. Using micromolded non-adhesive agarose hydrogels containing 822 concave recesses (800 λm deep x 400 λm wide), we demonstrate that neonatal rat ventricular CM and CF, when seeded alone or in combination, self-assembled into viable (Live/Dead® stain) spherical-shaped microtissues. Importantly, when CM and CF were seeded simultaneously or sequentially, they self-sorted to be highly interspersed, reminiscent of their distribution in ventricular tissue, as shown by cell type-specific CellTracker TM or antibody labeling. Depending on their cellular composition, microtissues expressed extracellular matrix and calcium handling proteins (including SERCA2a), featured typical I K1 and I Ca-L current densities and IV relationships, and formed functional cell-cell connections (as evidenced by spontaneous action potentials, contractions and connexin 43 expression). Microelectrode recordings and optical mapping showed characteristic triangular action potentials (AP) with a resting membrane potential of -66±7 mV (n=4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100±30 ms and 18.0±1.9 cm/s, respectively (n=5 each). The presence of CF led to a prolongation of both AP and calcium transients in CM:CF microtissues (1:1). Furthermore, cell-type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we have developed a novel scaffold-free cardiac 3D culture model, in which more than 800 homotypic (CM or CF) or heterotypic (CM:CF) microtissues can be formed in a 6-well with ease, analyzed functionally and subjected to cell-type-specific gene transfer, paving the way for future in vitro investigations of CM and CF behavior and their interactions in a 3D environment.

The in vitro modeling of cardiac development and cardiomyopathies in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) provides opportunities to aid the discovery of genetic, molecular, and developmental changes that are causal to, or influence, cardiomyopathies and related diseases. To better understand the functional and disease modeling potential of iPSC-differentiated CMs and to provide a proof of principle for large, epidemiological-scale disease gene discovery approaches into cardiomyopathies, well-characterized CMs, generated from validated iPSCs of 12 individuals who belong to four sibships, and one of whom reported a major adverse cardiac event (MACE), were analyzed by genome-wide mRNA sequencing. The generated CMs expressed CM-specific genes and were highly concordant in their total expressed transcriptome across the 12 samples (correlation coefficient at 95% CI =0.92 ± 0.02). The functional annotation and enrichment analysis of the 2116 genes that were significantly upregulated in CMs suggest that generated CMs have a transcriptomic and functional profile of immature atrial-like CMs; however, the CMs-upregulated transcriptome also showed high overlap and significant enrichment in primary cardiomyocyte (p-value = 4.36 × 10−9), primary heart tissue (p-value = 1.37 × 10−41) and cardiomyopathy (p-value = 1.13 × 10−21) associated gene sets. Modeling the effect of MACE in the generated CMs-upregulated transcriptome identified gene expression phenotypes consistent with the predisposition of the MACE-affected sibship to arrhythmia, prothrombotic, and atherosclerosis risk.

Simple SummaryCardiomyopathies modeling is greatly smoothened by the technological advances made in the use of human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs). Despite the advantages of allowing to model patient specific disease, hiPSC–CMs still show a degree of maturity comparable to fetal CMs. In this perspective, we discuss different methods to improve hiPSC-CMs maturity, and to create cardiomyopathy-specific models, allowing the assessment of relevant phenotypes. In addition, current limitations and required evolutions in cardiomyopathy disease modeling are addressed.The stem cell technology and the induced pluripotent stem cells (iPSCs) production represent an excellent alternative tool to study cardiomyopathies, which overcome the limitations associated with primary cardiomyocytes (CMs) access and manipulation. CMs from human iPSCs (hiPSC–CMs) are genetically identical to patient primary cells of origin, with the main electrophysiological and mechanical features of CMs. The key issue to be solved is to achieve a degree of structural and functional maturity typical of adult CMs. In this perspective, we will focus on the main differences between fetal-like hiPSC-CMs and adult CMs. A viewpoint is given on the different approaches used to improve hiPSC-CMs maturity, spanning from long-term culture to complex engineered heart tissue. Further, we outline limitations and future developments needed in cardiomyopathy disease modeling.

<strong>Purpose</strong>. Induced pluripotent stem cells (iPS) can be generated by patients intro- ducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. However, the various iPS cell lines are characterized by different properties such as differentiation efficiency and potential safety hazards. Among several readily available primary human somatic cell types, keratinocytes can be isolated easily from human skin or hair follicle, and therefore represent an attractive cell source for reprogramming. The aims of the present study were: 1. To assess the cardiomyogenic potential of human keratynocytes-derived iPS. 2. To increase the differentiation efficiency in order to obtain a homogeneous population of beating cardiomyocytes, also overcoming the limitation of embryonic body formation.<br><strong style="font-size: 10px;">Methods</strong>. Established iPS cell line obtained from human keratinocytes was cultured in monolayer and exposed sequentially to Ascorbic Acid, 5-Azacytidine, BMP4, ActivinA, VEGF up to 20 days. Differentiation was evaluated monitoring the expression of Nkx2.5, Gata4, sarcomeric α-actinin, α cardiac myosin heavy chain, cardiac T-troponin and Connexin43 and β-adrenoceptors as cardiac markers, by Western Blot, immunofluorescent and cytometric analyses. An ImageStream analysis for a simulta- neous quantitative and morphological evaluation was also performed.<br><strong>Results</strong>. During the differentiation induction, iPS became positive to all the analyzed cardiac markers. In particular, we observed a rapid and significant increase of Nkx2.5 and Gata4 expression (14.7±0.7% vs 43.2±2.1% Nkx2.5+ and 21.7±1.2 vs 65.9±3.6% Gata4+ in iPS and 15 days after the induction respectively, p&lt;.005, analy- sis by ImageStream), with a significant nuclear translocation (nuclear+ cells:7.2±0.8% vs 18.8±2.1% of the Nkx2.5+ cells in iPS and 15 days after the induction respectively, p&lt;.005, analysis by ImageStream). The differentiation process was accompanied by a modulation of β1- and β2-adrenoceptors expression. Foci of spontaneous contraction were observed after only 5 days from the induction and after 15 days the beating cells represented about the 60-90% of the entire population. Moreover, differentiated cardio- myocytes responded to adrenergic stimulation by increasing the contraction rate. <strong>Conclusion</strong>. iPS obtained from hKeratinocytes efficiently differentiate into cardi- omyocytes giving rise to a homogenous population with cardiac-specific molecular and functional properties.

Pathogenic variants in NOTCH1 have been implicated in multiple types of congenital heart defects, such as bicuspid aortic valve, Tetralogy of Fallot, and hypoplastic left heart syndrome (HLHS). However, the mechanisms by which NOTCH1 pathogenic variants cause abnormalities in human embryonic heart development are largely unknown. Here, we used CRISPR/Cas9-mediated genome editing to genetically delete NOTCH1 in human induced pluripotent stem cells (iPSCs). We found that NOTCH1 was dispensable for mesodermal and vascular endothelial differentiation of human iPSCs. Disruption of NOTCH activity promoted venous-specific gene expression but suppressed arterial-specific gene expression in iPSC-derived endothelial cells (iPSC-ECs). Intriguingly, NOTCH1 deletion significantly impaired the cardiac differentiation efficiency. In NOTCH1 homozygous knockout ( NOTCH1 -/- ) iPSC-derived cardiomyocytes (iPSC-CMs), atrial-specific genes ( NR2F2, KCNJ3 , and MYL7 ) were upregulated whereas ventricular-specific genes ( MYL2, IRX4 , and MYH7 ) were downregulated. Electrophysiological analysis by patch clamp and optical mapping indicated that atrial-like cardiomyocytes were dominant whereas the percentage of ventricular-like iPSC-CMs was dramatically reduced (&lt;1%) in NOTCH1 -/- iPSC-CMs. In addition, mitochondrial respiration was reduced in NOTCH1 deficient iPSC-CMs compared to wild-type controls, which was likely attributed to the reduction of ventricular cardiomyocytes in NOTCH1 -/- iPSC-CMs. As NOTCH1 is primarily expressed in endothelial cells rather than cardiomyocytes, we conclude that NOTCH1 affects ventricular cardiomyocyte lineage commitment possibly through controlling cell fate determination of cardiac progenitors during human iPSC differentiation. Our study may provide novel insights into the mechanisms by which NOTCH1 mutations lead to left ventricular hypoplasia in HLHS patients.