Cardiac Differentiation Process Research Articles

Seamless and non-destructive monitoring of extracellular microRNAs during cardiac differentiation from human pluripotent stem cells.

Monitoring cardiac differentiation and maturation from human pluripotent stem cells (hPSCs) and detecting residual undifferentiated hPSCs are indispensable for the development of cardiac regenerative therapy. MicroRNA (miRNA) is secreted from cells into the extracellular space, and its role as a biomarker is attracting attention. Here, we performed an miRNA array analysis of supernatants during the process of cardiac differentiation and maturation from hPSCs. We demonstrated that the quantification of extracellular miR-489-3p and miR-1/133a-3p levels enabled the monitoring of mesoderm and cardiac differentiation, respectively, even in clinical-grade mass culture systems. Moreover, extracellular let-7c-5p levels showed the greatest increase with cardiac maturation during long-term culture. We also verified that residual undifferentiated hPSCs in hPSC-derived cardiomyocytes (hPSC-CMs) were detectable by measuring miR-302b-3p expression, with a detection sensitivity of 0.01%. Collectively, we demonstrate that our method of seamlessly monitoring specific miRNAs secreted into the supernatant is non-destructive and effective for the quality evaluation of hPSC-CMs.

Cardiomyocyte differentiation from human induced pluripotent stem cells is delayed following knockout of Bcl-2.

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) regulates a wide array of cellular functions involved in cell death, cell survival and autophagy. Less known is its involvement in the differentiation of cardiomyocytes. As a consequence, mechanisms by which Bcl-2 contributes to cardiac differentiation remain to be elucidated. To address this, we used CRISPR/Cas9 to knockout (KO) BCL2 in human induced pluripotent stem cells (hiPSCs) and investigated the consequence of this KO for differentiation towards cardiomyocytes. Our results indicate that differentiation of hiPSCs to cardiomyocytes was delayed following BCL2 KO. This was not related to the canonical anti-apoptotic function of Bcl-2. This delay led to reduced expression and activity of the cardiomyocyte Ca2+ toolkit. Finally, Bcl-2 KO reduced c-Myc expression and nuclear localization in the early phase of the cardiac differentiation process, which accounts at least in part for the observed delay in the cardiac differentiation. These results suggest that there is a central role for Bcl-2 in cardiomyocyte differentiation and maturation.

Formation of human cardiomyocytes is impaired by fibroblastic paracrine signalling: breaking down the mechanisms involved in rebuilding the heart

Abstract To date (pre-)clinical effects of inducing cardiac regeneration by cardiac cell therapy have been disappointing. This lack of success may result from the fact that it remains largely unclear how the receiving pathological microenvironment affects this process of cardiac differentiation of implanted cells, and thereby may (negatively) influence the therapeutic outcome. We have recently generated lines of conditionally immortalized human CMCs (ciCMCs) through doxycycline-dependent expression of SV40 large T antigen. In these cells, proliferation and differentiation can be tightly controlled. The aim of this study is to improve our understanding of cardiac differentiation of transplanted cells in the context of the receiving microenvironment from a paracrine perspective. To investigate the role of fibroblastic paracrine signalling on cardiomyogenic differentiation, transwell experiments were conducted. Cardiac differentiation was induced in human ciCMCs while cardiac fibroblasts were cultured in the top inserts in different ratios (human ciCMCs:cardiac fibroblasts 1:1, 2:1 and 3:1). Cardiomyogenic differentiation was determined by immunostaining for cardiac specific markers, quantitative RT-PCR, and electrophysiological analysis. After 12 days of differentiation, under the influence of the fibroblastic paracrine signaling, the amount of ciCMCs that expressed the sarcomeric protein α-actinin and myosin light chain 2a was significantly and increasingly reduced (P<0.01) in the groups cultured under the influence of the cardiac fibroblasts (4.8±0.6, 8.1±1.9, 22.4±4.2 for 1:1, 2:1 and 3:1 human ciCMCs:cardiac fibrolasts respectively [%, mean±SD]) while in absence of the cardiac fibroblasts (control group) 41% (±9.8) human ciCMCs were formed. In corroboration with these results, quantitative RT-PCR showed significant reduction (P<0.05) of all cardiac specific markers on a mRNA level in 1:1 human ciCMC: cardiac fibroblasts cultures compared to the control group. Electrophysiological analysis showed a significantly reduced (P<0.01) conduction velocity in the group cultured under the influence of the highest amount of cardiac fibroblasts compared to the control group (13.1±1.0 vs 10.6±0.9 control group vs 1:1 human ciCMCs:cardiac fibroblasts [cm/s, mean±SD]). Additionally, action potential duration was significantly prolonged (P<0.05) in the 1:1 human ciCMC:cardiac fibroblast cultures compared to the control group (93.6±4.8 vs 129.9±3.5 control group vs 1:1 human ciCMCs:cardiac fibroblasts [ms, mean±SD]). In conclusion, fibroblastic paracrine signalling has a negative effect on the structural and electrical differentiation of human cardiomyocytes. This not only emphasizes the need to consider the interaction between the transplanted cells and the receiving microenvironment in cardiac regenerative medicine, but also offers new leads to increase the therapeutic potential of this strategy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Leiden University Fund

High-throughput 3D Spheroid Formation and Effective Cardiomyocyte Differentiation from Human iPS Cells Using the Microfabric Vessels EZSPHERETM

High-throughput 3D spheroid formation from human induced pluripotent stem cells (hiPSCs) can be easily performed using the unique microfabric vessels EZSPHERE, resulting in effective and large scale generation of differentiated cells such as cardiomyocytes or neurons. Such hiPSC-derived cardiomyocytes (hiPSC-CMs) or neurons are very useful in the fields of regenerative medicine or cell-based drug safety tests. Previous studies indicated that 3D spheroids arising from hiPSCs are effectively differentiated into high quality hiPSC-CMs by controlling Wnt signals through utilization of the microfabric vessels EZSPHERE. Here, we describe a simple and highly efficient protocol for generating a large number of uniformly sized hiPSC spheroids and inducing them for cardiac differentiation using the EZSPHERE. This method comprises the collection and dissociation of the spheroids from cardiac differentiation medium, in the middle stage of the whole cardiac differentiation process, and re-seeding the obtained single cells into the EZSPHERE to re-aggregate them into uniform hiPSC-CM spheroids with controlled size. This re-aggregation process promotes non-canonical Wnt signal-related cardiac development and improves the purity and maturity of the hiPSC-CMs generated. Graphic abstract: Overview of cardiac differentiation from iPSCs by spheroid formation and reaggregation using EZSPHERE.

Non-Invasive and Continuous Monitoring of Differentiation Process into Cardiomyocytes By 2D-SPR Observation

Introduction Two-dimensional surface plasmon resonance (2D-SPR) measurement method has the advantages of noninvasive and real-time for observation of intracellular reactions. We have already applied this attractive method to monitor cell differentiation noninvasively.P19CL6, mouse embryonal carcinoma cells were cultured in a medium containing DMSO and 5-azacytidine to induce cardiac differentiation. 2D-SPR observation of the cells was then performed every two days to monitor intracellular refractive index change. The resonance angle of cell regions increased as the differentiation progressed. However, in previous study, cells in differentiation process were once detached from the well plate with trypsin treatment and replated on a gold chip and 2D-SPR measurement was done after one day. Therefore, we considered that the cells might be damaged by trypsin treatment exactly. Here, we aimed to realize noninvasive and continual 2D-SPR observation of the cells during cardiac differentiation process. In this experiment, the sensor chip chamber was covered with a gas permeable membrane and cardiac differentiation process from day 4 to day 14 was continually observed with a high resolution of SPR imager. Experimental method In this experiment, P19CL6 cells were cultured on a well plate during the period for formation of embryoid bodies from day 0 to day 4 in the presence of DMSO, and cardiac differentiation was induced on a gold chip from day 4 to day 14 in the presence of DMSO and 5-Azacitidine.The gold sensor chip chamber was covered with a gas permeable membrane to avoid contamination. 2D-SPR observations were carried out every two days to obtain the SPR images of cell regions and to monitor how the resonance angle changes at the cell regions during cardiac differentiation process. Result and discussion It was demonstrated that cardiac differentiation of P19CL6 cells on a gold chip was possible and the SPR images of cell regions were continually observed from day 4 to day 14 without contamination. Though the average of the resonance angles at cell regions decreased from day 4 to day 8, but it changed to increase from day 8 to day 12 and reached the maximum on day 12 and day 14. It is considered that intracellular proteins may be reformed to differentiate from embryonic cells into cardiomyocytes from the day 4 to day 8. The cells became cardiomyocyte-like on the day 10 and beating could be observed on a well plate from day 12.From these results we conclude that continual observation with a SPR imager is very promising method for noninvasive monitoring of cardiac differentiation. Figure 1

Abstract 264: Upstream and Downstream Regulators of Lysophosphatidic Acid Receptor 4 are Critical for Cardiac Differentiation and Repair

The therapeutic application of cell therapy to repair the damaged heart requires a comprehensive understanding of the process of cardiac differentiation of stem cells. We discovered a cardiac-specific marker, lysophosphatidic acid receptor 4 (LPAR4), which is G protein-coupled receptor (GPCR) and demonstrated its functional significance during cardiac differentiation. We screened GPCR expressing on mouse cardiac progenitor cells at differentiation day 3 compared to mouse undifferentiated pluripotent stem cells (PSCs). Among candidates, we identified LPAR4. We have found that in both mouse and human PSCs, LPAR4 has a transient expression pattern during cardiac differentiation. During in vitro differentiation of mouse and human PSCs toward cardiomyocytes, LPAR4 expression peaked for 3-5 days and then and declined immediately. Treatment with ODP (LPAR4 specific agonist) followed by p38MAPK blocker (SB203580) in the cardiac differentiation protocol significantly increased cardiac differentiation efficiency. Then we performed RNA sequencing to find transcription factors that regulate LPAR4. We screened 2-fold up-regulated transcription factors that related to cardiac differentiation during human iPSCs toward cardiomyocytes. The expression of LPAR4 is regulated by SOX17 expression, demonstrating that SOX17 is an upstream regulator of LPAR4. During cardiac differentiation, when knocking down SOX17 by siRNA, the expression of LPAR4 decreased as the expression of SOX17 mRNA decreased, resulting in no progression of cardiac differentiation. Besides, cardiac differentiation did not progress when SOX17 was overexpressed. SOX17 plays a role early in cardiac differentiation and that the expression should be reduced. However, it is assumed that sox17 lentivirus was gene-integrated and continued expression without decreasing expression. SOX17 overexpression needs further study. In conclusion, we demonstrated that LPAR4 is a novel cardiac progenitor cell marker and modulation of the upstream and downstream regulators shown functional significance during cardiac differentiation. Furthermore, our findings provide new insight into cell-free cardiac repair by the modulation of LPAR4 positive cells in the heart. Funding Source: This study was supported by "Strategic Center of Cell and Bio Therapy for Heart, Diabetes & Cancer" (HI17C2085) through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea.

Intrinsic Color Sensing System Allows for Real-Time Observable Functional Changes on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Stem-cell based in vitro differentiation for disease modeling offers great value to explore the molecular and functional underpinnings driving many types of cardiomyopathy and congenital heart diseases. Nevertheless, one major caveat in the application of in vitro differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) involves the immature phenotype of the CMs. Most of the existing methods need complex apparatus and require laborious procedures in order to monitor the cardiac differentiation/maturation process and often result in cell death. Here we developed an intrinsic color sensing system utilizing a microgroove structural color methacrylated gelatin film, which allows us to monitor the cardiac differentiation process of hiPSC-derived cardiac progenitor cells in real time. Subsequently this system can be employed as an assay system to live monitor induced functional changes on hiPSC-CMs stemming from drug treatment, the effects of which are simply revealed through color diversity. Our research shows that early intervention of cardiac differentiation through simple physical cues can enhance cardiac differentiation and maturation to some extent. Our system also simplifies the previous complex experimental processes for evaluating the physiological effects of successful differentiation and drug treatment and lays a solid foundation for future transformational applications.

A novel cardiac differentiation method of a large number and uniformly-sized spheroids using microfabricated culture vessels

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) have great potential for regenerative medicine and drug discovery. In this study, we developed a novel protocol to more reproducibly and efficiently induce cardiomyocytes. A large quantity of uniformly sized spheroids were generated from hiPSCs using microfabricated vessels and induced into cardiac differentiation. In the middle of the cardiac differentiation process, spheroids were then dissociated into single cells and reaggregated into smaller spheroids using the microfabricated vessels. This reaggregation process raised WNT5A and WNT11 expression levels and improved the quality of cardiomyocyte population compared to that in a control group in which dissociation and reaggregation were not performed.

Cardiac differentiation of induced pluripotent stem cells derived from mesenchymal stem cells

Background & Aim Induced pluripotent stem cells (iPSCs) are commonly studied for clinical use due to its flexibility in differentiating into any desirable cellular products. iPSCs are commonly derived from fibroblasts or cells from peripheral blood due to its accessibility. Although not common, we have derived iPSCs from mesenchymal stem cells (MSC) for several purposes. We are developing an allogenic combinatory cellular therapy for heart disease using both MSCs with iPSC derived cardiomyocyte (CM) or cardiac progenitor cells (CPCs). We theorize, by utilizing iPSC derived from MSCs, it provides more compatibility when transplanting the two final cellular products from one donor into patients, thus yielding better therapeutic effects. Also, recent changes in donor qualification increase the financial burden when launching new cellular therapeutics due to the increase of required testing on donor and final cellular product. In this scenario, by reprogramming the MSCs into iPSCs then differentiate into CM, we eliminated the additional cost of qualifying the parent cells of iPSCs. Methods, Results & Conclusion Although cardiac differentiation of iPSC is well studied, the optimal differentiation condition needed to be tested for each cell line used. The initial evaluation must be performed to determine the optimal cell density, the concentration of CHIR99021, and the time point of switching to IWP2. After determining these factors, the cardiac differentiation was performed on both IMR90 (lung fibroblast) and MSC derived iPSCs in order to evaluate the efficiency of differentiation of the latter iPSC. The iPSCs were plated onto Vitronectin coated plates until the desired confluence reached using iPSC culture medium. At day 0 of the cardiac differentiation process, the iPSC culture medium was replaced with CHIR99021in differentiation medium (RPMI-1640 with B-27 without insulin). Two or three days later, IWP2 replaced CHIR99021 in the differentiation medium for another two days. After day 7 of the cardiac differentiation process, cells were maintained with CM culture medium (RPMI-1640 with B-27 with insulin) for another seven days. The medium was changed every three to four days. In summary, only the optimized combination of conditions yield beating CM culture at day 14 from both MSC and IMP90 derived iPSCs. CM derived from both iPSCs stained positive for Troponin T. At day 7 of the cardiac differentiation, the cultures were stained positive for GHRHR, which will be used as a marker for CPC selection. Induced pluripotent stem cells (iPSCs) are commonly studied for clinical use due to its flexibility in differentiating into any desirable cellular products. iPSCs are commonly derived from fibroblasts or cells from peripheral blood due to its accessibility. Although not common, we have derived iPSCs from mesenchymal stem cells (MSC) for several purposes. We are developing an allogenic combinatory cellular therapy for heart disease using both MSCs with iPSC derived cardiomyocyte (CM) or cardiac progenitor cells (CPCs). We theorize, by utilizing iPSC derived from MSCs, it provides more compatibility when transplanting the two final cellular products from one donor into patients, thus yielding better therapeutic effects. Also, recent changes in donor qualification increase the financial burden when launching new cellular therapeutics due to the increase of required testing on donor and final cellular product. In this scenario, by reprogramming the MSCs into iPSCs then differentiate into CM, we eliminated the additional cost of qualifying the parent cells of iPSCs. Although cardiac differentiation of iPSC is well studied, the optimal differentiation condition needed to be tested for each cell line used. The initial evaluation must be performed to determine the optimal cell density, the concentration of CHIR99021, and the time point of switching to IWP2. After determining these factors, the cardiac differentiation was performed on both IMR90 (lung fibroblast) and MSC derived iPSCs in order to evaluate the efficiency of differentiation of the latter iPSC. The iPSCs were plated onto Vitronectin coated plates until the desired confluence reached using iPSC culture medium. At day 0 of the cardiac differentiation process, the iPSC culture medium was replaced with CHIR99021in differentiation medium (RPMI-1640 with B-27 without insulin). Two or three days later, IWP2 replaced CHIR99021 in the differentiation medium for another two days. After day 7 of the cardiac differentiation process, cells were maintained with CM culture medium (RPMI-1640 with B-27 with insulin) for another seven days. The medium was changed every three to four days.

Hsa-miR-335 regulates cardiac mesoderm and progenitor cell differentiation

BackgroundWNT and TGFβ signaling pathways play critical regulatory roles in cardiomyocyte fate determination and differentiation. MiRNAs are also known to regulate different biological processes and signaling pathways. Here, we intended to find candidate miRNAs that are involved in cardiac differentiation through regulation of WNT and TGFβ signaling pathways.MethodsBioinformatics analysis suggested hsa-miR-335-3p and hsa-miR-335-5p as regulators of cardiac differentiation. Then, RT-qPCR, dual luciferase, TOP/FOP flash, and western blot analyses were done to confirm the hypothesis.ResultsHuman embryonic stem cells (hESCs) were differentiated into beating cardiomyocytes, and these miRNAs showed significant expression during the differentiation process. Gain and loss of function of miR-335-3p and miR-335-5p resulted in BRACHYURY, GATA4, and NKX2-5 (cardiac differentiation markers) expression alteration during the course of hESC cardiac differentiation. The overexpression of miR-335-3p and miR-335-5p also led to upregulation of CNX43 and TNNT2 expression, respectively. Our results suggest that this might be mediated through enhancement of WNT and TGFβ signaling pathways.ConclusionOverall, we show that miR-335-3p/5p upregulates cardiac mesoderm (BRACHYURY) and cardiac progenitor cell (GATA4 and NKX2-5) markers, which are potentially mediated through activation of WNT and TGFβ signaling pathways. Our findings suggest miR-335-3p/5p to be considered as a regulator of the cardiac differentiation process.

Arsenic trioxide blocked proliferation and cardiomyocyte differentiation of human induced pluripotent stem cells: Implication in cardiac developmental toxicity

Arsenic trioxide (ATO) has been recommended as the first-line agent for the treatment of acute promyelocytic leukaemia (APL), due to its substantial anticancer effect. Numerous clinical reports have indicated that ATO is a developmental toxicant which can result in birth defects of human beings. But whether arsenic trioxide can lead to human cardiac developmental toxicity remains largely unknown. So the present study aims to explore the influence and mechanisms of ATO on human cardiac development by using a vitro cardiac differentiation model of human induced pluripotent stem cells (hiPSCs). Here we found that clinically achievable concentrations (0.1, 0.5 and 1 μM) of ATO resulted in a significant inhibition of proliferation during the whole process of cardiac differentiation of hiPSCs. Meanwhile, TUNEL assay revealed that ATO could cause cell apoptosis during cardiac differentiation in a concentration-dependent manner. Consistently, we found that ATO reduced the expressions of mesoderm markers Brachyury and EOMES, cardiac progenitor cell markers GATA-4, MESP-1 and TBX-5, and cardiac specific marker α-actinin in differentiated hiPSCs. Furthermore, ATO treatment had caused DNA damage which was shown in the upregulation of γH2AX, a sensitive marker for DNA double-strand breaks. Taken together, ATO blocked cardiomyocyte differentiation, induced apoptosis and cell growth arrest during cardiac differentiation of hiPSCs, which might be associated with DNA damage.

Discovery of Natural Compounds Promoting Cardiomyocyte Differentiation.

The commitment of pluripotent stem cells to the cardiac lineage has enormous potential in regenerative medicine interventions for several cardiac diseases. Thus, it is necessary to understand and regulate this differentiation process for potential clinical application. In this study, we developed defined conditions with chemical inducers for effective cardiac lineage commitment and elucidated the mechanism for high-efficiency differentiation. First, we designed a robust reporter-based platform to screen chemical inducers of cardiac differentiation in the mouse P19 teratocarcinoma cell line. Using this system, we identified two natural alkaloids, lupinine and ursinoic acid, which enhanced cardiomyocyte differentiation of P19 cells in terms of beating colony numbers with respect to oxytocin, and confirmed their activity in mouse embryonic stem cells. By analyzing the expression of key markers, we found that this enhancement can be attributed to the early and rapid induction of the Wnt signaling pathway. We also found that these natural compounds could not only supersede the action of the Wnt3a ligand but also had a very quick response time, allowing them to act as efficient cardiac mesoderm inducers that subsequently promoted cardiomyocyte differentiation. Thus, this study offers a way to develop chemical-based differentiation strategy for high-efficiency cardiac lineage commitment, which has an advantage over currently available methods with complex medium composition and parameters. Furthermore, it also provides an opportunity to pinpoint the key molecular mechanisms pivotal to the cardiac differentiation process, which are necessary to design an efficient strategy for cardiomyocyte differentiation.

The status of MAPK cascades contributes to the induction and activation of Gata4 and Nkx2.5 during the stepwise process of cardiac differentiation

Cardiac differentiation in vitro is a complex, stepwise process that is rigidly governed by a subset of transcription factors and signaling cascades. In this study, we investigated the cooperation of cardiac-specific transcription factors Gata4 and Nkx2.5, as well as mitogen-activated protein kinase (MAPK) cascades. P19 embryonic carcinoma cells were induced into spontaneously beating cardiomyocytes utilizing a two-step protocol that comprised an early stage and a late stage of differentiation. During early-stage differentiation in suspension culture, P19 cells aggregated to form embryoid bodies (EBs), and the Gata4 and Nkx2.5 genes were induced. However, Gata4 expressed at the early stage of differentiation was incapable of activating downstream gene expression, as it was localized in the cytoplasm and prone to degradation. After EBs were plated for late-stage differentiation in adherent culture, the MAPK cascades were highly activated and contributed to the activation of Gata4 and Nkx2.5. Specifically, we revealed that p38 signaling participated in regulating the localization and stabilization of Gata4 and Nkx2.5. Additionally, the JNK cascade regulated late-stage cardiac differentiation; JNK kinase reduced Gata4 stabilization and conversely alleviated Nkx2.5 degradation by direct interaction and phosphorylation of Nkx2.5. Finally, we found that the C-terminal domain of Nkx2.5 was required for its stabilization under conditions of oxidative stress and JNK activation. Overall, our results indicated that the induction and activation of Gata4 and Nkx2.5 during early- and late-stage cardiac differentiation was closely associated with the function of the MAPK signaling cascades.

Hsa-miR-497 as a new regulator in TGFβ signaling pathway and cardiac differentiation process

Cardiosphere-derived cells (CDCs) contain cardiac stem cell subpopulations and are introduced as useful source for cardiac differentiation and therapy. Furthermore, research has highlighted miRNAs important role in various biological processes and cardiogenesis. Here, we intended to investigate the effect of hsa-miR-497 (miR-497) on TGFβ signaling pathway genes expression during the process of CDCs differentiation to cardiomyocytes. CDCs were successfully differentiated to the cardiac-like cells. In this study, we found that after cardiac differentiation induction, miR-497 expression was significantly decreased. Computational miRNA target prediction analyses revealed that TGFβ signaling pathway is a possible target of miR-497. Therefore, miR-497 was overexpressed in CDCs before the induction of differentiation. TGFβ1, TGFβR1, TGFβR2, and SMAD3 genes expression level was decreased after miR-497 overexpression. Also, immunocytochemistry and cell morphology analysis indicated that miR-497 overexpression affecting cardiac differentiation process. Finally, direct interaction of miR-497 with 3′-UTR sequence of TGFβR1 was supported through dual luciferase assay, consistent with miR-497 reported negative effect on SMAD3 expression. Accordingly, here a model of miR-497 involvement in regulation of TGFβ signaling pathway is introduced in which, side branches of TGFβ signaling pathway downregulate miR-497 to ensure upregulation of TGFβR1 and TGFβR2 and finally stronger TGFβ signaling.

Fndc5 knockdown induced suppression of mitochondrial integrity and significantly decreased cardiac differentiation of mouse embryonic stem cells.

Fibronectin type III domain-containing 5 protein (Fndc5) is a glycosylated protein with elevated expression in high energy demanded tissues as heart, brain, and muscle. It has been shown that upregulation of Fndc5 is regulated by peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α), which is known as a master regulator of mitochondrial function and biogenesis. Also, our group indicated that Fndc5 expression increases gradually during cardiac differentiation of mouse embryonic stem cells (mESCs). In this paper, to clarify the importance of Fndc5 in cardiac differentiation, we south to knock down Fndc5 expression by generation a stably transduced mESC line that derives the expression of a short hairpin RNA (shRNA) against Fndc5 gene following doxycycline (Dox) induction. Knock-down of Fndc5 demonstrated a considerable decrease in expression of cardiac progenitor and cardiomyocyte markers. Considering the fact that mitochondria play a crucial role in cardiac differentiation of ESCs, we investigated the role of Fndc5, as a downstream target of PGC1-α, on mitochondrial indices. Results showed that expression of nuclear encoded mitochondrial genes including PGC1-α, Atp5b, Ndufb5, and SOD2 significantly decreased. Moreover, mitochondrial membrane potential (ΔΨm) and relative ATP content of cardiomyocytes decreased markedly with relative ROS level increase. Together, our results suggest that Fndc5 attenuates process of cardiac differentiation of mESCs which is associated with modulation of mitochondrial function and gene expression.

Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM, including lack of T-tubules, immature excitation-contraction coupling, and inefficient Ca-induced Ca release remain major limitations. Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote T-tubule development and mature excitation-contraction coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress). hiPSC-CM were generated using a standard chemical differentiation method supplemented with T3 (triiodothyronine) and/or Dex (dexamethasone) during days 16 to 30 followed by single-cell culture for 5 days on Matrigel mattress. hiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive T-tubule network. Notably, Matrigel mattress was necessary for T-tubule formation. Compared with adult human ventricular cardiomyocytes, T-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of excitation-contraction coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks and promoted RyR2 (ryanodine receptor) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex-treated cells. Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process, which when coupled with further maturation on Matrigel mattress, is sufficient for T-tubule development, enhanced Ca-induced Ca release, and more ventricular-like excitation-contraction coupling. This new hormone maturation method could advance the use of hiPSC-CM for disease modeling and cell-based therapy.

Role of Nitric Oxide, Nitric Oxide Synthase, Soluble Guanylyl Cyclase, and cGMP‐Dependent Protein Kinase I in Mouse Stem Cell Cardiac Development

Introduction and Aim. Nitric oxide (NO) can trigger cardiac differentiation of embryonic stem cells (ESCs), indicating a cardiogenic function of the NO synthetizing enzyme(s) (NOS). However, the involvement of the NO/NOS downstream effectors soluble guanylyl cyclase (sGC) and cGMP activated protein kinase I (PKG-I) is less defined. Therefore, we assess the involvement of the entire NO/NOS/sGC/PKG-I pathway during cardiac differentiation process. Methods. Mouse ESCs were differentiated toward cardiac lineages by hanging drop methodology for 21 days. NOS/sGC/PKG-I pathway was studied quantifying genes, proteins, enzymatic activities, and effects of inhibition during differentiation. Percentages of beating embryoid bodies (mEBs) were evaluated as an index of cardiogenesis. Results and Discussion. Genes and protein expression of enzymes were increased during differentiation with distinctive kinetics and proteins possessed their enzymatic functions. Exogenous administered NO accelerated whereas the blockade of PKG-I strongly slowed cardiogenesis. sGC inhibition was effective only at early stages and NOS blockade ineffective. Of NOS/sGC/PKG-I pathway, PKG-I seems to play the prominent role in cardiac maturation. Conclusion. We concluded that exogenous administered NO and other pharmacological strategies able to increase the activity of PKG-I provide new tools to investigate and promote differentiation of cardiogenic precursors.

Cardiac differentiation of mouse embryonic stem cells is influenced by a PPAR γ/PGC-1α—FNDC5 pathway during the stage of cardiac precursor cell formation

Peroxisome proliferator-activated receptor (PPAR) γ co-activator 1α (PGC-1α) up-regulation induces FNDC5 expression in muscle and consequently causes browning of white adipose tissue (WAT). In addition to skeletal muscle, FNDC5 is mainly expressed in heart and brain tissues. Here, we demonstrate that FNDC5 expression increased during the process of cardiac differentiation of mouse embryonic stem cells (mESCs) similar to PGC-1α and PPARα. To testify the correlation between PGC-1α and FNDC5 in cardiac cell differentiation of mESCs, we utilized specific PPARγ agonist and antagonist in two stages of cardiac differentiation, during and post-cardiac precursor cells (CPCs) formation. Our results indicated that a reduction in PGC-1α expression, via treatment with GW9662 during CPCs formation stage, down-regulated FNDC5 transcript levels as well as mitochondrial markers which negatively influenced on the whole process of cardiac differentiation efficiency. On the other hand, increase PGC-1α expression during CPCs formation stage via rosiglitazone treatment increase FNDC5 and mitochondrial markers transcript levels which enhanced cardiac differentiation efficiency. Importantly, such alteration in PGC-1α expression at post-CPCs formation stage did not affect overall cardiac differentiation rate as expression of FNDC5 and mitochondrial markers were not significantly changed. We concluded that PPARγ agonist and antagonist induced up and down-regulation of PGC-1α and subsequently modulated the process of CPCs formation through an alteration in FNDC5 and mitochondrial markers expression.

Autophagy eliminates cytoplasmic β-catenin and NICD to promote the cardiac differentiation of P19CL6 cells

Autophagy plays important roles in adipogenesis and neuron development. However, how autophagy contributes to cardiac development is not well understood. The main aim of our study was to determine the association between autophagy and myocardial differentiation and its roles in this process. Using a well-established in vitro cardiomyocyte differentiation system, P19CL6 cells, we found that autophagy occurred from the early stage of cardiac differentiation. Blocking autophagy by knocking-down of autophagy-related gene Atg7 or Atg5 inhibited the cardiac differentiation of P19CL6 cells. Further investigation demonstrated that LC3 and P62 could form a complex with β-catenin and NICD, respectively, and promoted the degradation of β-catenin and NICD. Enhancing autophagy promoted the formation of complex, whereas blocking autophagy attenuated the degradation of β-catenin and NICD. Taken together, autophagy could facilitate P19CL6 cells to complete the cardiac differentiation process through blocking Wnt and Notch signaling pathways.

Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient–derived cardiomyocytes

Duchenne muscular dystrophy (DMD)–associated cardiac diseases are emerging as a major cause of morbidity and mortality in DMD patients, and many therapies for treatment of skeletal muscle failed to improve cardiac function. The reprogramming of patients’ somatic cells into pluripotent stem cells, combined with technologies for correcting the genetic defect, possesses great potential for the development of new treatments for genetic diseases. In this study, we obtained human cardiomyocytes from DMD patient–derived, induced pluripotent stem cells genetically corrected with a human artificial chromosome carrying the whole dystrophin genomic sequence. Stimulation by cytokines was combined with cell culturing on hydrogel with physiological stiffness, allowing an adhesion-dependent maturation and a proper dystrophin expression. The obtained cardiomyocytes showed remarkable sarcomeric organization of cardiac troponin T and α-actinin, expressed cardiac-specific markers, and displayed electrically induced calcium transients lasting less than 1 second. We demonstrated that the human artificial chromosome carrying the whole dystrophin genomic sequence is stably maintained throughout the cardiac differentiation process and that multiple promoters of the dystrophin gene are properly activated, driving expression of different isoforms. These dystrophic cardiomyocytes can be a valuable source for in vitro modeling of DMD-associated cardiac disease. Furthermore, the derivation of genetically corrected, patient-specific cardiomyocytes represents a step toward the development of innovative cell and gene therapy approaches for DMD.