Dose response effects of phtalates on stem cell differentiation towards cardiomyocytes
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ORGANISM(S): Mus musculus
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PROVIDER: S-DIXA-071 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.
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Project description:Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca(2+)) imaging, and intracellular recording techniques, we found that intracellular Ca(2+)-cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca(2+)-handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to influence early cardiac development. Our study is the first to demonstrate that non-cardiomyocytes influence electrophysiological maturation of early hESC-CMs in cultures. Defining the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.
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Project description:Myc is activated in many tumours, yet, paradoxically, stimulates differentiation in mammalian epidermis. To test whether the epidermis responds differently to different levels of Myc, we treated K14MycER transgenic mice with a range of concentrations of the inducing agent, 4-hydroxy-tamoxifen (4OHT). Proliferation was stimulated at all levels of Myc activity; sebocyte differentiation was stimulated at low and intermediate levels; and interfollicular epidermal differentiation at intermediate and high levels. Mutational inactivation of the Myc p21 activated kinase 2 (PAK2) phosphorylation sites increased Myc activity and further enhanced epidermal differentiation. We conclude that Myc induced differentiation acts as a fail-safe device to prevent uncontrolled proliferation and neoplastic conversion of epidermal stem cells expressing high levels of Myc.
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2019-12-16 | PXD013426 | Pride
Project description:Chronic arsenic exposure is associated with increased morbidity and mortality for cardiovascular diseases. Arsenic increases myocardial infarction mortality in young adulthood, suggesting that exposure during foetal life correlates with cardiac alterations emerging later. Here, we investigated the mechanisms of arsenic trioxide (ATO) cardiomyocytes disruption during their differentiation from mouse embryonic stem cells. Throughout 15 days of differentiation in the presence of ATO (0.1, 0.5, 1.0??M) we analysed: the expression of i) marker genes of mesoderm (day 4), myofibrillogenic commitment (day 7) and post-natal-like cardiomyocytes (day 15); ii) sarcomeric proteins and their organisation; iii) Connexin 43 and iv) the kinematics contractile properties of syncytia. The higher the dose used, the earlier the stage of differentiation affected (mesoderm commitment, 1.0??M). At 0.5 or 1.0??M the expression of cardiomyocyte marker genes is altered. Even at 0.1??M, ATO leads to reduction and skewed ratio of sarcomeric proteins and to a rarefied distribution of Connexin 43 cardiac junctions. These alterations contribute to the dysruption of the sarcomere and syncytium organisation and to the impairment of kinematic parameters of cardiomyocyte function. This study contributes insights into the mechanistic comprehension of cardiac diseases caused by in utero arsenic exposure.
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Project description:Burgeoning applications of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in disease modeling, regenerative medicine, and drug screening have broadened the usage of hiPSC-CMs and entailed their long-term storage. Cryopreservation is the most common approach to store hiPSC-CMs. However, the effects of cryopreservation and recovery on hiPSC-CMs remain poorly understood. Here, we characterized the transcriptome, electro-mechanical function, and drug response of fresh hiPSC-CMs without cryopreservation and recovered hiPSC-CMs from cryopreservation. We found that recovered hiPSC-CMs showed upregulation of cell cycle genes, similar or reduced contractility, Ca2+ transients, and field potential duration. When subjected to treatment of drugs that affect electrophysiological properties, recovered hiPSC-CMs showed an altered drug response and enhanced propensity for drug-induced cardiac arrhythmic events. In conclusion, fresh and recovered hiPSC-CMs do not always show comparable molecular and physiological properties. When cryopreserved hiPSC-CMs are used for assessing drug-induced cardiac liabilities, the altered drug sensitivity needs to be considered.
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Project description:Human pluripotent stem cells were aggregated in mTeSR medium for 4 days and subjected to an optimzed small molecule-based cardiac differentiation protocol (modified from Kempf et. al., Stem Cell Reports, 2014) in agitated Erlenmeyer flasks. Differentiation was induced using 7.5µM CHIR99021 (day 0). Cardiac mesoderm specification was carried out using 5µM IWP2 either directly after CHIR99021 exposure after 24h (day 1; optimized protocol) or after 72h hours (day 3; standard procedure). RNA samples were harvested at undifferentiated state (day 0), after differentiation induction (day 1) and after 3 days with and without IWP2 treatment.
2019-01-13 | E-MTAB-6828 | biostudies-arrayexpress
Project description:Identification of factors that direct embryonic stem (ES) cell (ESC) differentiation into functional cardiomyocytes is essential for successful use of ESC-based therapy for cardiac repair. Neuregulin-1 (NRG1) and microRNA play important roles in the cardiac differentiation of ESCs. Understanding how NRG1 regulates microRNA will provide new mechanistic insights into the role of NRG1 on ESCs. It may also lead to the discovery of novel microRNAs that are important for ESC cardiac differentiation. The objective of this study was to assess the microRNA expression profile during NRG1-induced ESC cardiac differentiation. Murine ESCs were incubated with a recombinant NRG1? or an inhibitor of ErbB2 or ErbB4 during hanging drop-induced cardiac differentiation. The expression of cardiac-specific markers and microRNAs was analyzed by RT-PCR and microRNA array, respectively. We found that the expression of NRG1 and the ErbB receptors was increased during hanging drop-induced cardiac differentiation of ESCs. NRG1 stimulation during a specific developmental window enhanced, while inhibition of the ErbB2 or ErbB4 receptor inhibited, cardiac differentiation of ESCs. NRG1 increased the expression of mmu-miR-296-3p and mmu-miR-200c*, and decreased mmu-miR-465b-5p. Inhibition of mmu-miR-296-3p or mmu-miR-200c* decreased, while inhibition of mmu-miR-465-5p increased, the differentiation of ESCs into the cardiac lineage. This is the first report demonstrating that microRNAs are differentially regulated by NRG1-ErbB signaling during cardiac differentiation of ESCs. This study has also identified new microRNAs that are important for ESC cardiac differentiation.
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Project description:AIM: Ghrelin is involved in regulating the differentiation of mesoderm-derived precursor cells. The aim of this study was to investigate whether ghrelin modulated the differentiation of human embryonic stem (hES) cells into cardiomyocytes and, if so, whether the effect was mediated by growth hormone secretagogue receptor 1α (GHS-R1α). METHODS: Cardiomyocyte differentiation from hES cells was performed according to an embryoid body (EB)-based protocol. The cumulative percentage of beating EBs was calculated. The expression of cardiac-specific markers including cardiac troponin I (cTnI) and α-myosin heavy chain (α-MHC) was detected using RT-PCR, real-time PCR and Western blot. The dispersed beating EBs were examined using immunofluorescent staining. RESULTS: The percentage of beating EBs and the expression of cTnI were significantly increased after ghrelin (0.1 and 1 nmol/L) added into the differentiation medium. From 6 to 18 d of differentiation, the increased expression of cTnI and α-MHC by ghrelin (1 nmol/L) was time-dependent, and in line with the alteration of the percentages of beating EBs. Furthermore, the dispersed beating EBs were double-positively immunostained with antibodies against cTnI and α-actinin. However, blockage of GHS-R1α with its specific antagonist D-[lys(3)]-GHRP-6 (1 μmol/L) did not alter the effects of ghrelin on cardiomyocyte differentiation. CONCLUSION: Our data show that ghrelin enhances the generation of cardiomyocytes from hES cells, which is not mediated via GHS-R1α.
| S-EPMC4010076 | biostudies-other
Project description:AimsThe differentiation of embryonic stem (ES) cells into energetically efficient cardiomyocytes contributes to functional cardiac repair and is envisioned to ameliorate progressive degenerative cardiac diseases. Advanced cell maturation strategies are therefore needed to create abundant mature cardiomyocytes. In this study, we tested whether the redox-sensitive heme oxygenase-1/carbon monoxide (HO-1/CO) system, operating through mitochondrial biogenesis, acts as a mechanism for ES cell differentiation and cardiomyocyte maturation.ResultsManipulation of HO-1/CO to enhance mitochondrial biogenesis demonstrates a direct pathway to ES cell differentiation and maturation into beating cardiomyocytes that express adult structural markers. Targeted HO-1/CO interventions up- and downregulate specific cardiogenic transcription factors, transcription factor Gata4, homeobox protein Nkx-2.5, heart- and neural crest derivatives-expressed protein 1, and MEF2C. HO-1/CO overexpression increases cardiac gene expression for myosin regulatory light chain 2, atrial isoform, MLC2v, ANP, MHC-β, and sarcomere α-actinin and the major mitochondrial fusion regulators, mitofusin 2 and MICOS complex subunit Mic60. This promotes structural mitochondrial network expansion and maturation, thereby supporting energy provision for beating embryoid bodies. These effects are prevented by silencing HO-1 and by mitochondrial reactive oxygen species scavenging, while disruption of mitochondrial biogenesis and mitochondrial DNA depletion by loss of mitochondrial transcription factor A compromise infrastructure. This leads to failure of cardiomyocyte differentiation and maturation and contractile dysfunction.InnovationThe capacity to augment cardiomyogenesis via a defined mitochondrial pathway has unique therapeutic potential for targeting ES cell maturation in cardiac disease.ConclusionOur findings establish the HO-1/CO system and redox regulation of mitochondrial biogenesis as essential factors in ES cell differentiation as well as in the subsequent maturation of these cells into functional cardiac cells.
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