Toxicity of ecstasy (MDMA) towards embryonic stem cell-derived cardiac and neural cells

“Ecstasy” or methylenedioxymethamphetamine (MDMA) is primarily a recreational drug commonly used during the child bearing period, thus, there is a major concern regarding the embryonic and fetal toxicity of this drug. Here, we report the cardio- and neuro-toxic effects of MDMA on beating embryoid bodies (EBs) and neural cell-containing EBs derived from mouse embryonic stem cell (ESCs). Based on our linear discriminate function, MDMA is considered to be a moderate or weak teratogen. Moreover, the generation of EBs with neural cell morphology and the expression of MAP2, a mature neuron marker, decrease more when MDMA is administered during the EB formation stage rather than post-plated EBs. In addition, the ID50 (inhibition of differentiation) of EBs with neural cell morphology is less than cardiac cells. In conclusion, MDMA causes a marked reduction in beating cardiomyocytes and neurons in ESC cultures, and this drug has a more potent toxicity on neural rather than cardiac cell differentiation.     

Ghrelin promotes differentiation of human embryonic stem cells into cardiomyocytes

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-[lys³]-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.     

Improvement of the embryonic stem cell test endpoint analysis by use of field potential detection

The embryonic stem cell test (EST) is a validated in vitro method to assess the embryotoxic potential of compounds and is a promising tool for drug screening. EST requires microscopic observation of beating cardiomyocytes differentiated from embryonic stem cells as a toxicological endpoint. However, this process is time-consuming and lacks throughput performance. To improve the analysis, we introduced an electrophysiological method with a microelectrode array system for the evaluation of differentiated cardiomyocytes. Embryotoxic (valproic acid, verapamil, and 5-fluorouracil) and non-embryotoxic (penicillin G, d-camphor, and isoniazid) compounds were assessed with the system. Mouse embryonic stem cells were differentiated into cardiomyocytes and treated with each compound during the differentiation process. The embryotoxicity of each compound was then assessed by measuring the field potentials of differentiated cardiomyocytes using the microelectrode array system, as well as by microscopic evaluation. All the embryotoxic compounds dose-dependently inhibited the field potential formation and the myocardial beating of differentiated cells, while the non-embryotoxic compounds did not affect either endpoint. The detection capabilities of the two assay methods were similar. These results indicated that the field potential measurements can be used as an alternative endpoint of EST. Moreover, the field potential can be measured automatically, introducing a high throughput performance compared to the conventional microscopic observation. We therefore concluded that the endpoint analysis with the microelectrode array system improves the original EST and can be useful for the assessment of the embryotoxic potential of compounds.     

光电成像系统在评价胚胎干细胞体外定向分化为心肌细胞中的应用

目的采用简易光电成像系统记录胚胎干细胞(ES细胞)体外定向分化心肌细胞的搏动频率,为药物诱导ES细胞体外定向分化心肌细胞提供量化评价指标。方法以简易光电成像系统记录心肌细胞搏动频率,并以维A酸和淫羊藿苷诱导ES细胞定向分化心肌细胞为例,收集分化过程中发育依赖性基因(α-肌球蛋白重链、心室肌球蛋白轻链及β-肾上腺素受体)和心肌特异性蛋白(α-辅肌动蛋白和肌钙蛋白T)表达情况,同步分析光电成像信号与分子生物学指标的一致性。结果光电成像系统可灵敏反映分化心肌细胞的搏动频率,在与维A酸和淫羊藿苷共培养体系中,搏动频率与心肌发育依赖性基因、蛋白表达和β-肾上腺素受体形成等一致,可体现心肌分化的不同时间段。结论简易光电成像系统能灵敏记录ES细胞定向分化心肌细胞的搏动频率,此搏动频率与细胞分化成熟程度一致,可望成为药物诱导分化心肌细胞初步评价的量化指标。     

Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test

The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a reliable, scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body (EB) outgrowths compared to cytotoxic effects on undifferentiated murine ES cells and differentiated 3T3 fibroblasts. In order to identify more objective endpoints of differentiation other than the microscopic evaluation of "beating areas" and to adapt the EST to applications in high-throughput screening systems we improved and expanded the EST protocol by establishing molecular endpoints of differentiation. The quantitative expression of sarcomeric myosin heavy chain (MHC) and alpha-actinin genes under the influence of test compounds was studied employing intracellular flow cytometry. Strong embryotoxicants exerted a dose-dependent effect on both the expression levels of MHC and alpha-actinin and the differentiation into beating cardiomyocytes. Furthermore, quantitative FACS (fluorescence-activating cell sorting) analysis showed the same sensitivity for the classification of substances as the conventional endpoint but allowed a significant reduction of the test period. Within 7 days, maximal expression of sarcomeric marker proteins was observed. Our findings indicate that structural proteins of the sarcomere apparatus, alpha-actinin and myosin heavy chain (MHC), seem to be promising candidates to predict developmental toxicity in vivo from in vitro data. Thus, the improved EST holds promise as a new predictive screen for risk assessment with respect to developmental toxicity using stem cell technology and technological advances in the field of gene expression analysis.     

Embryotoxic effects of the marine biotoxin okadaic acid on murine embryonic stem cells

Okadaic acid (OA), a marine toxin produced by dinoflagellates, can accumulate in various bivalve molluscs. In humans, consumption of OA induces acute toxic effects like diarrhoea, nausea, vomiting and abdominal pain. OA is a potent inhibitor of protein phosphatase 1 (PP1) and 2A (PP2A), enzymes that are known to be critical regulators of embryonic development. To determine the embryotoxic potential of OA, we performed two independent cellular in-vitro assays, both of which are applicable for the detection of teratogenic compounds: (i) the validated embryonic stem cell test (EST) based on the morphological analysis of beating cardiomyocytes in embryoid bodies and (ii) the F9 cell assay quantifying the induction of cell differentiation by measuring the emitted luminescence of a reporter gene. In the presence of OA, beating cardiomyocytes in the EST were inhibited and the reporter gene in transiently transfected F9 cells was activated. Furthermore, OA treatment led to rapid morphological changes including cell rounding, the loss of cell-cell contacts and changed electrical impedance as monitored in real time by the xCELLigence system. The two independent bioassays (EST and F9 cell test) detected OA as a potential embryotoxic compound, since OA influences the differentiation process of cultured murine embryonic cells.     

Digital movie analysis for quantification of beating frequencies, chronotropic effects, and beating areas in cardiomyocyte cultures

Software, named Cardio Analyser, was developed for digital movie analysis of beating frequencies, drug-induced chronotropic effects, and quantification of beating areas of contracting cardiomyocyte cultures. A major novelty of the software is the introduction of automated noise filtering and automated movie analysis of beating frequencies and areas of contracting cardiomyocyte cultures. The software was based on the observation that the intensity of light transmitted through a contractive tissue changes periodically in a way that correlates with the contractions. We provided proof of principle for the method by derivation of relevant data from movies of multicellular cardiomyocyte cultures derived from embryonic stem cells. Moreover, we compared the data to equivalent results obtained by extracellular electric field potential recordings. The comparison demonstrated higher sensitivity to chronotropic effects of the beta-adrenoceptor agonist isoprenaline, and hence implied that more embryonic stem cells underwent differentiation into beta-adrenoceptor-responding cardiomyocytes, in the experimental setup applied for movie analysis than in the setup used for extracellular electric field potential recordings. Our study indicates that the movie analysis method may have potential to be optimized for screening in early drug discovery, aiming to identify cardiac drug candidates or to alert for adverse effects on heart functionality or embryonic heart development.     

Usefulness of field potential as a marker of embryonic stem cell-derived cardiomyocytes, and endpoint analysis of embryonic stem cell test

The embryonic stem cell test (EST) is a validated method and a useful screening tool for drug discovery. EST requires microscopic observation of beating cells to be considered cardiomyocytes as an endpoint assay. However, this procedure is time-consuming and limits the throughput performance. Instead of microscopic observation, we previously established a novel assay method based on cardiac field potential as an endpoint. However, cardiac specificity of this field potential is not yet clarified, because beating cells have not been rigorously evaluated as skeletal or cardiomyocyte. Here, we investigated the relationships between field potential, beating, and cardiac troponin T (cTnT) expression, selected as a cardiomyocyte-specific marker, and evaluated suitability of the field potential as a marker for cardiomyocyte in vehicle or 5-fluorouracil treated embryo bodies. Embryoid bodies of mouse embryonic stem cells (D3) were differentiated in a chamber with multi-electrode array for 5 days, and field potential and beating were measured at the end of differentiation. In addition, these chambers were immunohistochemically stained with anti-cTnT antibody, and the correlation between field potential, beating, and cTnT expression was examined. These results indicated the area of field potential or beating mainly coincided with that of cTnT expression. 5-fluorouracil treatment decreased not only the number of field potential detecting electrodes and beating area, but also cTnT expression, and the area of these parameters was also nearly identical. These results indicate that field potential can be used as a suitable cardiac differentiation marker, and can be a promising parameter of EST.     

Stirred suspension culture improves embryoid body formation and cardiogenic differentiation of genetically modified embryonic stem cells

Embryonic stem cells (ESCs) can propagate unlimitedly in vitro and differentiate into cardiomyocytes, which have been proposed as unlimited cell sources for cardiac cell therapy. This was limited by difficulties in large-scale generation of pure cardiomyocytes. In this study, we used stirred bioreactors to optimize the differentiation condition for mass production of embryoid bodies (EBs) derived from genetically modified mouse ESCs. Stirred suspension culture could more efficiently produce EBs and have a more uniform EB population without large necrotic centers, compared with the conventional static culture. Importantly, the cardiac-specific gene expressions (GATA binding protein 4, α-cardiac myosin heavy chain and myosin light chain-2v) were increased within EBs cultured in stirred bioreactor. Stirred suspension culture significantly increased the proportion of spontaneously contracting EBs, yielded a greater percentage of α-sarcomeric actinin-positive cells detected via flow cytometry, and harvested relatively more cardiomyocytes after G418 selection. Stirred suspension culture provided a more ideal culture condition facilitating the growth of EBs and enhancing the cardiogenic differentiation of genetically modified ESCs, which may be valuable in large-scale generation of pure cardiomyocytes.     

Simvastatin modulates mouse embryonic stem cell-derived chondrogenesis in vitro

It has been studied in detail that cellular differentiation during chondrogenesis can be recapitulated in vitro by differentiation of embryonic stem (ES) cells as embryoid bodies (EBs). We here used this model system of cartilage development to analyze the effect of simvastatin, a potentially embryotoxic substance. Statins are a group of drugs used to treat hypercholesterolaemia. We found that simvastatin activated cartilage nodule formation during EB differentiation. Extended application of simvastatin resulted in enhanced expression of cartilage marker molecules and prolonged persistence of cartilage nodules. Expression of collagen type II was upregulated during simvastatin-induced chondrogenic ES cell differentiation as demonstrated by quantitative real time PCR. However, immunostaining for cartilage marker molecules revealed that cartilage nodules within simvastatin-treated EBs were defective, bearing cavities of cell loss. Furthermore, caspase activity was reduced in comparison to untreated controls indicating reduced apoptosis. Taken together, we may speculate that simvastatin prolongs survival of chondrocytes and disrupts cellular integrity of cartilage nodules during EB development by affecting apoptotic mechanisms. The study underlines that ES cell-derived EBs are a useful in vitro model to screen substances for their embryotoxic and teratogenic potential.     

Effect of Panax ginseng components on the differentiation of mouse embryonic stem cells into cardiac-like cells

Bioactive compounds that may control the specific differentiation from mouse embryonic stem (ES) cells into cardiac-like cells have been screened from herbal medicines. Among seven preparations, Panax ginseng was found to promote the differentiation into beating cells and to sustain their beating for longer than the control. Active compounds were found in its water-soluble fraction. Although they were not isolated, their candidates were surveyed in 42 compounds selected from the database of P. ginseng. Finally we found that vitamin B12 (VB12) and methionine were active. VB12 accelerated the differentiation into beating cells and made the beating rate constantly 100%. Moreover, VB12 was effective in the recovery of beating that was inhibited by spermine action. The mechanism of action of VB12 is discussed in termo of the relevance of intercellular electrical signal transduction.     

Expression profiles of histone lyslne demethylases during cardiomyocyte differentiation of mouse embryonic stem cells

Aim： Histone lysine demethylases （KDMs） control the lineage commitments of stem cells. However, the KDMs involved in the determination of the cardiomyogenic lineage are not fully defined. The aim of this study was to investigate the expression profiles of KDMs during the cardiac differentiation of mouse embryonic stem cells （mESCs）. Methods： An in vitro cardiac differentiation system of mESCs with Brachyury （a mesodermal specific marker） and FIk-1^＋/Cxcr4^＋ （dual cell surface biomarkers） selection was used. The expression profiles of KDMs during differentiation were analyzed using Q-PCR. To understand the contributions of KDMs to cardiomyogenesis, the mESCs on differentiation d 3.5 were sorted by FACS into Brachyury^＋ cells and Brachyury cells, and mESCs on d 5.5 were sorted into FIk-1^＋/Cxcr4^＋ and FIk-1-/Cxcr4- cells. Results： mESCs were differentiated into spontaneously beating cardiomyocytes that were visible in embryoid bodies （EBs） on d 7. On d 12-14, all EBs developed spontaneously beating cardiomyocytes. Among the 16 KDMs examined, the expression levels of Phf8, Jaridla, Jhdmld, Utx, and Jmjd3 were increased by nearly 2-6-fold on d 14 compared with those on d O. Brachyury^＋ cells showed higher expression levels of Jmjd3, Jmjd2a and Jhdmld than Brachyury- cells. A higher level of Jmjd3 was detected in FIk-1^＋/Cxcr4^＋ cells, whereas the level of Jmjd2c was lower in both Brachyury^＋ cells and FIk-1^＋/Cxcr4^＋ cells. Conclusion： KDMs may play important roles during cardiomyogenesis of mESCs. Our results provide a clue for further exploring the roles of KDMs in the cardiac lineage commitment of mESCs and the potential interference of cardiomyogenesis.     

Disruption of cardiogenesis in human embryonic stem cells exposed to trichloroethylene

Trichloroethylene (TCE) is ubiquitous in our living environment, and prenatal exposure to TCE is reported to cause congenital heart disease in humans. Although multiple studies have been performed using animal models, they have limited value in predicting effects on humans due to the unknown species‐specific toxicological effects. To test whether exposure to low doses of TCE induces developmental toxicity in humans, we investigated the effect of TCE on human embryonic stem cells (hESCs) and cardiomyocytes (derived from the hESCs). In the current study, hESCs cardiac differentiation was achieved by using differentiation medium consisting of StemPro‐34. We examined the effects of TCE on cell viability by cell growth assay and cardiac inhibition by analysis of spontaneously beating cluster. The expression levels of genes associated with cardiac differentiation and Ca²⁺ channel pathways were measured by immunofluorescence and qPCR. The overall data indicated the following: (1) significant cardiac inhibition, which was characterized by decreased beating clusters and beating rates, following treatment with low doses of TCE; (2) significant up‐regulation of the Nkx2.5/Hand1 gene in cardiac progenitors and down regulation of the Mhc‐7/cTnT gene in cardiac cells; and (3) significant interference with Ca²⁺ channel pathways in cardiomyocytes, which contributes to the adverse effect of TCE on cardiac differentiation during early embryo development. Our results confirmed the involvement of Ca²⁺ turnover network in TCE cardiotoxicity as reported in animal models, while the inhibition effect of TCE on the transition of cardiac progenitors to cardiomyocytes is unique to hESCs, indicating a species‐specific effect of TCE on heart development. This study provides new insight into TCE biology in humans, which may help explain the development of congenital heart defects after TCE exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1372–1380, 2016.     

Monitoring of gene expression in differentiation of embryoid bodies from cynomolgus monkey embryonic stem cells in the presence of bisphenol A

An embryonic stem (ES) cell differentiation model would facilitate analysis of developmental processes at the cellular level and the effects of embryotoxic and teratogenic factors in vitro. We explored the use of differentiation of embryoid bodies (EBs) from cynomolgus monkey ES cells for embryotoxicity testing. We determined the mRNA expression of various genes using real-time RT-PCR. Oct-3/4 expression was almost completely suppressed on day 14, suggesting that ES cells reached differentiated status in around 14 days. mRNA expression of E-cadherin, connexin 43, caveolin-1, and argininosuccinate synthetase was reproducibly suppressed during EB differentiation in 7-32% of ES cells in three separate experiments. Although these may not be "general stemness marker genes" such as Oct-3/4, they could play a role in readying stem cells for differentiation in response to deletion of signals from feeder cells. Next, we examined the effects of bisphenol A (BPA) on the mRNA expression of several differentiation marker genes for ES cells. That of PAX-6, an ectoderm marker, with 0, 0.1, and 10 microM BPA in 21-day EBs was 3,500%, 6,668%, and 8,394%, respectively, compared with ES cells. The difference between doses of 0 and 10 microM BPA in 21-day EBs was statistically significant (p=0.049). Pax-6 activation in the presence of BPA may interfere with the development of eyes, sensory organs, and certain neural and epidermal tissues usually derived from ectodermal tissues. Differentiation of EBs from cynomolgus monkey ES cells could be a useful model for detecting gene expression changes in response to chemical exposure.     

An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. II. Testing of compounds

The embryonic stem cell test (EST) developed by Spielmann et al. [Spielmann, H., Pohl, I., Doering, B., Liebsch, M., Moldenhauer, F., 1997. The embryonic stem cell test, an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. In Vitro. Toxicol. 10, 119–127] is currently the most promising in vitro assay to predict the embryotoxic potential of compounds. In this assay the disturbance of the differentiation of embryonic stem (ES) cells into contracting cardiomyocytes by test compounds as well as the direct cytotoxicity of the test compounds on ES cells and 3T3 fibroblasts is analyzed. On the basis of these results and by applying a biostatistical prediction model (PM) [Genschow, E., Scholz, G., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., Spielmann, H., 2000. Development of prediction models for three in vitro embryotoxicity tests in an ECVAM validation study. In Vitr. Mol. Toxicol. 13, 51–66; Genschow, E., Spielmann, H., Scholz, G., Pohl, I., Seiler, A., Clemann, N., Bremer, S., Becker, K., 2004. Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern. Lab. Anim. 32, 209–244; Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern. Lab. Anim. 30, 151–176] test compounds can be classified as non-embryotoxic, weakly or strongly embryotoxic. In order to introduce a further endpoint into the EST, the disturbance of vasculogenesis and/or angiogenesis, a protocol to differentiate ES cells into endothelial cells, was established in the accompanying paper. PECAM-1 and VE-Cadherin gene expressions, quantified by real-time TaqMan^® PCR, were shown to be appropriate molecular markers for the differentiation of ES cells into endothelial cells. In the present study, the disturbance of the differentiation of ES cells into endothelial cells (i.e. the reduction in the expression of PECAM-1 and VE-Cadherin) by six test compounds with known embryotoxic potential was investigated: all-trans-retinoic acid (RA) and 5-fluorouracil (5-FU) are strongly embryotoxic, diphenylhydantoin (DPH) and valproic acid (Val) are weakly embryotoxic and saccharin (Sacch) and penicillin G (Pen G) are non-embryotoxic. In a first step the concentration of the test compound resulting in a 50% inhibition of PECAM-1 and VE-Cadherin gene expression and the concentration leading to a 50% decrease in the viability of ES cells and 3T3 fibroblasts were determined. In a second step and in a first attempt to assess the predictive potential of the newly developed test system the concentration values obtained were applied in the PM of the established EST to classify the selected test compounds. All six test compounds were correctly classified (i.e. the data obtained in vitro correlated with their known embryotoxic potential in vivo). Taken together it can be concluded that the disturbance of the differentiation of murine ES cells into endothelial cells represents a very promising new endpoint in a broadened EST with PECAM-1 and VE-Cadherin as specific differentiation marker genes.     

Impedance-based detection of beating rhythm and proarrhythmic effects of compounds on stem cell-derived cardiomyocytes

The xCELLigence real time cell analyzer Cardio system offers a new system for real-time cell analysis that measures impedance-based signals in a label-free noninvasive manner. The aim of this study was to test whether impedance readings are a useful tool to detect compound effects on beating frequency (beats per minute, bpm) and arrhythmias of human induced pluripotent stem cell- and a mouse embryonic stem cell-derived cardiomyocyte line (hiPSC-CM and mESC-CM, respectively). Baseline values for control wells were 45±3 and 179±6 bpm, respectively (n=6). Correspondingly, isoproterenol increased beating frequency by 77% and 71%, whereas carbachol decreased frequency by 11% and 100% (stopped in 5/6 mESC-CM wells). E-4031 decreased beating rate and caused arrhythmias in both cell types, however, more pronounced in the human iPSC-CMs. Amlodipine inhibited contractions in both models, and T-type calcium channel block strongly reduced beating rate and eventually stopped beating in mESC-CM but caused a smaller effect in hiPSC-CM. The results of this initial study show that, under the right conditions, the beating frequency of a monolayer of cells can be stably recorded over several days. Additionally, the system detects changes in beating frequency and amplitude caused by added reference compounds. This assay system has the potential to enable medium-throughput screening, but for implementation into routine daily work, extended validation, testing of additional batches of cardiomyocytes, and further assay optimization (e.g., frequency of media exchange, growth matrix, seeding density, age of cells after plating, and temperature control) will be needed.     

Comparison of Matrigel and gelatin substrata for feeder-free culture of undifferentiated mouse embryonic stem cells for toxicity testing.

Murine embryonic stem (mES) cells have been used to evaluate cytotoxicity and developmental injury following exposure to embryotoxic agents. However, maintaining a homogeneous population of undifferentiated mES cells for this purpose has been complicated by the need for continuous co-culture with murine embryonic fibroblast (mEF) cells or limited passaging on plastic surfaces coated with gelatin. Here, we compare the synthetic basement membrane Matrigel with 0.1% gelatin substratum for feeder-free propagation of undifferentiated mES cells. Biomarkers of pluripotentiality, chromosome number, caspase-3 expression, and cardiomyocyte differentiation were monitored for mES cells cultured on Matrigel or 0.1% gelatin up to passage 7 (P7). Our results suggest that choice of substratum had no significant effect on population doubling time, cell viability, stage-specific embryonic antigen-1 (SSEA-1) expression, or early passage formation of beating cardiomyocytes (all P>or=0.09). In other comparisons, however, Matrigel supported significantly higher synthesis of alkaline phosphatase (7.7x10(-3)+/-0.8 vs 6.6x10(-3)+/-0.8 units/liter/cell, respectively, P=0.012), overall expression of activated caspase-3 following exposure to 5, 10, 50, 100 and 500 parts per billion (ppb) sodium arsenite (P<0.0001), and percent development to beating cardiomyocytes at P7 (P=0.01). Together, our findings suggest that Matrigel shows promise as a substrate for feeder-free propagation of undifferentiated mES cells for embryotoxicity endpoints.     

Oxytocin in the heart regeneration

We have demonstrated that entire oxytocin (OT) system is synthesized in the rat and human heart and this hormone is implicated in several cardiac functions including stem cells differentiation into cardiomyocytes. These observations led us to the invention of OT as an inducer of cardiomyogenesis (US20060205636A1). We also proposed the use of OT, its functional derivatives, and/or physiological precursors as well as nucleic acids capable of encoding OT as cell-differentiating and useful agents for treating or preventing diseases, such as heart diseases associated with loss of cardiomyocytes. The invention is relevant to the use of OT or OT-related compounds. OT is claimed as an inducer that promotes the differentiation of non-cardiomyocytes (e.g. stem/progenitor cells) in situ, which can be used to repair, restore or fortify damaged cardiac tissue or in cell culture in order to provide material for cell or tissue grafting in the heart. Recent reports documented the significant progress in the research on the role of OT in cardiovascular regulation. Most of the results are consistent with the postulated cardioprotective role of OT. In this review, new data are discussed in the context of the main points presented in the invention.