Embryonic stem cell-derived cardiac, neuronal and pancreatic cells as model systems to study toxicological effects

Primary cultures or established cell lines of vertebrates are commonly used to analyse the cytotoxic potential of chemical factors, drugs and xenobiotics in vitro. An alternative approach will be provided by permanent lines of pluripotent embryonic stem (ES) cells, which are able to differentiate into specialised somatic cell types in vitro. Here, we demonstrate the capacity of ES cells to generate functional cardiac, neuronal and pancreatic cells. We show that during ES cell differentiation, tissue-specific genes, proteins as well as functional properties are expressed in a developmentally regulated manner recapitulating processes of early embryonic development. We present data that show the use of ES-derived cardiomyocytes and dopaminergic neurons in toxicological studies and the potential of ES-derived pancreatic beta-like cells in future in vitro assays. The application of these differentiation systems to human ES cells opens up new perspectives in basic and applied toxicology.     

Embryonic stem cells in predictive cardiotoxicity: laser capture microscopy enables assay development.

Embryonic stem (ES) cells offer unprecedented opportunities for in vitro drug discovery and safety assessment of compounds. Cardiomyocytes derived from ES cells enable development of predictive cardiotoxicity models to increase the safety of novel drugs. Heterogeneity of differentiated ES cells limits the development of reliable in vitro models for compound screening. We report an innovative and robust approach to isolate ES-derived cardiomyocytes using laser microdissection and pressure catapulting (LMPC). LMPC cells were readily applied onto 96-well format in vitro pharmacology assays. The expression of developmental and functional cardiac markers, Nkx 2.5, MLC2V, GATA-4, Connexin 43, Connexin 45, Serca-2a, cardiac alpha actin, and phospholamban, among others, was confirmed in LMPC ES-derived cardiomyocytes. Functional assays exhibited cardiac-like response to increased extracellular calcium (5.4 mM extracellular Ca2+) and L-type calcium channel antagonist (1 microM nifedipine). In conclusion, laser microdissection and pressure catapulting is a robust technology to isolate homogeneous ES-derived cell types from heterogeneous populations applicable to assay development.     

Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum

Aim： There is increasing evidence indicating that embryonic stem （ES） cells are capable of differentiating into hepatocyte-like cells in vitro. However, it is neces- sary to improve the differentiation efficiency so as to promote the clinical application. Here, we report an efficient culture system to support hepatocyte differentiation from ES cells by utilizing cholestatic serum. Methods： One week after the induction of El4 mouse ES cells into hepatocytes with sodium butyrate, cholestatic serum was added into the culture system at various concentrations and hepatocyte-like cells were induced to proliferate. The morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunocytochemistry, and RT-PCR, respectively. The function of glycogen stor- age of the differentiated cells was detected by Periodic acid-Schiff （PAS） reaction, and the ratio of hepatic differentiation was determined by counting the albumin and PAS-positive cells. Results： In the presence of conditional selective medium containing cholestatic serum, numerous epithelial cells resembling hepatocytes were observed. The RT-PCR analysis showed that undifferentiated ES cells did not express any hepatic-specific markers; however, in the presence of sodium butyrate and conditional selective medium containing cholestatic serum, hepatic differentiation markers were detected. Immunofluorescence staining showed that those ES-derived hepatocytes were αfetoprotein, albumin, and cytokeratin 18 positive, with the ability of storing glycogen. Further determination of the hepatic differentiation ratio showed that the application of cholestatic serum efficiently enriched ES-derived hepatocyte-like cells by inducing lineage differentiation and enhancing lineage proliferation. Conclusion： The conditional selective medium containing cholestatic serum is optimal to selectively enrich hepatocyte-like cells from mixed differentiated ES cells, which may provide a novel method to improve the hepatic differentiation ratio of ES cells.     

Transplanting stem cells: potential targets for immune attack. Modulating the immune response against embryonic stem cell transplantation

The curative promise of stem cells and their descendants for tissue regeneration and repair is currently the subject of an intense research effort worldwide. If it proves feasible to differentiate stem cells into specific tissues reliably and safely, this approach will be invaluable in the treatment of diseases that lead to organ degeneration or failure, providing an alternative or supplementary source of tissue for transplantation. Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of a pre-implantation blastocyst that can produce all cells and tissues of the foetus. In recent years, several laboratories have described the directed differentiation of ES cells into multiple mature cell types including: cardiomyocytes; haemopoietic cells; hepatocytes; neurones; muscle cells and both endocrine and exocrine cells of the pancreas. How the immune system of the host will respond when these ES cell-derived mature cells are transplanted is ill defined. This review will focus on the potential mechanisms that the immune system could use to target ES cell-derived transplants and how unwanted responses might be prevented.     

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。     

Optimization of adenovirus vectors for transduction in embryonic stem cells and induced pluripotent stem cells

Because embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can differentiate into various types of cells in vitro, they are considered as a valuable model to understand the processes involved in the differentiation into functional cells as well as an unlimited source of cells for therapeutic applications. Efficient gene transduction method is one of the powerful tools for the basic researches and for differentiating ES and iPS cells into lineage-committed cells. Recently, we have developed an adenovirus (Ad) vector for efficient transduction into ES and iPS cells. We showed that Ad vectors containing the cytomegalovirus enhancer/β-actin promoter with β-actin intron (CA) promoter or the elongation factor (EF)-1α promoter were the appropriate for the transduction into ES and iPS cells. We also found that enforced expression of a PPARγ gene or a Runx2 gene into mouse ES and iPS cells by an optimized Ad vector markedly augmented the differentiation of adipocytes or osteoblasts, respectively. Thus, a gene transfer technique using an Ad vector could be an advantage for the regulation of stem cell differentiation and could be applied to regenerative medicine based on ES and iPS cells.     

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.     

Myocardial adenosine does not correlate with the protection mediated by ischaemic or pharmacological preconditioning in rat heart

1. We tested the hypothesis that ischaemic preconditioning of the rat heart activates cardiovascular adenosine formation to provide enhanced cardioprotection. 2. Rat isolated perfused hearts were either non-preconditioned, preconditioned with 5 min ischaemia or treated for 5 min with the alpha1-adrenoceptor agonist phenylephrine (50 micro mol/L) before being subjected to 30 min sustained ischaemia followed by 30 min reperfusion. Isolated cardiomyocytes were either non-preconditioned, subjected to 10 min simulated ischaemia or treated for 10 min with phenylephrine (50 micro mol/L) before being subjected to 30 min simulated ischaemia. Functional recovery of hearts and cell viability were used as indices of the effects of ischaemia. 3. Myocardial adenosine, as well as intracellular pH, was determined at the end of the preconditioning period and at 10, 20 and 30 min of sustained ischaemia. Intracellular pH was also determined during the reperfusion. 4. Ischaemic or pharmacological preconditioning with phenylephrine correlated with an improved functional recovery of perfused hearts during reperfusion and increased cell viability during ischaemia. 5. In perfused hearts, ischaemic preconditioning resulted in increased adenosine production in the myocardium during the following sustained ischaemia. However, in isolated cardiomyocytes, adenosine levels during sustained ischaemia were lower in ischaemically preconditioned cells compared with the respective non-preconditioned cardiomyocytes. 6. The increase in adenosine production was not observed in hearts preconditioned with phenylephrine instead of transient ischaemia. Similarly, pharmacological preconditioning resulted in decreased adenosine levels during sustained ischaemia in isolated cardiomyocytes. 7. Intracellular pH was preserved during ischaemia to the same extent in both ischaemically or pharmacologically preconditioned hearts and cardiomyocytes, indicating that less acidosis during ischaemia is related to protection. 8. Taken together, the results suggest that cardioprotection does not necessarily correlate with increased adenosine production. Thus, adenosine concentration is not crucial to the beneficial effects of preconditioning in rat heart.     

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.     

Stimulation of P19CL6 with multiple reagents induces pulsating particles in vivo

OBJECTIVE: Injection of stem cells into ischaemic areas of the heart is expected to be an effective method for myocardial regeneration. The embryogenic carcinoma (EC) cell line P19CL6 is known to differentiate into cardiomyocytes when cultured with dimethyl sulfoxide (DMSO) and is expected to be a promising source for regenerative therapy in cardiac disease. To establish a high-yield method of cardiomyocyte differentiation, P19CL6 cells were double-stimulated with 5-azacytidine. Double stimulation-induced cardiomyocytes were also transplanted into ectopic sites in mice and their function evaluated. METHODS AND RESULTS: To induce differentiation under adherent conditions, P19CL6 cells were incubated in growth medium with 10 microM 5-azacytidine for 24 h. After 5-azacytidine treatment, P19CL6 cells were incubated with 1% DMSO for nine days until they began to pulsate. Prior to transplantation, cells were treated again with 5-azacytidine. Differentiated cells were injected into the greater omentum, para-aorta region of the retroperitoneum and peri-femoral artery of adult BALB/c nude mice. Nine days after transplantation, irregularly pulsating tissues at a rate slower than the host heart were observed in the transplanted sites. Light microscopy showed formation of cardiac muscle tissues originating from P19CL6 cells. Differentiated cardiomyocytes were positive for cardiac troponin I, cadherin and alpha-smooth muscle actin, and the expressions of Csx/Nkx2.5 and GATA4 mRNAs were up-regulated. Electron microscopy demonstrated components specific to cardiomyocytes, such as Z-bands, desmosomes, fasciae adherens, myofibrils and mitochondria, which confirmed successful heterotopic cardiac muscle differentiation from P19CL6 cells. CONCLUSION: This study demonstrated high-yield cardiac muscle differentiation of P19CL6 by 5-azacytidine and DMSO double stimulation and successful formation of cardiac muscle-like tissue by ectopic transplantation of cardiomyocytes derived from P19CL6 into the retroperitoneal area as well as into the peripheral vessel area.     

Differentiation of bone marrow mesenchymal stem cells induced by myocardial medium under hypoxic conditions

AIM: To explore whether bone marrow mesenchymal stem cells (MSC) can differentiate into myocardial-like cells induced by myocardial medium, especially the hypoxia/reoxygenation-conditioned medium of cardiomyocytes. METHODS: Myocardial cells obtained from neonatal Sprague-Dawley rat ventricles were isolated and cultured in vitro and a hypoxia reoxygenation model was established. The MSC isolated from adult Sprague-Dawley rats were purified and then incubated with 3 different mediums: medium A- the conditioned medium of normal cardiomyocytes; medium B - the conditioned medium of cardiomyocytes after hypoxia reoxygenation; and the control medium - ordinary medium. The expressions of the cardiac myosin heavy chain (MHC), troponin T (TnT) and connexin 43 were investigated in the MSC after 24 h, 48 h and 72 h cultivation, respectively. RESULTS: The MSC expressed MHC and TnT when incubated with the conditioned medium of cardiomyocytes after hypoxia reoxygenation, but did not express connexin 43. None of MHC, TnT and connexin 43 was detected in the MSC incubated with the conditioned medium of normal cardiomyocytes. CONCLUSION: The results indicate for the first time that myocardial medium for hypoxic preconditioning can induce MSC differentiation into myocardial-like cells.     

Peptidomimetic small-molecule compounds promoting cardiogenesis of stem cells

Embryonic stem (ES) cells may be used as an alternative source of functionally intact cardiomyocytes for ischemic heart disease. Several natural and synthetic small molecules have been identified as useful tools for controlling and manipulating stem cell renewal and differentiation. Currently, there is an urgent requirement for novel small molecules that specifically induce differentiation of stem cells into cardiomyocytes. To identify compounds that promote cardiomyogenesis of stem cells, cell-based screening of a peptidomimetic small-molecule library was carried out. A series of ??-turn peptidomimetic compounds, including CW209E, increased the expression of ??-MHC promoter-driven enhanced green fluorescent protein (EGFP) and ratio of beating embryoid bodies (EBs) without inducing cytotoxicity in mouse embryonic stem cells. CW209E also increased the number of beating EBs in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Thus, this chemical compound should be useful for elucidation of the molecular pathway of cardiogenesis and generation of cardiomyocytes ex vivo, which can be further applied for experimental or clinical cell therapy for ischemic heart diseases.     

心脏移植的供心切除及心肌保护

目的总结心脏移植的供心切除及心肌保护。方法 6例终末期扩张型心肌病移植,1例风湿性心脏病联合瓣膜病变晚期。前3例供心缺血时间采用4℃改良st.Thoms液顺行灌注快速心脏停搏,取出心脏后再灌注st.Thoms液1000ml,然后置4℃生理盐水浸泡保存、运输。后4例供心缺血时间采用4℃UW液（unives-risty of Wisconsin solution）。取出心脏后,再灌注UW液1000ml,在4℃UW液内保存、运输。结果 7例心脏移植完毕后,6例心脏自动复跳,1例电击复跳,术后心脏功能恢复好,未出现低心排综合征。术后1月左心室射血分数（LVEF）恢复到60%~65%。结论此方法可获得良好的供心心肌保护。     

Mesenchymal stem cells promote cardiomyocyte hypertrophy in vitro through hypoxia-induced paracrine mechanisms

1. Mesenchymal stem cell (MSC) therapy for myocardial infarction has received increased attention since transplanted MSC were shown to improve cardiac function by transdifferentiating into cardiomyocytes and endothelial cells. However, recent studies have demonstrated that other mechanisms may contribute to the improvement in cardiac function observed after transplantation of MSC. The paracrine effect of MSC on cardiomyocyte is not clear. Thus, in the present study, we investigated the paracrine effect of MSC on the growth of neonatal rat cardiomyocytes in vitro. 2. Samples of MSC-conditioned medium (MSC-CM) were collected after rat MSC had been cultured under conditions of hypoxia and serum deprivation for 0, 3, 6, 9 or 24 h. Cardiomyocytes were then stimulated with the MSC-CM for 48 h. Then, the protein content, cell area, [(3)H]-leucine incorporation and atrial natriuretic factor-luciferase (ANF-Luc) expression of cardiomyocytes were measured. 3. The data showed that MSC-CM collected at different time points had different effects and that MSC-CM collected at 6 h significantly promoted cardiomyocyte hypertrophy by increasing total protein content, cell area, [(3)H]-leucine incorporation and ANF gene expression. 4. In conclusion, MSC-CM promoted cardiomyocyte hypertrophy in a paracrine manner. The results provide a better understanding of the mechanisms underlying the improvement in heart function after MSC transplantation.     

Involvement of dendritic cells in allograft rejection new implications of dendritic cell-endothelial cell interactions

For almost half a century immunologists have tried to tear down the MHC barrier, which separates two unrelated individuals during transplantation. Latest experimental data suggest that a breakthrough in vitro is imminent. Dendritic cells (DCs), which activate na?ve allo-reactive T-cells (TCs), play a central role in the establishment of allo-antigen-specific immunity. Allograft solid organ rejection is initiated at the foreign endothelial cell (EC) layer, which forms an immunogenic barrier for migrating DCs. Thus, DC/EC interactions might play a crucial role in antigen-specific allograft rejection. Organ rejection is mediated by host allo-reactive TCs, which are activated by donor DCs (direct activation) or host DCs (indirect activation). Direct allo-antigen presentation by regulatory dendritic cells (DCreg) can play an instructive role towards tolerance induction. Several groups established that, DCregs, if transplanted beforehand, enter host thymus, spleen, or bone marrow where they might eventually establish allo-antigen-specific tolerance. A fundamental aspect of DC function is migration throughout the entire organism. After solid organ transplantation, host DCs bind to ECs, invade allograft tissues, and finally transmigrate into lymphoid vessels and secondary lymphoid organs, where they present allo-antigens to na?ve host TCs. Recent data suggest that in vitro manipulated DCregs may mediate allo-transplantation tolerance induction. However, the fundamental mechanisms on how such DCregs cause host TCs in the periphery towards tolerance remain unclear. One very promising experimental concept is the simultaneous manipulation of DC direct and indirect TC activation/suppression, towards donor antigen-specific allo-transplantation tolerance. The allo-antigen-specific long-term tolerance induction mediated by DCreg pre-transplantation (with simultaneous short-term immunosuppression) has become reproducible in the laboratory animal setting. Despite the shortcomings of laboratory animal studies, strong promises are deriving from these studies for clinical kidney, heart, and liver transplantation.     

Stimulation of P19CL6 with multiple reagents induces pulsating particles in vivo

ABSTRACT

Objective: Injection of stem cells into ischaemic areas of the heart is expected to be an effective method for myocardial regeneration. The embryogenic carcinoma (EC) cell line P19CL6 is known to differentiate into cardiomyocytes when cultured with dimethyl sulfoxide (DMSO) and is expected to be a promising source for regenerative therapy in cardiac disease. To establish a high-yield method of cardiomyocyte differentiation, P19CL6 cells were double-stimulated with 5‐azacytidine. Double stimulation-induced cardiomyocytes were also transplanted into ectopic sites in mice and their function evaluated.

Methods and results: To induce differentiation under adherent conditions, P19CL6 cells were incubated in growth medium with 10?µM 5‐azacytidine for 24?h. After 5‐azacytidine treatment, P19CL6 cells were incubated with 1% DMSO for nine days until they began to pulsate. Prior to transplantation, cells were treated again with 5‐azacytidine. Differentiated cells were injected into the greater omentum, para-aorta region of the retroperitoneum and peri-femoral artery of adult BALB/c nude mice. Nine days after transplantation, irregularly pulsating tissues at a rate slower than the host heart were observed in the transplanted sites. Light microscopy showed formation of cardiac muscle tissues originating from P19CL6 cells. Differentiated cardiomyocytes were positive for cardiac troponin I, cadherin and α-smooth muscle actin, and the expressions of Csx/Nkx2.5 and GATA4 mRNAs were up-regulated. Electron microscopy demonstrated components specific to cardiomyocytes, such as Z-bands, desmosomes, fasciae adherens, myofibrils and mitochondria, which confirmed successful heterotopic cardiac muscle differentiation from P19CL6 cells.

Conclusion: This study demonstrated high-yield cardiac muscle differentiation of P19CL6 by 5‐azacytidine and DMSO double stimulation and successful formation of cardiac muscle-like tissue by ectopic transplantation of cardiomyocytes derived from P19CL6 into the retroperitoneal area as well as into the peripheral vessel area.