Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types

Maltsev, Victor A.; Rohwedel, Jürgen; Hescheler, J.; Wobus, Anna M.

doi:10.1016/0925-4773(93)90015-p

Cited by 444 publications

(296 citation statements)

References 26 publications

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“…After 2 days as hanging-drops, EBs aggregated in either high or low glucose had a similar morphological appearance and size (not shown), indicating that the medium glucose concentration did not perturb simple EB formation. To discern serum-driven cardiac differentiation, EBs were placed individually into tissue culture plates, and cardiac development was monitored by examination of rhythmically contracting focal areas [26]. In high glucose, beating focal areas within individual HM-1-differentiated EBs were observed from day 7, with >70% of EBs containing beating areas at the end of the experiment ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Our results suggest that p38 is downstream of Nox4 in this pathway because AA was unable to relieve the cardiomyocyte block in p38 dominant-negative ES cells under high glucose, whereas expression of constitutive p38 was sufficient to by-pass the necessity for AA supplementation for cardiomyocyte differentiation under low glucose conditions. Differentiation of ES cells toward the cardiac lineage creates a characteristic gene profile, which includes the early expression of a network of mesodermal-cardiac transcription factors such as Brachyury, Nkx2.5, Gata4, and Mef2c, among others, which activate the expression of structural genes such as aMHC, Mlc2v, and troponin I [26,32]. We found that low glucose-cultured ES cells had an accelerated mesodermal differentiation, failed to upregulate the expression of Nkx2.5, showed mislocalized (cytosolic) expression of Gata4 and Mef2c, and at the same time failed to express the cardiac structural genes aMHC and Mlc2v.…”

Section: Discussionmentioning

confidence: 99%

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Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose

et al. 2010

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Accumulating evidence points to reactive oxygen species (ROS) as important signaling molecules for cardiomyocyte differentiation in embryonic stem (ES) cells. Given that ES cells are normally maintained and differentiated in medium containing supraphysiological levels of glucose (25 mM), a condition which is known to result in enhanced cellular ROS formation, we questioned whether this high glucose concentration was necessary for cardiomyocyte lineage potential. We show here that ES cells cultured in physiological glucose (5 mM), maintained their general stemness qualities but displayed an altered mitochondrial metabolism, which resulted in decreased ROS production. Furthermore, ES and induced pluripotent stem (iPS) cells differentiated in lower glucose concentrations failed to generate cardiomyocyte structures; an effect mimicked with antioxidant treatments using catalase, N-acetyl cysteine and mitoubiquinone, under high glucose conditions in ES cells. Molecular analysis revealed that ES cells differentiated in 5 mM glucose had reduced expression of the pro-cardiac NOX4 gene and diminished phosphorylation of p38 mitogen-activated protein kinase (MAPK), together with specific changes in the cardiac transcriptional network. These outcomes could be reversed by supplementation of low glucose cultures with ascorbic acid, paradoxically acting as a pro-oxidant. Furthermore, forced expression of an upstream p38 MAPK kinase (MKK6) could bypass the requirement for ROS during differentiation to cardiomyocytes under low glucose conditions, illustrating a key role for p38 in the cardiac differentiation program. Together these data demonstrate that endogenous ROS control is important for cardiomyocyte formation from ES cells, and furthermore that supraphysiological glucose, by supplying ROS, is absolutely required.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose

et al. 2010

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“…Based on the morphology and classification of AP properties ( Table 1) [1,26], three major types of AP (nodal-like, atriallike, and ventricular-like) were observed in our study ( Figure 4A). However, the ratios of the three major types of AP were different between the Noggin+RA-and Noggin+RAi-treated cultures.…”

Section: Electrophysiological Characterization Identifies Embryonic Amentioning

confidence: 93%

Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals

et al. 2010

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Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs) are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the panretinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, but not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiological studies indicated that with 64.7 ± 0.88% (mean ± s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca 2+ sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial-and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin and retinoid signals.

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“…Electrophysiological studies revealed that cells with characteristics of atrial, ventricular, and sinus-nodal cardiomyocytes were present following terminal differentiation of ES-derived cells (Maltsev et al 1993), a result that was confirmed via molecular analyses (Miller-Hance et al 1993). The developmental changes in the electrophysiological properties of ES-derived cardiomyocytes from initial cardiomyoblast commitment (Kolossov et al 1998) through formation of three-dimensional, spontaneously contracting structures (Maltsev et al 1993;Banach et al 2003) have been well characterized. (Thomson et al 1998) permitted analyses of in vitro generated human cardiomyocytes.…”

Section: Cardiomyogenic Differentiation In Es Cellsmentioning

confidence: 77%

“…If cultured at too high of a cell concentration, large aggregates formed, which subsequently failed to differentiate. Maltsev and colleagues developed the hanging drop technique, which permitted reproducible generation of uniformly sized EBs (Maltsev et al 1993). Dang and colleagues subsequently showed that cell-cell interactions in EBs was largely mediated by E-cadherin, and furthermore that E-cadherin expression decreased as EBs began to differentiate (Dang et al 2002(Dang et al , 2004).…”

Section: Large-scale Generation Of Embryonic Stem-derived Cardiomyocytesmentioning

confidence: 99%

Cardiac Repair by Embryonic Stem-Derived Cells

Rubart

Field

2006

Stem Cells

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Cell transplantation approaches offer the potential to promote regenerative growth of diseased hearts. It is well established that donor cardiomyocytes stably engraft into recipient hearts when injected directly into the myocardial wall. Moreover, the transplanted donor cardiomyocytes participate in a functional syncytium with the host myocardium. Thus, transplantation of donor cardiomyocytes resulted in at least partial restoration of lost muscle mass. It is also well established that embryonic stem (ES) cells differentiate into cells of ecto-, endo-, and mesodermal lineages when cultured under appropriate conditions in vitro. Robust cardiomyogenic differentiation was frequently observed in spontaneously differentiating ES cultures. Cellular, molecular and physiologic analyses indicated that ES-derived cells were bona fide cardiomyocytes, with in vitro characteristics typical for cells obtained from early stages of cardiac development. Thus, ES-derived cardiomyocytes constitute a viable source of donor cells for cell transplantation therapies.

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Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types

Cited by 444 publications

References 26 publications

Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose

Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose

Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals

Cardiac Repair by Embryonic Stem-Derived Cells

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