Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes

Assessment of early ultrastructural development and cell-cycle regulation in human cardiac tissue is significantly hampered by the lack of a suitable in vitro model. Here we describe the possible utilization of human embryonic stem cell (ES) lines for investigation of these processes. With the use of the embryoid body (EB) differentiation system, human ES cell-derived cardiomyocytes at different developmental stages were isolated and their histomorphometric, ultrastructural, and proliferative properties were characterized. Histomorphometric analysis revealed an increase in cell length, area, and length-to-width ratio in late-stage EBs (>35 days) compared with early (10-21 days) and intermediate (21-35 days) stages. This was coupled with a progressive ultrastructural development from an irregular myofibrillar distribution to an organized sarcomeric pattern. Cardiomyocyte proliferation, assessed by double labeling with cardiac-specific antibodies and either [3H]thymidine incorporation or Ki-67 immunolabeling, demonstrated a gradual withdrawal from cell cycle. Hence, the percentage of positively stained nuclei in early-stage cardiomyocytes ([3H]thymidine: 60 +/- 10%, Ki-67: 54 +/- 23%) decreased to 36 +/- 7% and 9 +/- 16% in intermediate-stage EBs and to <1% in late-stage cardiomyocytes. In conclusion, a reproducible temporal pattern of early cardiomyocyte proliferation, cell-cycle withdrawal, and ultrastructural maturation was noted in this model. Establishment of this unique in vitro surrogate system may allow to examine the molecular mechanisms underlying these processes and to assess interventions aiming to modify these properties. Moreover, the detailed characterization of the ES cell-derived cardiomyocyte may be crucial for the development of future cell replacement strategies aiming to regenerate functional myocardium.     

Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes

To improve proarrhythmic predictability of preclinical models, we assessed whether human ventricular-like embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be selected following a standardized protocol. Also, we quantified their arrhythmogenic response and compared this to a contemporary used rabbit Purkinje fiber (PF) model. Multiple transmembrane action potentials (AP) were recorded from 164 hESC-CM clusters (9 different batches), and 12 isolated PFs from New Zealand White rabbits. AP duration (APD), early afterdepolarizations (EADs), triangulation (T), and short-term variability of repolarization (STV) were determined on application of the I_Kr blocker E-4031 (0.03/0.1/0.3/1 μM). Isoproterenol (0.1 μM) was used to assess adrenergic response. To validate the phenotype, RNA isolated from atrial- and ventricular-like clusters (n = 8) was analyzed using low-density Taqman arrays. Based on initial experiments, slow beating rate (< 50 bpm) and long APD (> 200 ms) were used to select 31 ventricular-like clusters. E-4031 (1 μM) prolonged APD (31/31) and induced EADs only in clusters with APD90 > 300 ms (11/16). EADs were associated with increased T (1.6 ± 0.2 vs 2.0 ± 0.3?) and STV (2.7 ± 1.5 vs 6.9 ± 1.9?). Rabbit PF reacted in a similar way with regards to EADs (5/12), increased T (1.3 ± 0.1 vs 1.9 ± 0.4?), and STV (1.2 ± 0.9 vs 7.1 ± 5.6?). According to ROC values, hESC-CMs (STV 0.91) could predict EADs at least equivalent to PF (STV 0.69). Isoproterenol shortened APD and completely suppressed EADs. Gene expression analysis revealed that HCN1/2, KCNA5, and GJA5 were higher? in atrial/nodal-like cells, whereas KCNJ2 and SCN1B were higher? in ventricular-like cells (?P < 0.05). Selection of hESC-CM clusters with a ventricular-like phenotype can be standardized. The proarrhythmic results are qualitatively and quantitatively comparable between hESC-CMs and rabbit PF. Our results indicate that additional validation of this new safety pharmacology model is warranted.     

Beta-adrenergic and muscarinic modulation of human embryonic stem cell-derived cardiomyocytes.

BACKGROUND: Embryonic stem cells provide the most promising tool for cell replacement therapy including transplantation of human embryonic stem (hES) cell- derived cardiomyocytes in the infarcted area of the heart. Here we provide data for differentiation of cardiomyocytes from hES cells and firstly describe their hormonal modulation. METHODS: Using Micro-Electrode Arrays as a novel electrical mapping technique of beating cardiomyocyte clusters within whole hES cell aggregates, we were able to measure the field potential generation and morphology changes during hormonal modulation. RESULTS: We found that isoproterenol provokes, similar to the mouse ES cell system, a strong positive chronotropic effect with an EC50 of around 10(-8) M. Moreover, isoproterenol stimulated with a higher EC50 value the slow field potential amplitude, FP(slow), indicating a stimulation of Ca2+ channels in ventricular-like ES cell-derived cardiomyocytes which is shown to be clearly independent from frequency modulation. In contrast, carbachol (10 microM) produced a transient negative chronotropic effect but had no effect on FP(slow). CONCLUSION: The Micro-Electrode system allows measurement of ionic channel modulation and chronotropic responsiveness in a pharmacological screening setup. Moreover, all our data indicate that cardiomyocytes derived from human embryonic stem cells exhibit a physiological response to the major hormones of the vegetative nervous system and might therefore serve as an ideal candidate for the use in cell replacement strategies.     

Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.     

Electrophysiological properties of embryonic stem cell-derived neurons

In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP(+) neurons in culture display functional neuronal properties even at early stages of differentiation.     

Myocardial improvement with human embryonic stem cell-derived cardiomyocytes enriched by p38MAPK inhibition

Background aimsWe have shown previously that inhibition of the p38 mitogen-activated protein kinase (p38MAPK) directs the differentiation of human embryonic stem cell (hESC)-derived cardiomyocytes (hCM). We investigated the therapeutic benefits of intramyocardial injection of hCM differentiated from hESC by p38MAPK inhibition using closed-chest ultrasound-guided injection at a clinically relevant time post-myocardial infarction (MI) in a mouse model.MethodsMI was induced in mice and the animals treated at day 3 with: (a) hCM, (b) human fetal fibroblasts (hFF) as cell control, or (c) medium control (n = 10 animals/group). Left ventricular ejection fraction (LVEF) was evaluated post-MI prior to therapy, and at days 28 and 60 post-cell therapy. Hearts were analyzed at day 60 for infarct size, angiogenesis, cell fate and teratoma formation.ResultsLVEF was improved in the hCM-treated animals compared with both hFF and medium control-treated animals at day 28 (39.03 ± 1.79% versus 27.89 ± 1.27%, P < 0.05, versus 32.90 ± 1.46%, P < 0.05, respectively), with sustained benefit until day 60. hCM therapy resulted in significantly smaller scar size, increased capillary bed area, increased number of arterioles, less native cardiomyocyte (CM) apoptosis, and increased CM proliferation compared with the other two groups. These benefits were achieved despite a very low retention rate of the injected cells at day 60, as assessed by immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR). Therapy with hCM did not result in intramyocardial teratoma formation at day 60.ConclusionsThis study demonstrates that hCM derived from p38MAPK-treated hESC have encouraging therapeutic potential.     

Characterization and regulation of T-type Ca2+ channels in embryonic stem cell-derived cardiomyocytes

T-type Ca2+ channels may play a role in cardiac development. We studied the developmental regulation of the T-type currents (ICa,T) in cardiomyocytes (CMs) derived from mouse embryonic stem cells (ESCs). ICa,T was studied in isolated CMs by whole cell patch clamp. Subsequently, CMs were identified by the myosin light chain 2v-driven green fluorescent protein expression, and laser capture microdissection was used to isolate total RNA from groups of cells at various developmental time points. ICa,T showed characteristics of Cav3.1, such as resistance to Ni2+ block, and a transient increase during development, correlating with measures of spontaneous electrical activity. Real-time RT-PCR showed that Cav3.1 mRNA abundance correlated (r2 = 0.81) with ICa,T. The mRNA copy number was low at 7+4 days (2 copies/cell), increased significantly by 7+10 days (27/cell; P < 0.01), peaked at 7+16 days (174/cell), and declined significantly at 7+27 days (25/cell). These data suggest that ICa,T is developmentally regulated at the level of mRNA abundance and that this regulation parallels measures of pacemaker activity, suggesting that ICa,T might play a role in the spontaneous contractions during CM development.     

Cardiac disease modeling using induced pluripotent stem cell-derived human cardiomyocytes

Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alterationhas been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes(CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells(i PSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since i PSC preserve the entire patients’ genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with i PSC.     

Characterization of human embryonic stem cell-derived hematopoietic progenitor phenotype

Differentiation of human embryonic stem cells (hESCs) into hematopoietic lineages using various methods has been reported. However, the phenotype that precisely defines the hematopoietic progenitor compartment with clonogenic activities has yet to be determined. Here, we measured and characterized progenitor function of subfractions of cells prospectively isolated from human embryoid bodies (hEBs) during hematopoietic differentiation basing on surface markers CD45, CD34, CD43, and CD38. We report that hematopoietic progenitors predominantly resided in the CD45⁺ subset. CD43⁺ cells lacking CD45 expression were largely devoid of progenitor activity. However, progenitor activity and multipotentiality was more enriched in CD45⁺ cells co-expressing CD43. CD45⁺ subset co-expressing CD34 but lacking CD38 expression (CD45⁺CD34⁺CD38^-) were further enriched for CFU capacity compared to the CD45⁺CD34⁺CD38⁺ subset. Our study demonstrates a role of CD43 in enriching hematopoietic progenitors derived from hEBs and reveals a hierarchical organization of hESC-derived hematopoietic progenitor compartments defined by phenotypic markers.     

Structural differentiation, proliferation, and association of human embryonic stem cell-derived cardiomyocytes in vitro and in their extracardiac tissues

The proliferation, structural differentiation, and capacity of association of human ES cell-derived cardiomyocytes were assessed in culture and in extracardiac graft tissues. Embryoid body (EB) outgrowths having cardiomyocytes, and their transplants in mice retroperitoneum or renal subcapsular region were analyzed mainly by immunochemistry. During the culture of EB outgrowths, colonies of cardiomyocytes grew in size exhibiting synchronized beatings. Subcellular structures of those cardiomyocytes involved in the contraction, hormone production, and intercellular integration differentiated with distinct immunoreactivity for constituent proteins/peptides. Judging from PCNA staining, proliferation potential was maintained in part for more than 70 days. In teratoma tissues on post-transplantation Day 7, cardiomyocytes maintained their integration with connexin 43 and cadherin at their junctions. They partly exhibited strong PCNA reactivity. On Day 28, large part of the cardiomyocytes lost their association, dispersing among non-cardiac cells without discernible cadherin reactivity. Proliferation potential was generally low irrespective of their tissue diversity. From these results, structural differentiation and active proliferation of human ES cell-derived cardiomyocytes occurred in vitro, maintaining their association. When developed in extracardiac tissues, however, the cardiomyocytes showed low proliferation potential and reduced cellular integration. This leads to the proposal that some procedure will be necessary to accelerate or maintain the proliferation of cardiomyocytes in vivo.     

Effect of cardioactive drugs on action potential generation and propagation in embryonic stem cell-derived cardiomyocytes.

Extracellular recordings of spontaneous electrical activity in contracting cardiac clusters differentiated from murine embryonic stem cells enable to study electrophysiological features of this in-vitro cardiac-like tissue as well as effects of pharmacological compounds on its chronotropy and electrical conduction. To test if the microelectrode array (MEA) system could serve as a basis for development of a pharmacological screening tool for cardioactive drugs, we used spontaneously beating outgrowths of three-dimensional ES cell aggregates ("embryoid bodies", EBs) plated onto substrate-integrated MEAs. The effects of the L-type Ca(2+) channel antagonist verapamil and Na(+) and K(+) channel blockers (tetrodotoxin, 4-aminopyridine, and sparfloxacin) on the deduced interrelated cardiac network function were investigated. Application of 10(-6) M verapamil led to arrhythmic spiking with a burst-like pattern; at a higher concentration (10(-5) M) the drug caused a sustained negative chronotropy up to complete stop of beating. In the presence of tetrodotoxin a conduction block was observed. Since modulation of K(+) channel activity can cause anti- or proarrhythmic effects, the influence of K(+) channel blockers, namely 4-aminopyridine and sparfloxacin, was investigated. 4-aminopyridine (2x10(-3) M) significantly stabilized beating frequency, while the field potential duration (FPD) was concentration-dependently prolonged up to 2.7-fold. Sparfloxacin (3x10(-6) M) stabilized the beating frequency as well. At a higher concentration of sparfloxacin (3x10(-5) M), a significant prolongation of the spike duration was registered; application of the drug caused also early afterdepolarizations. The results demonstrate a suitability of the studied in-vitro cardiac cell model for pharmacological drug testing in cardiovascular research.     

A novel purification method of murine embryonic stem cell- and human-induced pluripotent stem cell-derived cardiomyocytes by simple manual dissociation

Cardiomyocytes derived from embryonic stem cells (ES-CMs) and induced pluripotent stem cells (iPS-CMs) are useful for toxicity and pharmacology screening. In the present study, we found that cardiomyocyte-rich beating cell clusters (CCs) emerged from murine embryonic stem cell (mESC)-derived beating EBs and from human-induced pluripotent stem cell (hiPSC)-derived beating EBs dissociated by gentle pipetting with a thin glass pipette. The percentage of cardiac troponin T (cTnT)-positive cells in the beating CCs obtained from mESC-derived and hiPSC-derived beating EBs was higher (81.5% and 91.6%, respectively) than in beating-undissociated EBs (13.7% and 67.1%, respectively). For mESCs, the yield of cTnT-positive cells from beating CCs was estimated to be 1.6 times higher than that of beating EBs. The bromodeoxyuridine labeling index of mouse ES-CMs and human iPS-CMs in beating CCs was 1.5- and 3.2-fold, respectively, greater than those in beating EBs. To investigate the utility of the cells in toxicity assessment, we showed that doxorubicin, a cardiotoxic drug, induced myofilament disruption in cardiomyocytes isolated by this method. This simple method enables preparation of mouse ES-CMs and human iPS-CMs with better proliferative activity than beating EBs not dissociated by pipetting, and the cardiomyocytes are useful for drug-induced myocardial toxicity testing.     

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance

Patients with type 2 diabetes (T2D) and/or insulin resistance (IR) have an increased risk for the development of heart failure (HF). Evidence indicates that this increased risk is linked to an altered cardiac substrate preference of the insulin resistant heart, which shifts from a balanced utilization of glucose and long-chain fatty acids (FAs) towards an almost complete reliance on FAs as main fuel source. This shift leads to a loss of endosomal proton pump activity and increased cardiac fat accumulation, which eventually triggers cardiac dysfunction. In this review, we describe the advantages and disadvantages of currently used in vitro models to study the underlying mechanism of IR-induced HF and provide insight into a human in vitro model: human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Using functional metabolic assays we demonstrate that, similar to rodent studies, hESC-CMs subjected to 16 h of high palmitate (HP) treatment develop the main features of IR, i.e., decreased insulin-stimulated glucose and FA uptake, as well as loss of endosomal acidification and insulin signaling. Taken together, these data propose that HP-treated hESC-CMs are a promising in vitro model of lipid overload-induced IR for further research into the underlying mechanism of cardiac IR and for identifying new pharmacological agents and therapeutic strategies. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.