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.     

Atomic force mechanobiology of pluripotent stem cell-derived cardiomyocytes

We describe a method using atomic force microscopy (AFM) to quantify the mechanobiological properties of pluripotent, stem cell-derived cardiomyocytes, including contraction force, rate, duration, and cellular elasticity. We measured beats from cardiomyocytes derived from induced pluripotent stem cells of healthy subjects and those with dilated cardiomyopathy, and from embryonic stem cell lines. We found that our AFM method could quantitate beat forces of single cells and clusters of cardiomyocytes. We demonstrate the dose-responsive, inotropic effect of norepinephrine and beta-adrenergic blockade of metoprolol. Cardiomyocytes derived from subjects with dilated cardiomyopathy showed decreased force and decreased cellular elasticity compared to controls. This AFM-based method can serve as a screening tool for the development of cardiac-active pharmacological agents, or as a platform for studying cardiomyocyte biology.     

Maturation strategies and limitations of induced pluripotent stem cell-derived cardiomyocytes

Induced pluripotent stem cells (iPSCs) have the ability to differentiate into cardiomyocytes (CMs). They are not only widely used in cardiac pharmacology screening, human heart disease modeling, and cell transplantation-based treatments, but also the most promising source of CMs for experimental and clinical applications. However, their use is largely restricted by the immature phenotype of structure and function, which is similar to embryonic or fetal CMs and has certain differences from adult CMs. In order to overcome this critical issue, many studies have explored and revealed new strategies to induce the maturity of iPSC-CMs. Therefore, this article aims to review recent induction methods of mature iPSC-CMs, related mechanisms, and limitations.     

Human pluripotent stem cell-derived cardiomyocytes for studying energy metabolism

Cardiomyocyte energy metabolism is altered in heart failure, and primary defects of metabolic pathways can cause heart failure. Studying cardiac energetics in rodent models has principal shortcomings, raising the question to which extent human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) can provide an alternative. As metabolic maturation of CM occurs mostly after birth during developmental hypertrophy, the immaturity of hiPSC-CM is an important limitation. Here we shortly review the physiological drivers of metabolic maturation and concentrate on methods to mature hiPSC-CM with the goal to benchmark the metabolic state of hiPSC-CM against in vivo data and to see how far known abnormalities in inherited metabolic disorders can be modeled in hiPSC-CM. The current data indicate that hiPSC-CM, despite their immature, approximately mid-fetal state of energy metabolism, faithfully recapitulate some basic metabolic disease mechanisms. Efforts to improve their metabolic maturity are underway and shall improve the validity of this model.     

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.     

Disease modeling of desmosome-related cardiomyopathy using induced pluripotent stem cell-derived cardiomyocytes

Cardiomyopathy is a pathological condition characterized by cardiac pump failure due to myocardial dysfunction and the major cause of advanced heart failure requiring heart transplantation. Although optimized medical therapies have been developed for heart failure during the last few decades, some patients with cardiomyopathy exhibit advanced heart failure and are refractory to medical therapies. Desmosome, which is a dynamic cell-to-cell junctional component, maintains the structural integrity ...     

Therapeutic potential of human-induced pluripotent stem cell-derived endothelial cells in a bleomycin-induced scleroderma mouse model

Vascular injury and destruction of endothelial cells (ECs) are the early events in scleroderma (SSc) patients. This study aims to investigate the therapeutic potential of human-induced pluripotent stem cell-derived ECs (hiPSC-ECs) to treat SSc. We have assessed the functional differentiation of hiPSC-ECs and compared them with human embryonic stem cell-derived ECs (hESC-ECs) by a variety of in vitro experimental approaches. Additionally, we evaluated the therapeutic potential of hiPSC-ECs in a bleomycin-induced SSc mouse model. Our results demonstrated that hiPSC-ECs and hESC-ECs showed similar maximum expressions of FLK1 (early EC marker) at day five during differentiation. After sorting and culturing, the FLK1-positive cells exhibited spindle and subsequent endothelial cobblestone morphology in EGM2 medium. The hESC-ECs and hiPSC-ECs also expressed late EC markers CD31 (68% and 75%), CD144 (50% and 61%), CD146 (46% and 61%), and DiI-labeled acetylated low-density lipoprotein (DiI-ac-LDL) uptake (55% and 63%), respectively. They additionally formed capillary-like structures on Matrigel. Analyses of the transplantation of sorted CD31-positive hiPSC-ECs into the bleomycin-induced SSc mouse model demonstrated that these cells participate in recovery of the damaged vessels. There was a reduction in collagen content; the number of total and degranulated mast cells returned to their normal state, and bleomycin-induced wounds as well as skin fibrosis improved four weeks after transplantation of hiPSC-ECs. Our findings have shown that the differentiation process from hESCs and hiPSCs to vascular cell components is similar. Additionally, this is the first study to determine the therapeutic potential of vascular cells from hiPSCs in the treatment of an SSc model. In the future, with further validation, these may be used as an appropriate source for the treatment of SSc patients.     

PPARdelta activation induces metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes

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Clinical potential of human-induced pluripotent stem cells

The recent establishment of induced pluripotent stem (iPS) cells promises the development of autologous cell therapies for degenerative diseases, without the ethical concerns associated with human embryonic stem (ES) cells. Initially, iPS cells were generated by retroviral transduction of somatic cells with core reprogramming genes. To avoid potential genotoxic effects associated with retroviral transfection, more recently, alternative non-viral gene transfer approaches were developed. Before a potential clinical application of iPS cell-derived therapies can be planned, it must be ensured that the reprogramming to pluripotency is not associated with genome mutagenesis or epigenetic aberrations. This may include direct effects of the reprogramming method or “off-target” effects associated with the reprogramming or the culture conditions. Thus, a rigorous safety testing of iPS or iPS-derived cells is imperative, including long-term studies in model animals. This will include not only rodents but also larger mammalian model species to allow for assessing long-term stability of the transplanted cells, functional integration into the host tissue, and freedom from undifferentiated iPS cells. Determination of the necessary cell dose is also critical; it is assumed that a minimum of 1 billion transplantable cells is required to achieve a therapeutic effect. This will request medium to long-term in vitro cultivation and dozens of cell divisions, bearing the risk of accumulating replication errors. Here, we review the clinical potential of human iPS cells and evaluate which are the most suitable approaches to overcome or minimize risks associated with the application of iPS cell-derived cell therapies.     

Molecular and pharmacological properties of human embryonic stem cell-derived cardiomyocytes

Human embryonic stem cells (hESCs) can be coaxed to differentiate into specific cell types, including cardiomyocyte-like cells. These cells express cardiac-specific markers and display functional similarities to their adult counterparts. Based on these properties, hESC-derived cardiomyocytes have the potential to be extremely useful in various in vitro applications and to provide the opportunity for cardiac cell replacement therapies. However, before this can become a reality, the molecular and functional characteristics of these cells need to be investigated in more detail. In the present study we differentiate hESCs into cardiomyocyte-like cells via embryoid bodies (EBs). The fraction of spontaneously beating clusters obtained from the EBs averaged approximately 30% of the total number of EBs used. These cell clusters were isolated, dissociated into single-cell suspensions, and frozen for long-term storage. The cryopreserved cells could be successfully thawed and subcultured. Using electron microscopy, we observed Z discs and tight junctions in the hESC-derived cardiomyocytes, and by immunohistochemical analysis we detected expression of cardiac-specific markers (cTnI and cMHC). Notably, using BrdU labeling we also could demonstrate that some of the hESC-derived cardiomyocytes retain a proliferative capacity. Furthermore, pharmacological stimulation of the cells resulted in responses indicative of functional adrenergic and muscarinic receptor coupling systems. Taken together, these results lend support to the notion that hESCs can be used as a source for the procurement of cardiomyocytes for in vitro and in vivo applications.     

Long-term electrophysiological activity and pharmacological response of a human induced pluripotent stem cell-derived neuron and astrocyte co-culture

Human induced pluripotent stem cell (hiPSC)-derived neurons may be effectively used for drug discovery and cell-based therapy. However, the immaturity of cultured human iPSC-derived neurons and the lack of established functional evaluation methods are problematic. We here used a multi-electrode array (MEA) system to investigate the effects of the co-culture of rat astrocytes with hiPSC-derived neurons on the long-term culture, spontaneous firing activity, and drug responsiveness effects. The co-culture facilitated the long-term culture of hiPSC-derived neurons for >3 months and long-term spontaneous firing activity was also observed. After >3 months of culture, we observed synchronous burst firing activity due to synapse transmission within neuronal networks. Compared with rat neurons, hiPSC-derived neurons required longer time to mature functionally. Furthermore, addition of the synapse antagonists bicuculline and 6-cyano-7-nitroquinoxaline-2,3-dione induced significant changes in the firing rate. In conclusion, we used a MEA system to demonstrate that the co-culture of hiPSC-derived neurons with rat astrocytes is an effective method for studying the function of human neuronal cells, which could be used for drug screening.     

The course of ionic transmembrane currents during cardiac action potentials.

The course of the total transmembrane ionic current (Ii) during a natural action potential (AP) was reconstructed from a family of current traces recorded for single voltage clamp depolarization steps to various levels. The experiments were performed on 9 papillary cat muscles driven at 0.5 per second in oxygenated 31 degrees C Tyrode. Under varying experimental conditions very good agreement was found between the resulting Ii curve and another indicator of Ii, the first time derivative of the AP (dV/dt). Furthermore, the coefficient needed to adjust dV/dt to reconstructed Ii may serve as an indicator of the membrane capacity. The results suggest the validity of the employed approximation and, in general, the adequacy of the sucrose gap technique applied to cardiac muscle.     

Role of somatic cell sources in the maturation degree of human induced pluripotent stem cell-derived cardiomyocytes

BackgroundInduced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) are a unique source of human cardiomyocytes for cardiac disease modeling. Incomplete functional maturation remains a major limitation, however. One of the determinants of iPSC-CM maturation is somatic cell origin. We therefore compared iPSC-CMs derived from different somatic cell sources.MethodsCardiac-derived mesenchymal progenitor cells (CPCs), bone marrow-derived mesenchymal stem cells (BMCs), and human dermal fibroblasts (HDFs) from same patients were reprogrammed into iPSCs and differentiated into iPSC-CMs. Expression of cardiac-specific genes, caffeine-responsive cells, and electrophysiological properties of differentiated cells were analyzed. To assess the contribution of epigenetic memory toward differences in gene expression observed during cardiac differentiation, DNA methylation patterns were determined in the early mesodermal cardiac promoter NKX2–5 and KCNQ1, which encodes for the pore-forming α-subunit of the slow component of delayed-rectifier potassium current (I_Ks).ResultsCardiac genes (MYH6, TNNI3, KCNQ1, KCNE1) were upregulated in CPC-vs. BMC- and HDF-iPSC-CMs. At early differentiation stages, CPC-iPSC-CMs displayed higher numbers of caffeine-responsive cells than BMC- and HDF-iPSC-CMs. The hERG1 (KV11.1) blocker, E4031, followed by the IKs blocker, JNJ303, increased extracellular field potential duration in CPC-iPSC-CMs to a greater extent than in BMC- and HDF-iPSC-CMs. The promoter region of NKX2–5 was more highly methylated in BMCs and HDFs compared to CPCs, and to a lesser extent in BMC-iPSCs compared to CPC-iPSCs.ConclusionsThese results suggest that human iPSCs from cardiac somatic cell sources may display enhanced capacity toward cardiac re-differentiation compared to non-cardiac cell sources, and that epigenetic mechanisms may play a role in this regard.     

Conditioned media from human pluripotent stem cell-derived cardiomyocytes inhibit the growth and migration of lung cancer cells

Conditioned media (CM) from various cell types contain significant levels of paracrine factors. Recently, therapeutic properties of CM derived from stem cells have been revealed. Based on the fact that heart cancer is extremely rarely, we hypothesized that the CM obtained from human pluripotent stem cell-derived cardiomyocytes might inhibit cancer cell growth and survival. To this end, lung cancer cell line A549 along with human foreskin fibroblasts (HFF) were treated with serial concentrations of cardiomyocyte CM (CCM) or fibroblast CM (FCM). We found that CCM markedly reduced the viability of lung cancer cells, while FCM did not compromise the viability of neither cancer cells nor HFF cells. Furthermore, we determined an optimized CCM concentration, 30 mg/mL, at which the growth, clonogenicity, and migration of A549 and Calu6 lung cancer cell lines were substantially impaired, whereas FCM did not influence these properties. Moreover, lung cancer cells exhibited cell cycle regulation upon treatment with CCM and the rate of apoptosis was markedly increased by cardiomyocyte CM in both lung cancer cell lines tested. Finally, in response to CCM treatment, A549 and Calu6 cells expressed lower levels of antiapoptotic and stemness genes, but higher levels of proapoptotic genes. In conclusion, this study provides cellular and molecular evidence for the antitumor ability of secretome obtained from stem cell-derived cardiomyocytes.     

Zinc-induced changes in ionic currents of cardiomyocytes

The whole-cell voltage-clamp technique was applied to isolated ventricular myocytes to investigate the effects of extracellular and intracellular zinc application on L-type Ca²⁺ channel currents (I _Ca). Extracellular zinc exposure at micromolar concentration induced a reversible (with washout of ZnCl₂) reduction (30%) of I _Ca with no change in current-voltage relationship. On the other hand, an increase of intracellular free-zinc concentration, [Zn²⁺]_i, from normal (less than 1 nM) to approx 7 nM with 10 μM Zn-pyrithione exposure caused an inhibition of 33±6% in the peak of the I _Ca and altered the voltage dependency of L-type Ca²⁺ channels with a 10-mV left shift and a hump at around −40 mV in its current-voltage relation. In contrast, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly inhibited the I _Ca (42±2%), with only a small but detectable outward shift of the holding current measured at the end of the pulses. Zn-pyrithione and TPEN caused a reproducible decrease of the I _Ca. Interestingly, TPEN application, without Zn-pyrithione pretreatment, inhibited the I _Ca (35±2%) with no change in voltage dependency. Taken together, the results suggest that both extracellular and intracellular zinc increases under pathological conditions in cardiomyocytes can alter the I _Ca, but their effects are not in the same order and same manner. One should consider these possible side effects when it is suggested to be vital to cardiovascular cell integrity and functions.     

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.     

Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.     

Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl₂ (n = 22). Patch-clamp techniques were used to record I_Kr and I_K1 from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl₂, at concentrations that selectively block I_K1 (50–100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I_K1 in 53% (20/38) of the cells studied, but presence of I_Kr in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I_K1 in the majority of cells, but robust expression of I_Kr. In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I_Kr due to the absence of I_K1. Thus, efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications.     

Selenium-induced alterations in ionic currents of rat cardiomyocytes

In the present study, rats were treated with sodium selenite (5 micromol/kg body weight/day, ip) for 4 weeks and the parameters of contractile activity, action potential, L-type Ca2+-current (ICaL), as well as transient outward (Ito), inward rectifier (IK1), and steady state (Iss) K+-currents were investigated. Sodium selenite treatment increased rat blood glucose level and lowered plasma insulin level, significantly. This treatment also caused slightly prolongation in action potential with no significant effects on spontaneous contraction parameters and intracellular Ca2+ transients of the heart preparations. These effects were associated with marked alterations in the kinetics of both ICaL and Ito including a significant slowing in both inactivation time constants of ICaL and a significant shift to negative potential at half-inactivation of these channels without any change in the current density. Also, there was a significantly faster inactivation of Ito and no shift in half-inactivation of this channel without any change in its current density. Consequently, there was a approximately 50% increase in total charges carried by Ca2+ current and approximately 50% decrease in total charges carried by K+ currents of the treated rat cardiomyocytes. Additionally we observed a significant inhibition in IK1 density in treated rat cardiomyocytes. Oxidized glutathione level was significantly increased (70%) while the observed decrease in reduced glutathione was much less. Since a shift in redox state of regulatory proteins is related with cell dysfunction, selenium-induced increase in blood glucose and decrease in plasma insulin may correlate these alterations. These alterations, in the kinetics of the channels and in IK1 density, might lead to proarrhythmic effect of chronic selenium supplementation.