Leon G.J. Tertoolen

Differentiation of human embryonic stem cells to cardiomyocytes

Background—Cardiomyocytes derived from human embryonic stem (hES) cells could be useful in restoring heart function after myocardial infarction or in heart failure. Here, we induced cardiomyocyte differentiation of hES cells by a novel method and compared their electrophysiological properties and coupling with those of primary human fetal cardiomyocytes. Methods and Results—hES cells were cocultured with visceral-endoderm (VE)–like cells from the mouse. This initiated differentiation to beating muscle. Sarcomeric marker proteins, chronotropic responses, and ion channel expression and function were typical of cardiomyocytes. Electrophysiology demonstrated that most cells resembled human fetal ventricular cells. Real-time intracellular calcium measurements, Lucifer yellow injection, and connexin 43 expression demonstrated that fetal and hES-derived cardiomyocytes are coupled by gap junctions in culture. Inhibition of electrical responses by verapamil demonstrated the presence of functional &agr;1c-calcium ion channels. Conclusions—This is the first demonstration of induction of cardiomyocyte differentiation in hES cells that do not undergo spontaneous cardiogenesis. It provides a model for the study of human cardiomyocytes in culture and could be a step forward in the development of cardiomyocyte transplantation therapies.

Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction

Regeneration of the myocardium by transplantation of cardiomyocytes is an emerging therapeutic strategy. Human embryonic stem cells (HESC) form cardiomyocytes readily but until recently at low efficiency, so that preclinical studies on transplantation in animals are only just beginning. Here, we show the results of the first long-term (12 weeks) analysis of the fate of HESC-derived cardiomyocytes transplanted intramyocardially into healthy, immunocompromised (NOD-SCID) mice and in NOD-SCID mice that had undergone myocardial infarction (MI). Transplantation of mixed populations of differentiated HESC containing 20-25% cardiomyocytes in control mice resulted in rapid formation of grafts in which the cardiomyocytes became organized and matured over time and the noncardiomyocyte population was lost. Grafts also formed in mice that had undergone MI. Four weeks after transplantation and MI, this resulted in significant improvement in cardiac function measured by magnetic resonance imaging. However, at 12 weeks, this was not sustained despite graft survival. This suggested that graft size was still limiting despite maturation and organization of the transplanted cells. More generally, the results argued for requiring a minimum of 3 months follow-up in studies claiming to observe improved cardiac function, independent of whether HESC or other (adult) cell types are used for transplantation.

Cardiomyocyte differentiation of mouse and human embryonic stem cells

Ischaemic heart disease is the leading cause of morbidity and mortality in the western world. Cardiac ischaemia caused by oxygen deprivation and subsequent oxygen reperfusion initiates irreversible cell damage, eventually leading to widespread cell death and loss of function. Strategies to regenerate damaged cardiac tissue by cardiomyocyte transplantation may prevent or limit post‐infarction cardiac failure. We are searching for methods for inducing pluripotent stem cells to differentiate into transplantable cardiomyocytes. We have already shown that an endoderm‐like cell line induced the differentiation of embryonal carcinoma cells into immature cardiomyoctyes. Preliminary results show that human and mouse embryonic stem cells respond in a similar manner. This study presents initial characterization of these cardiomyocytes and the mouse myocardial infarction model in which we will test their ability to restore cardiac function.

Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes

Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. Block of the human Ether-a-go go Related Gene (hERG) ion channel in particular is associated with life-threatening arrhythmias, such as Torsade de Pointes (TdP). Here we investigated human cardiomyocytes derived from pluripotent (embryonic) stem cells (hESC) as a renewable, scalable, and reproducible system on which to base cardiac safety pharmacology assays. Analyses of extracellular field potentials in hESC-derived cardiomyocytes (hESC-CM) and generation of derivative field potential duration (FPD) values showed dose-dependent responses for 12 cardiac and noncardiac drugs. Serum levels in patients of drugs with known effects on QT interval overlapped with prolonged FPD values derived from hESC-CM, as predicted. We thus propose hESC-CM FPD prolongation as a safety criterion for preclinical evaluation of new drugs in development. This is the first study in which dose responses of such a wide range of compounds on hESC-CM have been generated and shown to be predictive of clinical effects. We propose that assays based on hESC-CM could complement or potentially replace some of the preclinical cardiac toxicity screening tests currently used for lead optimization and further development of new drugs.

Epidermal growth factor—induced actin remodeling is regulated by 5-lipoxygenase and cyclooxygenase products

In a number of cell types, epidermal growth factor (EGF) evokes dramatic morphological changes, cortical actin polymerization, and stress fiber breakdown. The molecular processes by which increased EGF receptor tyrosine kinase activity results in actin reorganization and morphological changes are unresolved. Recently, we demonstrated that arachidonic acid metabolites function in EGF signal transduction. We now report that in A431 cells, HeLa cells, and rat-1 fibroblasts, the EGF-induced cortical actin polymerization is produced by lipoxygenase metabolism, whereas in these cells stress fiber breakdown is mediated by cyclooxygenase metabolites. Also, the EGF-provoked rounding up in A431 cells is dependent on arachidonic acid metabolism. We conclude that leukotrienes and prostaglandins act in concert, as second messengers, to produce morphological effects and actin reorganization, providing a novel mechanism for directing growth factor-induced cytoskeletal changes.

Receptor protein tyrosine phosphatase alpha activates pp60c-src and is involved in neuronal differentiation.

Here we report that protein tyrosine phosphatases (PTPases), like their enzymatic counterpart the protein tyrosine kinases, can play an important role in cell differentiation. Expression of the transmembrane PTPase receptor protein tyrosine phosphatase alpha (RPTP alpha) is transiently enhanced during neuronal differentiation of embryonal carcinoma (EC) and neuroblastoma cells. Retinoic acid induces wild type P19 cells to differentiate into endoderm‐ and mesoderm‐like cells. By contrast, retinoic acid treatment leads to neuronal differentiation of P19 cells, ectopically expressing functional RPTP alpha, as illustrated by their ability to generate action potentials. Endogenous pp60c‐src kinase activity is enhanced in the RPTP alpha‐transfected cells, which may be due to direct dephosphorylation of the regulatory Tyr residue at position 527 in pp60c‐src by RPTP alpha. Our results demonstrate that RPTP alpha is involved in neuronal differentiation and imply a role for pp60c‐src in the differentiation process.

Rac mediates growth factor-induced arachidonic acid release

Growth factor-induced stress fiber formation involves signal transduction through Rac and Rho proteins and production of leukotrienes from arachidonic acid metabolism. In exploring the relationship between these pathways, we found that Rac is essential for EGF-induced arachidonic acid production and subsequent generation of leukotrienes and that Rac V12, a constitutively activated mutant of Rac, generates leukotrienes in a growth factor-independent manner. Leukotrienes generated by EGF or Rac V12 are necessary and sufficient for stress fiber formation. Furthermore, leukotriene-dependent stress fiber formation requires Rho proteins. We have therefore identified elements of a pathway from growth factor receptors that includes Rac, arachidonic acid production, arachidonic acid metabolism to leukotrienes, and leukotriene-dependent Rho activation. This appears to be the major pathway by which Rac influences Rho-dependent cytoskeleton rearrangements.

Epidermal growth factor activates calcium channels by phospholipase A25-lipoxygenase-mediated leukotriene C4 production

Epidermal growth factor (EGF) induces a Ca2+ influx in many cell types, but the underlying mechanisms are so far unresolved. We report that: EGF-induced Ca2+ channel activity is eliminated by lipoxygenase inhibition and is mimicked by artificial induction of lipoxygenase activity; addition of leukotriene C4 can fully mimic EGF in its ability to activate Ca2+ channels; and EGF induces a rapid accumulation of intracellular leukotriene C4. In addition, we show that EGF-induced, Ca(2+)-dependent membrane hyperpolarization and junB proto-oncogene expression are dependent on lipoxygenase activity, whereas EGF-induced cytoplasmic alkalinization is not. We conclude that PLA2/5-lipoxygenase-mediated leukotriene C4 production constitutes a novel and specific signal transduction pathway in growth factor action.

Regulation of receptor protein-tyrosine phosphatase α by oxidative stress

The presence of two protein‐tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The function of the generally inactive membrane‐distal PTP domain (RPTP‐D2) is unknown. Here we report that an intramolecular interaction between the spacer region (Sp) and the C‐terminus in RPTPα prohibited intermolecular interactions. Interestingly, stress factors such as H2O2, UV and heat shock induced reversible, free radical‐dependent, intermolecular interactions between RPTPα and RPTPα‐SpD2, suggesting an inducible switch in conformation and binding. The catalytic site cysteine of RPTPα‐SpD2, Cys723, was required for the H2O2 effect on RPTPα. H2O2 induced a rapid, reversible, Cys723‐dependent conformational change in vivo, as detected by fluorescence resonance energy transfer, with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) flanking RPTPα‐SpD2 in a single chimeric protein. Importantly, H2O2 treatment stabilized RPTPα dimers, resulting in inactivation. We propose a model in which oxidative stress induces a conformational change in RPTPα‐D2, leading to stabilization of RPTPα dimers, and thus to inhibition of RPTPα activity.

Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long‐QT syndrome

Patient‐specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long‐QT syndromes (LQTS). To study the LQT2‐associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC‐derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC‐derived CMs. Further characterization of N996I‐HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.