Bidirectional ventricular tachycardia: a hallmark of catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia is a familial cardiac arrhythmia that is related to RYR2 or CASQ2 gene mutation. It occurs in patients with structurally normal heart and causes exercise-emotion triggered syncope and sudden cardiac death. We present a 13 year-old girl with recurrent episodes of exercise-related syncope and prior history of sudden death in a first degree relative.     

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.     

致心律失常性右室心肌病患者的诱导性多能干细胞系的建立

目的：建立致心律失常性右室心肌病（ARVC）患者特异性的诱导性多能干细胞（iPSCs）,为研究ARVC发病机制提供研究模型。方法：培养来源于ARVC患者皮肤成纤维细胞,并进行突变位点测序鉴定。通过仙台病毒转导入外源性多能转录因子,将ARVC患者皮肤细胞诱导为iPSCs,结合免疫荧光法,实时荧光定量PCR,以及体内外三胚层形成实验对iPS细胞全能型进行鉴定。通过调控Wnt信号通路诱导iPS细胞定向分化为心肌细胞。结果：ARVC患者来源的iPSCs显示碱性磷酸酶阳性,多能性相关基因高表达,胚胎干细胞标志物Oct4,SSEA4,TRA-1-81阳性。体外悬浮培养形成的拟胚体以及体内畸胎瘤形成实验均显示ARVC-iPSCs具有向3个胚层分化能力。经过体外心肌定向,ARVC-iPSC可诱导产生自主节律性搏动细胞团,免疫荧光显示cTnT阳性。结论：本研究使用仙台病毒,建立了无插入型ARVC患者特异的诱导性多能干细胞系,该细胞系具有多能分化特性,并可定向分化为心肌细胞,为研究ARVC的致病因素和药物筛选提供宝贵的实验模型。     

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.     

CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia

Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies, disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro ‘patient-specific cell-based system'' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited form of fatal arrhythmia. Here, we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs, both in resting conditions and after β-adrenergic stimulation, resembling the cardiac phenotype of the patients. Furthermore, treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), an antiarrhythmic drug that inhibits Ca²⁺/calmodulin-dependent serine–threonine protein kinase II (CaMKII), drastically reduced the presence of DADs in CVPT-CMs, rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition, intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients, whereas in the wild-type clusters, only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice, the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells, supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies.     

CRISPR/Cas9 Gene editing of RyR2 in human stem cell-derived cardiomyocytes provides a novel approach in investigating dysfunctional Ca2+ signaling

Type-2 ryanodine receptors (RyR2s) play a pivotal role in cardiac excitation-contraction coupling by releasing Ca²⁺ from sarcoplasmic reticulum (SR) via a Ca²⁺ -induced Ca²⁺ release (CICR) mechanism. Two strategies have been used to study the structure-function characteristics of RyR2 and its disease associated mutations: (1) heterologous cell expression of the recombinant mutant RyR2s, and (2) knock-in mouse models harboring RyR2 point mutations. Here, we establish an alternative approach where Ca²⁺ signaling aberrancy caused by the RyR2 mutation is studied in human cardiomyocytes with robust CICR mechanism. Specifically, we introduce point mutations in wild-type RYR2 of human induced pluripotent stem cells (hiPSCs) by CRISPR/Cas9 gene editing, and then differentiate them into cardiomyocytes. To verify the reliability of this approach, we introduced the same disease-associated RyR2 mutation, F2483I, which was studied by us in hiPSC-derived cardiomyocytes (hiPSC-CMs) from a patient biopsy. The gene-edited F2483I hiPSC-CMs exhibited longer and wandering Ca²⁺ sparks, elevated diastolic Ca²⁺ leaks, and smaller SR Ca²⁺ stores, like those of patient-derived cells. Our CRISPR/Cas9 gene editing approach validated the feasibility of creating myocytes expressing the various RyR2 mutants, making comparative mechanistic analysis and pharmacotherapeutic approaches for RyR2 pathologies possible.     

新物种诱导多能干细胞的研究进展

胚胎干细胞（embryonic stem cells,ESC）在人类遗传病学研究、疾病模型建立、器官再生以及动物物种改良和定向变异等方面的地位是其他类型的细胞不可取代的。但是,由于实验技术和体外培养条件的限制,除了小鼠、恒河猴和人之外,大鼠、猪、牛、羊等其他哺乳动物的ES细胞系被证明很难获得。先后有多个研究小组报道了他们利用新兴的诱导多能干细胞（induced pluripotent stem cells,iPS细胞）技术成功建立大鼠和猪的iPS细胞系的研究成果。迄今为止,这两个物种是在未成功建立ES细胞系之前利用iPS技术建立多能干细胞系的成功范例。这些研究对于那些还未建立ES细胞的物种建立多能干细胞系提供了一种新的方案,也将给这些物种的胚胎干细胞的建立、基因修饰动物的产生以及人类医疗事业的促进和发展带来新的希望。     

Regulation of Ca2+ signaling by acute hypoxia and acidosis in cardiomyocytes derived from human induced pluripotent stem cells

AimsThe effects of acute (100 s) hypoxia and/or acidosis on Ca²⁺ signaling parameters of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are explored here for the first time.Methods and results1) hiPSC-CMs express two cell populations: rapidly-inactivating I_Ca myocytes (τ_i<40 ms, in 4–5 day cultures) and slowly-inactivating I_Ca (τ_i ≥ 40 ms, in 6–8 day cultures). 2) Hypoxia suppressed I_Ca by 10–20% in rapidly- and 40–55% in slowly-inactivating I_Ca cells. 3) Isoproterenol enhanced I_Ca in hiPSC-CMs, but either enhanced or did not alter the hypoxic suppression. 4) Hypoxia had no differential suppressive effects in the two cell-types when Ba²⁺ was the charge carrier through the calcium channels, implicating Ca²⁺-dependent inactivation in O₂ sensing. 5) Acidosis suppressed I_Ca by ∼35% and ∼25% in rapidly and slowly inactivating I_Ca cells, respectively. 6) Hypoxia and acidosis suppressive effects on Ca-transients depended on whether global or RyR2-microdomain were measured: with acidosis suppression was ∼25% in global and ∼37% in RyR2 Ca²⁺-microdomains in either cell type, whereas with hypoxia suppression was ∼20% and ∼25% respectively in global and RyR2-microdomaine in rapidly and ∼35% and ∼45% respectively in global and RyR2-microdomaine in slowly-inactivating cells.ConclusionsVariability in I_Ca inactivation kinetics rather than cellular ancestry seems to underlie the action potential morphology differences generally attributed to mixed atrial and ventricular cell populations in hiPSC-CMs cultures. The differential hypoxic regulation of Ca²⁺-signaling in the two-cell types arises from differential Ca²⁺-dependent inactivation of the Ca²⁺-channel caused by proximity of Ca²⁺-release stores to the Ca²⁺ channels.     

Functional abnormalities in iPSC‐derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia characterized by syncope and sudden death occurring during exercise or acute emotion. CPVT is caused by abnormal intracellular Ca²⁺ handling resulting from mutations in the RyR2 or CASQ2 genes. Because CASQ2 and RyR2 are involved in different aspects of the excitation‐contraction coupling process, we hypothesized that these mutations are associated with different functional and intracellular Ca²⁺ abnormalities. To test the hypothesis we generated induced Pluripotent Stem Cell‐derived cardiomyocytes (iPSC‐CM) from CPVT1 and CPVT2 patients carrying the RyR2^R420Q and CASQ2^D307H mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC‐CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca²⁺]_i transient characteristics. Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC‐CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca²⁺]_i. Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC‐CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.     

Critical roles of hMAGEA2 in induced pluripotent stem cell pluripotency,proliferation, and differentiation

Induced pluripotent stem (iPS) cells are important for clinical application and stem cell research. Although human melanoma‐associated antigen A2 (hMAGEA2) expression is known to affect differentiation in embryonic stem cells, its specific role in iPS cells remains unclear. To evaluate the function of hMAGEA2 and its characteristics in iPS cells, we produced hMAGEA2‐overexpressing iPS cells from hMAGEA2‐overexpressing transgenic mice. Although the iPS cells with overexpressed hMAGEA2 did not differ in morphology, their pluripotency, and self‐renewal related genes (Nanog, Oct3/4, Sox2, and Stat3), expression level was significantly upregulated. Moreover, hMAGEA2 contributed to the promotion of cell cycle progression, thereby accelerating cell proliferation. Through embryoid body formation in vitro and teratoma formation in vivo, we demonstrated that hMAGEA2 critically decreases the differentiation ability of iPS cells. These data indicate that hMAGEA2 intensifies the self‐renewal, pluripotency, and degree of proliferation of iPS cells, while significantly repressing their differentiation efficiency. Therefore, our findings prove that hMAGEA2 plays key roles in iPS cells.     

Generation and characterization of functional cardiomyocytes using induced pluripotent stem cells derived from human fibroblasts

We have successfully developed both spontaneous and inductive cardiomyocyte differentiation of iPS cells reprogrammed from human foreskin fibroblasts. The reprogrammed iPS cells morphologically resemble human cardiomyocytes which can beat. RT-PCR and immunostaining show that cardiac markers are expressed that are comparable to the differentiation pattern of authentic human embryonic stem cells, indicating the existence of both immature and mature differentiated cardiomyocytes. 5-Azacytidine greatly enhanced the efficiency of cardiomyocyte differentiation, whereas dimethylsulfoxide had no effect. Low serum and bone morphogenetic protein-2 marginally improved differentiation efficiency. iPS cell-derived cardiomyocytes changed their beat frequency in response to cardiac drugs, which included ion channel blockers and α/β adrenergic stimulators. Derived cardiomyocytes look promising as an in vitro system for potential drug screen and/or toxicity, making this system closer to practical use in the near future.     

The effects of cardioactive drugs on cardiomyocytes derived from human induced pluripotent stem cells

Developing effective drug therapies for arrhythmic diseases is hampered by the fact that the same drug can work well in some individuals but not in others. Human induced pluripotent stem (iPS) cells have been vetted as useful tools for drug screening. However, cardioactive drugs have not been shown to have the same effects on iPS cell-derived human cardiomyocytes as on embryonic stem (ES) cell-derived cardiomyocytes or human cardiomyocytes in a clinical setting. Here we show that current cardioactive drugs affect the beating frequency and contractility of iPS cell-derived cardiomyocytes in much the same way as they do ES cell-derived cardiomyocytes, and the results were compatible with empirical results in the clinic. Thus, human iPS cells could become an attractive tool to investigate the effects of cardioactive drugs at the individual level and to screen for individually tailored drugs against cardiac arrhythmic diseases.     

Derivation of male germ cells from induced pluripotent stem cells by inducers: A review

Induced pluripotent stem cells (iPSCs) refer to stem cells that are artificially produced using a new technology known as cellular reprogramming, which can use gene transduction in somatic cells. There are numerous potential applications for iPSCs in the field of stem cell biology becauase they are able to give rise to several different cell features of lineages such as three-germ layers. Primordial germ cells, generated via in vitro differentiation of iPSCs, have been demonstrated to produce functional gametes. Therefore, in this review we discussed past and recent advances in the in vitro differentiation of germ cells using pluripotent stem cells with an emphasis on iPSCs. Although this domain of research is still in its infancy, exploring development mechanisms of germ cells is promising, especially in humans, to promote future reproductive and developmental engineering technologies. While few studies have evaluated the ability and efficiency of iPSCs to differentiate toward male germ cells in vitro by different inducers, the given effect was investigated in this review.     

The first reported generation of human induced pluripotent stem cells (iPS cells) and iPS cell-derived cardiomyocytes in the Netherlands

One of the recent breakthroughs in stem cell research has been the reprogramming of human somatic cells to an embryonic stem cell (ESC)-like state (induced pluripotent stem cells, iPS cells). Similar to ESCs, iPS cells can differentiate into derivatives of the three germ layers, for example cardiomyocytes, pancreatic cells or neurons. This technique offers a new approach to investigating disease pathogenesis and to the development of novel therapies. It may now be possible to generate iPS cells from somatic cells of patients who suffer from vascular genetic diseases, such as hereditary haemorrhagic telangiectasia (HHT). The iPS cells will have a similar genotype to that of the patient and can be differentiated in vitro into the cell type(s) that are affected in the patient. Thus they will serve as excellent models for a better understanding of mechanisms underlying the disease. This, together with the ability to test new drugs, could potentially lead to novel therapeutic concepts in the near future. Here we report the first derivation of three human iPS cell lines from two healthy individuals and one HHT patient in the Netherlands. The iPS cells resembled ESCs in morphology and expressed typical ESC markers. In vitro, iPS cells could be differentiated into cells of the three germ layers, including beating cardiomyocytes and vascular cells. With this technique it will be possible to establish human cardiovascular disease models from patient biopsies provided by the principal hospitals in the Netherlands. (Neth Heart J 2010;18:51-4.)     

The applications of induced pluripotent stem (iPS) cells in drug development

The introduction of induced pluripotent stem (iPS) cells has been a milestone in the field of regenerative medicine and drug discovery. iPS cells can provide a continuous and individualized source of stem cells and are considered to hold great potential for economically feasible personalized stem cell therapy. Various diseases might potentially be cured by iPS cell-based therapy including Parkinson’s disease, Alzheimer’s disease, Huntington disease, ischemic heart disease, diabetes and so on. Moreover, iPS cells derived from patients suffering from unique incurable diseases can be developed into patient- and disease-specific cell lines. These cells can be used as an effective approach to study the mechanisms of diseases, providing useful tools for drug discovery, development and evaluation. The development of suitable methods for the culture and expansion of iPS cells and their differentiated progenies make feasible modern drug discovery techniques such as high-throughput screening. Furthermore, iPS cells can be applied in the field of toxicological and pharmacokinetics tests. This review focuses on the applications of iPS cells in the field of pharmaceutical industry.