AFM nano‐mechanical study of the beating profile of hiPSC‐derived cardiomyocytes beating bodies WT and DM1

Myotonic Dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterized by a variety of multisystemic features and associated with cardiac anomalies. Among cardiac phenomena, conduction defects, ventricular arrhythmias, and dilated cardiomyopathy represent the main cause of sudden death in DM1 patients. Patient‐specific induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) represent a powerful in vitro model for molecular, biochemical, and physiological studies of disease in the target cells. Here, we used an Atomic Force Microscope (AFM) to measure the beating profiles of a large number of cells, organized in CM clusters (Beating Bodies, BBs), obtained from wild type (WT) and DM1 patients. We monitored the evolution over time of the frequency and intensity of the beating. We determined the variations between different BBs and over various areas of a single BB, caused by morphological and biomechanical variations. We exploited the AFM tip to apply a controlled force over the BBs, to carefully assess the biomechanical reaction of the different cell clusters over time, both in terms of beating frequency and intensity. Our measurements demonstrated differences between the WT and DM1 clusters highlighting, for the DM1 samples, an instability which was not observed in WT cells. We measured differences in the cellular response to the applied mechanical stimulus in terms of beating synchronicity over time and cell tenacity, which are in good agreement with the cellular behavior in vivo. Overall, the combination of hiPSC‐CMs with AFM characterization can become a new tool to study the collective movements of cell clusters in different conditions and can be extended to the characterization of the BB response to chemical and pharmacological stimuli.     

Serum supplemented culture medium masks hypertrophic phenotypes in human pluripotent stem cell derived cardiomyocytes

It has been known for over 20 years that foetal calf serum can induce hypertrophy in cultured cardiomyocytes but this is rarely considered when examining cardiomyocytes derived from pluripotent stem cells (PSC). Here, we determined how serum affected cardiomyocytes from human embryonic‐ (hESC) and induced pluripotent stem cells (hiPSC) and hiPSC from patients with hypertrophic cardiomyopathy linked to a mutation in the MYBPC3 gene. We first confirmed previously published hypertrophic effects of serum on cultured neonatal rat cardiomyocytes demonstrated as increased cell surface area and beating frequency. We then found that serum increased the cell surface area of hESC‐ and hiPSC‐derived cardiomyocytes and their spontaneous contraction rate. Phenylephrine, which normally induces cardiac hypertrophy, had no additional effects under serum conditions. Likewise, hiPSC‐derived cardiomyocytes from three MYBPC3 patients which had a greater surface area than controls in the absence of serum as predicted by their genotype, did not show this difference in the presence of serum. Serum can thus alter the phenotype of human PSC derived cardiomyocytes under otherwise defined conditions such that the effects of hypertrophic drugs and gene mutations are underestimated. It is therefore pertinent to examine cardiac phenotypes in culture media without or in low concentrations of serum.     

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.     

多能干细胞诱导分化及体细胞转分化为心肌细胞的研究进展

心血管疾病是威胁人类健康的重大疾病,而心肌细胞数量逐渐减少,甚至衰竭是其核心病变。心肌细胞补偿性替代治疗是未来用于治疗这类疾病的重要手段,因此,心肌细胞的来源和有效治疗将成为关键。目前,心肌细胞构建的主要方法有多能干细胞诱导分化成心肌祖细胞或心肌细胞、心源性心肌祖细胞,以及体细胞重编程等。其中,多能干细胞向心肌细胞分化是最常用的方法;而体细胞转分化技术相较于传统的诱导多潜能干细胞衍生心肌细胞缩短了时间窗,为潜在的心血管疾病治疗提供了另一种思路。随着获取心肌细胞效率及其质量的提升,未来心血管疾病的治疗将有望获得重大突破。     

Highly efficient derivation of ventricular cardiomyocytes from induced pluripotent stem cells with a distinct epigenetic signature

Cardiomyocytes derived from pluripotent stem cells can be applied in drug testing, disease modeling and cell-based therapy. However, without procardiogenic growth factors, the efficiency of cardiomyogenesis from pluripotent stem cells is usually low and the resulting cardiomyocyte population is heterogeneous. Here, we demonstrate that induced pluripotent stem cells (iPSCs) can be derived from murine ventricular myocytes (VMs), and consistent with other reports of iPSCs derived from various somatic cell types, VM-derived iPSCs (ViPSCs) exhibit a markedly higher propensity to spontaneously differentiate into beating cardiomyocytes as compared to genetically matched embryonic stem cells (ESCs) or iPSCs derived from tail-tip fibroblasts. Strikingly, the majority of ViPSC-derived cardiomyocytes display a ventricular phenotype. The enhanced ventricular myogenesis in ViPSCs is mediated via increased numbers of cardiovascular progenitors at early stages of differentiation. In order to investigate the mechanism of enhanced ventricular myogenesis from ViPSCs, we performed global gene expression and DNA methylation analysis, which revealed a distinct epigenetic signature that may be involved in specifying the VM fate in pluripotent stem cells.     

Embryonic stem cells and cardiomyocyte differentiation: phenotypic and molecular analyses

Embryonic stem (ES) cell lines, derived from the inner cell mass (ICM) of blastocyst-stage embryos, are pluripotent and have a virtually unlimited capacity for self-renewal and differentiation into all cell types of an embryoproper. Both human and mouse ES cell lines are the subject of intensive investigation for potential applications in developmental biology and medicine. ES cells from both sources differentiate in vitro into cells of ecto-, endoand meso-dermal lineages, and robust cardiomyogenic differentiation is readily observed in spontaneously differentiating ES cells when cultured under appropriate conditions. Molecular, cellular and physiologic analyses demonstrate that ES cell-derived cardiomyocytes are functionally viable and that these cell derivatives exhibit characteristics typical of heart cells in early stages of cardiac development. Because terminal heart failure is characterized by a significant loss of cardiomyocytes, the use of human ES cell-derived progeny represents one possible source for cell transplantation therapies. With these issues in mind, this review will focus on the differentiation of pluripotent embryonic stem cells into cardiomyocytes as a developmental model, and the possible use of ES cell-derived cardiomyocytes as source of donor cells.     

Coupling primary and stem cell–derived cardiomyocytes in an in vitro model of cardiac cell therapy

The efficacy of cardiac cell therapy depends on the integration of existing and newly formed cardiomyocytes. Here, we developed a minimal in vitro model of this interface by engineering two cell microtissues (μtissues) containing mouse cardiomyocytes, representing spared myocardium after injury, and cardiomyocytes generated from embryonic and induced pluripotent stem cells, to model newly formed cells. We demonstrated that weaker stem cell–derived myocytes coupled with stronger myocytes to support synchronous contraction, but this arrangement required focal adhesion-like structures near the cell–cell junction that degrade force transmission between cells. Moreover, we developed a computational model of μtissue mechanics to demonstrate that a reduction in isometric tension is sufficient to impair force transmission across the cell–cell boundary. Together, our in vitro and in silico results suggest that mechanotransductive mechanisms may contribute to the modest functional benefits observed in cell-therapy studies by regulating the amount of contractile force effectively transmitted at the junction between newly formed and spared myocytes.     

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy.     

Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch

IntroductionDilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates.MethodshiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes.ResultsSEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro.ConclusionsOverall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch.     

Human dental pulp stem cells: Applications in future regenerative medicine

Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells (MSCs) from various human tissues, peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells (DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine.     

Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers

Abstract. A defined cultivation system was developed for the differentiation of pluripotent embryonic stem cells of the mouse into spontaneously beating cardiomyocytes, allowing investigations of chronotropic responses, as well as electrophysiological studies of different cardioactive drugs in vitro.
The β-adrenoceptor agonists (—)isoprenaline and clenbuterol, the mediators of cAMP metabolism, forsko-lin and isobutylmethylxanthine (IBMX), the α₁-adreno-ceptor agonist (—)phenylephrine, and the heart glyco-side digitoxine induced a positive, the muscarinic cholin-oceptor agonist carbachol and L-type Ca²⁺ channel blockers nisoldipine, gallopamil and diltiazem induced a negative chronotropic response.
In early differentiated cardiomyocytes β₁-, α₁-, but not β₂-adrenoceptors, cholinoceptors, as well as L-type Ca²⁺ channels participated in the chronotropic response. In terminally differentiated cardiomyocytes β₂-adrenoceptors and digitoxine responses were also functionally expressed.
The contractions of spontaneously beating cardiomyocytes were concommitant with rhythmic action potentials very similar to those described for embryonic cardiomyocytes and sinusnode cells. We conclude that cardiomyocytes differentiating from pluripotent embryonic stem cells are able to develop adrenoceptors and cholinoceptors and signal transduction pathways as well as L-type Ca²⁺ channels as a consequence of cell-cell interactions during embryoid body formation in vitro, independent of the development in living organisms.
The cellular system described may be useful as in vitro assay for toxicological investigations of chronotropic drugs and a model system for studying commitment and cellular differentiation in vitro.     

Adipose‐derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.     

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.     

干细胞与心肌细胞替代治疗

胚胎干细胞及来源于骨髓、骨骼肌、血管、肝脏、皮肤、脂肪等组织器官的成体干细胞均有多向分化潜能。胚胎干细胞可分化为3个胚层的所有组织细胞。成体干细胞具有可塑性和转分化的潜能。在一定条件下,这些干细胞可被诱导分化为心肌细胞。成年心脏可能存在心肌干细胞,具有增殖和分化为包括跳动性心肌细胞的多种细胞的潜能。因此,干细胞可用于心肌细胞替代治疗,以替代死亡的心肌细胞,改善心脏功能,防治心肌梗塞后心衰、减少心肌重构等症状。本文对干细胞治疗心肌梗塞有关进展及问题作一综述。     

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.     

Ubiquitination and degradation of the anti-apoptotic protein ARC by MDM2

Current evidence shows that cardiomyocyte apoptosis plays a central role in the pathogenesis of myocardial disease and that reactive oxygen species is critically responsible for mediating cardiomyocyte apoptosis in both ischemia-reperfusion injury and dilated cardiomyopathy. ARC (Apoptosis Repressor with Caspase recruitment domain) is an anti-apoptotic protein that is found abundantly in terminally differentiated cells such as cardiomyocytes. The ARC knock-out mouse developed larger infarct in response to ischemia-reperfusion and transitioned more rapidly and severely to dilated cardiomyopathy following aortic constriction. In addition, ARC protein levels are decreased in human dilated cardiomyopathy and when cardiomyocytes are exposed to oxidative stress in vitro, but the mechanisms regulating ARC protein levels are not known. Here we show that degradation of ARC is dependent on the p53-induced ubiquitin E3 ligase, MDM2. Oxidative stress reduced ARC levels and up-regulated MDM2. MDM2 directly accelerated ARC protein turnover via ubiquitination and proteasomal-dependent degradation. This activity requires a functioning MDM2 ring finger domain because the MDM2(C464A) mutant was unable to direct ARC degradation. Furthermore, ARC degradation requires MDM2, because MDM2 knock-out fibroblasts showed defective ARC degradation that could be rescued by MDM2. Proteasomal inhibitors rescued both MDM2 and H(2)O(2)-induced degradation of ARC and inhibited cardiomyocyte apoptosis. Dilated cardiomyopathic hearts from mice that have undergone transverse aortic banding have increased MDM2 levels associated with decreased ARC levels. We conclude that MDM2 is a critical regulator of ARC levels in cardiomyocytes. Prevention of MDM2-induced degradation of ARC represents a potential therapeutic target to prevent cardiomyocyte apoptosis.     

Defining Differentially Methylated Regions Specific for the Acquisition of Pluripotency and Maintenance in Human Pluripotent Stem Cells via Microarray

Background

Epigenetic regulation is critical for the maintenance of human pluripotent stem cells. It has been shown that pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, appear to have a hypermethylated status compared with differentiated cells. However, the epigenetic differences in genes that maintain stemness and regulate reprogramming between embryonic stem cells and induced pluripotent stem cells remain unclear. Additionally, differential methylation patterns of induced pluripotent stem cells generated using diverse methods require further study.

Methodology

Here, we determined the DNA methylation profiles of 10 human cell lines, including 2 ESC lines, 4 virally derived iPSC lines, 2 episomally derived iPSC lines, and the 2 parental cell lines from which the iPSCs were derived using Illumina''s Infinium HumanMethylation450 BeadChip. The iPSCs exhibited a hypermethylation status similar to that of ESCs but with distinct differences from the parental cells. Genes with a common methylation pattern between iPSCs and ESCs were classified as critical factors for stemness, whereas differences between iPSCs and ESCs suggested that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cellular reprogramming. No significant differences were identified between virally and episomally derived iPSCs. This study determined in detail the de novo differential methylation signatures of particular stem cell lines.

Conclusions

This study describes the DNA methylation profiles of human iPSCs generated using both viral and episomal methods, the corresponding somatic cells, and hESCs. Series of ss-DMRs and ES-iPS-DMRs were defined with high resolution. Knowledge of this type of epigenetic information could be used as a signature for stemness and self-renewal and provides a potential method for selecting optimal pluripotent stem cells for human regenerative medicine.