Adipocyte-mediated electrophysiological remodeling of human stem cell - derived cardiomyocytes.

Adipocytes normally accumulate in the epicardial and pericardial layers around the human heart, but their infiltration into the myocardium can be proarrhythmic. METHODS AND RESULTS: Human adipose derived stem/stromal cells and human induced pluripotent stem cells (hiPSC) were differentiated, respectively into predominantly white fat-like adipocytes (hAdip) and ventricular cardiomyocytes (CMs). Adipocytes cultured in CM maintenance medium (CM medium) maintained their morphology, continued to express adipogenic markers, and retained clusters of intracellular lipid droplets. In contrast, hiPSC-CMs cultivated in adipogenic growth medium displayed abnormal cell morphologies and more clustering across the monolayer. Pre-plated hiPSC-CMs co-cultured in direct contact with hAdips in CM medium displayed prolonged action potential durations, increased triangulation, slowed conduction velocity, increased conduction velocity heterogeneity, and prolonged calcium transients. When hAdip-conditioned medium was added to monolayer cultures of hiPSC-CMs, results similar to those recorded with direct co-cultures were observed. Both co-culture and conditioned medium experiments resulted in increases in transcript abundance of SCN10A, CACNA1C, SLC8A1, and RYR2, with a decrease in KCNJ2. Human adipokine immunoblots revealed the presence of cytokines that were elevated in adipocyte-conditioned medium, including MCP-1, IL-6, IL-8 and CFD that could induce electrophysiological changes in cultured hiPSC-CMs. CONCLUSIONS: Co-culture of hiPSC-CMs with hAdips reveals a potentially pathogenic role of infiltrating human adipocytes on myocardial tissue. In the absence of structural changes, hAdip paracrine release alone is sufficient to cause CM electrophysiological dysfunction mirroring the co-culture conditions. These effects, mediated largely by paracrine mechanisms, could promote arrhythmias in the heart.

Understanding Arrhythmogenic Cardiomyopathy: Advances through the Use of Human Pluripotent Stem Cell Models.

Cardiomyopathies (CMPs) represent a significant healthcare burden and are a major cause of heart failure leading to premature death. Several CMPs are now recognized to have a strong genetic basis, including arrhythmogenic cardiomyopathy (ACM), which predisposes patients to arrhythmic episodes. Variants in one of the five genes (PKP2, JUP, DSC2, DSG2, and DSP) encoding proteins of the desmosome are known to cause a subset of ACM, which we classify as desmosome-related ACM (dACM). Phenotypically, this disease may lead to sudden cardiac death in young athletes and, during late stages, is often accompanied by myocardial fibrofatty infiltrates. While the pathogenicity of the desmosome genes has been well established through animal studies and limited supplies of primary human cells, these systems have drawbacks that limit their utility and relevance to understanding human disease. Human induced pluripotent stem cells (hiPSCs) have emerged as a powerful tool for modeling ACM in vitro that can overcome these challenges, as they represent a reproducible and scalable source of cardiomyocytes (CMs) that recapitulate patient phenotypes. In this review, we provide an overview of dACM, summarize findings in other model systems linking desmosome proteins with this disease, and provide an up-to-date summary of the work that has been conducted in hiPSC-cardiomyocyte (hiPSC-CM) models of dACM. In the context of the hiPSC-CM model system, we highlight novel findings that have contributed to our understanding of disease and enumerate the limitations, prospects, and directions for research to consider towards future progress.

Integrated modeling framework reveals co-regulation of transcription factors, miRNAs and lncRNAs on cardiac developmental dynamics.

AIMS: Dissecting complex interactions among transcription factors (TFs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are central for understanding heart development and function. Although computational approaches and platforms have been described to infer relationships among regulatory factors and genes, current approaches do not adequately account for how highly diverse, interacting regulators that include noncoding RNAs (ncRNAs) control cardiac gene expression dynamics over time. METHODS: To overcome this limitation, we devised an integrated framework, cardiac gene regulatory modeling (CGRM) that integrates LogicTRN and regulatory component analysis bioinformatics modeling platforms to infer complex regulatory mechanisms. We then used CGRM to identify and compare the TF-ncRNA gene regulatory networks that govern early- and late-stage cardiomyocytes (CMs) generated by in vitro differentiation of human pluripotent stem cells (hPSC) and ventricular and atrial CMs isolated during in vivo human cardiac development. RESULTS: Comparisons of in vitro versus in vivo derived CMs revealed conserved regulatory networks among TFs and ncRNAs in early cells that significantly diverged in late staged cells. We report that cardiac genes ("heart targets") expressed in early-stage hPSC-CMs are primarily regulated by MESP1, miR-1, miR-23, lncRNAs NEAT1 and MALAT1, while GATA6, HAND2, miR-200c, NEAT1 and MALAT1 are critical for late hPSC-CMs. The inferred TF-miRNA-lncRNA networks regulating heart development and contraction were similar among early-stage CMs, among individual hPSC-CM datasets and between in vitro and in vivo samples. However, genes related to apoptosis, cell cycle and proliferation, and transmembrane transport showed a high degree of divergence between in vitro and in vivo derived late-stage CMs. Overall, late-, but not early-stage CMs diverged greatly in the expression of "heart target" transcripts and their regulatory mechanisms. CONCLUSIONS: In conclusion, we find that hPSC-CMs are regulated in a cell autonomous manner during early development that diverges significantly as a function of time when compared to in vivo derived CMs. These findings demonstrate the feasibility of using CGRM to reveal dynamic and complex transcriptional and posttranscriptional regulatory interactions that underlie cell directed versus environment-dependent CM development. These results with in vitro versus in vivo derived CMs thus establish this approach for detailed analyses of heart disease and for the analysis of cell regulatory systems in other biomedical fields.

Adipose tissue-derived human mesenchymal stromal cells can better suppress complement lysis, engraft and inhibit acute graft-versus-host disease in mice.

BACKGROUND: Acute graft-versus-host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Transplantation of immunosuppressive human mesenchymal stromal cells (hMSCs) can protect against aGvHD post-HSCT; however, their efficacy is limited by poor engraftment and survival. Moreover, infused MSCs can be damaged by activated complement, yet strategies to minimise complement injury of hMSCs and improve their survival are limited. METHODS: Human MSCs were derived from bone marrow (BM), adipose tissue (AT) and umbilical cord (UC). In vitro immunomodulatory potential was determined by co-culture experiments between hMSCs and immune cells implicated in aGvHD disease progression. BM-, AT- and UC-hMSCs were tested for their abilities to protect aGvHD in a mouse model of this disease. Survival and clinical symptoms were monitored, and target tissues of aGvHD were examined by histopathology and qPCR. Transplanted cell survival was evaluated by cell tracing and by qPCR. The transcriptome of BM-, AT- and UC-hMSCs was profiled by RNA-sequencing. Focused experiments were performed to compare the expression of complement inhibitors and the abilities of hMSCs to resist complement lysis. RESULTS: Human MSCs derived from three tissues divergently protected against aGvHD in vivo. AT-hMSCs preferentially suppressed complement in vitro and in vivo, resisted complement lysis and survived better after transplantation when compared to BM- and UC-hMSCs. AT-hMSCs also prolonged survival and improved the symptoms and pathological features of aGvHD. We found that complement-decay accelerating factor (CD55), an inhibitor of complement, is elevated in AT-hMSCs and contributed to reduced complement activation. We further report that atorvastatin and erlotinib could upregulate CD55 and suppress complement in all three types of hMSCs. CONCLUSION: CD55, by suppressing complement, contributes to the improved protection of AT-hMSCs against aGvHD. The use of AT-hMSCs or the upregulation of CD55 by small molecules thus represents promising new strategies to promote hMSC survival to improve the efficacy of transplantation therapy. As complement injury is a barrier to all types of hMSC therapy, our findings are of broad significance to enhance the use of hMSCs for the treatment of a wide range of disorders.

Surfaceome mapping of primary human heart cells with CellSurfer uncovers cardiomyocyte surface protein LSMEM2 and proteome dynamics in failing hearts.

Cardiac cell surface proteins are drug targets and useful biomarkers for discriminating among cellular phenotypes and disease states. Here we developed an analytical platform, CellSurfer, that enables quantitative cell surface proteome (surfaceome) profiling of cells present in limited quantities, and we apply it to isolated primary human heart cells. We report experimental evidence of surface localization and extracellular domains for 1,144 N-glycoproteins, including cell-type-restricted and region-restricted glycoproteins. We identified a surface protein specific for healthy cardiomyocytes, LSMEM2, and validated an anti-LSMEM2 monoclonal antibody for flow cytometry and imaging. Surfaceome comparisons among pluripotent stem cell derivatives and their primary counterparts highlighted important differences with direct implications for drug screening and disease modeling. Finally, 20% of cell surface proteins, including LSMEM2, were differentially abundant between failing and non-failing cardiomyocytes. These results represent a rich resource to advance development of cell type and organ-specific targets for drug delivery, disease modeling, immunophenotyping and in vivo imaging.

Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.

Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have hindered disease treatments. Here, we show that ABCA4 knockout and induced pluripotent stem cell-derived RPE (STGD1-iRPE) from patients with Stargardt differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity-indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are lowered by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.

Altered Electrical, Biomolecular, and Immunologic Phenotypes in a Novel Patient-Derived Stem Cell Model of Desmoglein-2 Mutant ARVC.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive heart condition which causes fibro-fatty myocardial scarring, ventricular arrhythmias, and sudden cardiac death. Most cases of ARVC can be linked to pathogenic mutations in the cardiac desmosome, but the pathophysiology is not well understood, particularly in early phases when arrhythmias can develop prior to structural changes. Here, we created a novel human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of ARVC from a patient with a c.2358delA variant in desmoglein-2 (DSG2). These DSG2-mutant (DSG2Mut) hiPSC-CMs were compared against two wildtype hiPSC-CM lines via immunostaining, RT-qPCR, Western blot, RNA-Seq, cytokine expression and optical mapping. Mutant cells expressed reduced DSG2 mRNA and had altered localization of desmoglein-2 protein alongside thinner, more disorganized myofibrils. No major changes in other desmosomal proteins were noted. There was increased pro-inflammatory cytokine expression that may be linked to canonical and non-canonical NFκB signaling. Action potentials in DSG2Mut CMs were shorter with increased upstroke heterogeneity, while time-to-peak calcium and calcium decay rate were reduced. These were accompanied by changes in ion channel and calcium handling gene expression. Lastly, suppressing DSG2 in control lines via siRNA allowed partial recapitulation of electrical anomalies noted in DSG2Mut cells. In conclusion, the aberrant cytoskeletal organization, cytokine expression, and electrophysiology found DSG2Mut hiPSC-CMs could underlie early mechanisms of disease manifestation in ARVC patients.

Maturing heart muscle cells: Mechanisms and transcriptomic insights.

Cardiomyocyte (CM) maturation is the transformation of differentiated fetal CMs into adult CMs that involves changes in morphology, cell function and metabolism, and the transcriptome. This process is, however, incomplete and ultimately arrested in pluripotent stem cell-derived CMs (PSC-CMs) in culture, which hinders their broad biomedical application. For this reason, enormous efforts are currently being made with the goal of generating mature PSC-CMs. In this review, we summarize key aspects of maturation observed in native CMs and discuss recent findings on the factors and mechanisms that regulate the process. Particular emphasis is put on transcriptional regulation and single-cell RNA-sequencing analysis that has emerged as a key tool to study time-series gene regulation and to determine the maturation state. We then discuss different biomimetic strategies to enhance PSC-CM maturation and discuss their effects at the single cell transcriptomic and functional levels.

Cell surface markers for immunophenotyping human pluripotent stem cell-derived cardiomyocytes.

Human pluripotent stem cells (hPSC) self-renew and represent a potentially unlimited source for the production of cardiomyocytes (CMs) suitable for studies of human cardiac development, drug discovery, cardiotoxicity testing, and disease modelling and for cell-based therapies. However, most cardiac differentiation protocols yield mixed cultures of atrial-, ventricular-, and pacemaker-like cells at various stages of development, as well as non-CMs. The proportions and maturation states of these cell types result from disparities among differentiation protocols and time of cultivation, as well as hPSC reprogramming inconsistencies and genetic background variations. The reproducible use of hPSC-CMs for research and therapy is therefore limited by issues of cell population heterogeneity and functional states of maturation. A validated method that overcomes issues of cell heterogeneity is immunophenotyping coupled with live cell sorting, an approach that relies on accessible surface markers restricted to the desired cell type(s). Here we review current progress in unravelling heterogeneity in hPSC-cardiac cultures and in the identification of surface markers suitable for defining cardiac identity, subtype specificity, and maturation states.

Importance of evaluating protein glycosylation in pluripotent stem cell-derived cardiomyocytes for research and clinical applications.

Proper protein glycosylation is critical to normal cardiomyocyte physiology. Aberrant glycosylation can alter protein localization, structure, drug interactions, and cellular function. The in vitro differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM) has become increasingly important to the study of protein function and to the fields of cardiac disease modeling, drug testing, drug discovery, and regenerative medicine. Here, we offer our perspective on the importance of protein glycosylation in hPSC-CM. Protein glycosylation is dynamic in hPSC-CM, but the timing and extent of glycosylation are still poorly defined. We provide new data highlighting how observed changes in hPSC-CM glycosylation may be caused by underlying differences in the protein or transcript abundance of enzymes involved in building and trimming the glycan structures or glycoprotein gene products. We also provide evidence that alternative splicing results in altered sites of glycosylation within the protein sequence. Our findings suggest the need to precisely define protein glycosylation events that may have a critical impact on the function and maturation state of hPSC-CM. Finally, we provide an overview of analytical strategies available for studying protein glycosylation and identify opportunities for the development of new bioinformatic approaches to integrate diverse protein glycosylation data types. We predict that these tools will promote the accurate assessment of protein glycosylation in future studies of hPSC-CM that will ultimately be of significant experimental and clinical benefit.

Cardiac Na+-Ca2+ exchanger 1 (ncx1h) is critical for the ventricular cardiomyocyte formation via regulating the expression levels of gata4 and hand2 in zebrafish.

Ca2+ signaling is critical for heart development; however, the precise roles and regulatory pathways of Ca2+ transport proteins in cardiogenesis remain largely unknown. Sodium-calcium exchanger 1 (Ncx1) is responsible for Ca2+ efflux in cardiomyocytes. It is involved in cardiogenesis, while the mechanism is unclear. Here, using the forward genetic screening in zebrafish, we identified a novel mutation at a highly-conserved leucine residue in ncx1 gene (mutantLDD353/ncx1hL154P) that led to smaller hearts with reduced heart rate and weak contraction. Mechanistically, the number of ventricular but not atrial cardiomyocytes was reduced in ncx1hL154P zebrafish. These defects were mimicked by knockdown or knockout of ncx1h. Moreover, ncx1hL154P had cytosolic and mitochondrial Ca2+ overloading and Ca2+ transient suppression in cardiomyocytes. Furthermore, ncx1hL154P and ncx1h morphants downregulated cardiac transcription factors hand2 and gata4 in the cardiac regions, while overexpression of hand2 and gata4 partially rescued cardiac defects including the number of ventricular myocytes. These findings demonstrate an essential role of the novel 154th leucine residue in the maintenance of Ncx1 function in zebrafish, and reveal previous unrecognized critical roles of the 154th leucine residue and Ncx1 in the formation of ventricular cardiomyocytes by at least partially regulating the expression levels of gata4 and hand2.

Induced pluripotent stem cell-derived vascular smooth muscle cells.

The reproducible generation of human-induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (vSMCs) in vitro has been critical to overcoming many limitations of animal and primary cell models of vascular biology and disease. Since this initial advance, research in the field has turned toward recapitulating the naturally occurring subtype specificity found in vSMCs throughout the body, and honing functional models of vascular disease. In this review, we summarize vSMC derivation approaches, including current phenotype and developmental origin-specific methods, and applications of vSMCs in functional disease models and engineered tissues. Further, we discuss the challenges of heterogeneity in hiPSC-derived tissues and propose approaches to identify and isolate vSMC subtype populations.

Functional Properties of Engineered Heart Slices Incorporating Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for cardiac studies, but their structural and functional immaturity precludes their use as faithful models of adult myocardium. Here we describe engineered heart slices (EHS), preparations of decellularized porcine myocardium repopulated with hiPSC-CMs that exhibit structural and functional improvements over standard culture. EHS exhibited multicellular, aligned bundles of elongated CMs with organized sarcomeres, positive inotropic responses to isoproterenol, anisotropic conduction of action potentials, and electrophysiological functionality for more than 200 days. We developed a new drug assay, GRIDS, that serves as a "fingerprint" of cardiac drug sensitivity for a range of pacing rates and drug concentrations. GRIDS maps characterized differences in drug sensitivity between EHS and monolayers more clearly than changes in action potential durations or conduction velocities. EHS represent a tissue-like model for long-term culture, structural, and functional improvement, and higher fidelity drug response of hiPSC-CMs.

Are These Cardiomyocytes? Protocol Development Reveals Impact of Sample Preparation on the Accuracy of Identifying Cardiomyocytes by Flow Cytometry.

Several protocols now support efficient differentiation of human pluripotent stem cells to cardiomyocytes (hPSC-CMs) but these still indicate line-to-line variability. As the number of studies implementing this technology expands, accurate assessment of cell identity is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity has not yet been established. Therefore, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in day 25 hPSC-CMs and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges to interpreting data generated by published methods and inform the development of a robust protocol for routine assessment of hPSC-CMs. The data, workflow for antibody evaluation, and standardized protocol described here should benefit investigators new to this field and those with expertise in hPSC-CM differentiation.