CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GCs), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-of-function experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GCresistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GCs against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

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.

Pharmacogenomic Profiling of Pediatric Acute Myeloid Leukemia to Identify Therapeutic Vulnerabilities and Inform Functional Precision Medicine.

Despite the expanding portfolio of targeted therapies for adults with acute myeloid leukemia (AML), direct implementation in children is challenging due to inherent differences in underlying genetics. Here we established the pharmacologic profile of pediatric AML by screening myeloblast sensitivity to approved and investigational agents, revealing candidates of immediate clinical relevance. Drug responses ex vivo correlated with patient characteristics, exhibited age-specific alterations, and concorded with activities in xenograft models. Integration with genomic data uncovered new gene-drug associations, suggesting actionable therapeutic vulnerabilities. Transcriptome profiling further identified gene-expression signatures associated with on- and off-target drug responses. We also demonstrated the feasibility of drug screening-guided treatment for children with high-risk AML, with two evaluable cases achieving remission. Collectively, this study offers a high-dimensional gene-drug clinical data set that could be leveraged to research the unique biology of pediatric AML and sets the stage for realizing functional precision medicine for the clinical management of the disease. SIGNIFICANCE: We conducted integrated drug and genomic profiling of patient biopsies to build the functional genomic landscape of pediatric AML. Age-specific differences in drug response and new gene-drug interactions were identified. The feasibility of functional precision medicine-guided management of children with high-risk AML was successfully demonstrated in two evaluable clinical cases. This article is highlighted in the In This Issue feature, p. 476.

R4 RGS proteins as fine tuners of immature and mature hematopoietic cell trafficking.

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors. They are involved in almost every physiologic process and consequently have a pivotal role in an extensive number of pathologies, including genetic, neurologic, and immune system disorders. Indeed, the vast array of GPCRs mechanisms have led to the development of a tremendous number of drug therapies and already account for about a third of marketed drugs. These receptors mediate their downstream signals primarily via G proteins. The regulators of G-protein signaling (RGS) proteins are now in the spotlight as the critical modulatory factors of active GTP-bound Gα subunits of heterotrimeric G proteins to fine-tune the biologic responses driven by the GPCRs. Also, they possess noncanonical functions by multiple mechanisms, such as protein-protein interactions. Essential roles and impacts of these RGS proteins have been revealed in physiology, including hematopoiesis and immunity, and pathologies, including asthma, cancers, and neurologic disorders. This review focuses on the largest subfamily of R4 RGS proteins and provides a brief overview of their structures and G-proteins selectivity. With particular interest, we explore and highlight, their expression in the hematopoietic system and the regulation in the engraftment of hematopoietic stem/progenitor cells (HSPCs). Distinct expression patterns of R4 RGS proteins in the hematopoietic system and their pivotal roles in stem cell trafficking pave the way for realizing new strategies for enhancing the clinical performance of hematopoietic stem cell transplantation. Finally, we discuss the exciting future trends in drug development by targeting RGS activity and expression with small molecules inhibitors and miRNA approaches.

Temporal Control of the WNT Signaling Pathway During Cardiac Differentiation Impacts Upon the Maturation State of Human Pluripotent Stem Cell Derived Cardiomyocytes.

Inefficient differentiation and insufficient maturation are barriers to the application of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) for research and therapy. Great strides have been made to the former, and multiple groups have reported cardiac differentiation protocol that can generate hPSC-CMs at high efficiency. Although many such protocols are based on the modulation of the WNT signaling pathway, they differ in their timing and in the WNT inhibitors used. Little is currently known about whether and how conditions of differentiation affect cardiac maturation. Here we adapted multiple cardiac differentiation protocols to improve cost-effectiveness and consistency, and compared the properties of the hPSC-CMs generated. Our results showed that the schedule of differentiation, but not the choice of WNT inhibitors, was a critical determinant not only of differentiation efficiency, which was expected, but also CM maturation. Among cultures with comparable purity, hPSC-CMs generated with different differentiation schedules vary in the expression of genes which are important for developmental maturation, and in their structural, metabolic, calcium transient and proliferative properties. In summary, we demonstrated that simple changes in the schedule of cardiac differentiation could promote maturation. To this end, we have optimized a cardiac differentiation protocol that can simultaneously achieve high differentiation efficiency and enhanced developmental maturation. Our findings would advance the production of hPSC-CMs for research and therapy.

Mitochondrial-Targeted Therapy for Doxorubicin-Induced Cardiotoxicity.

Anthracyclines, such as doxorubicin, are effective chemotherapeutic agents for the treatment of cancer, but their clinical use is associated with severe and potentially life-threatening cardiotoxicity. Despite decades of research, treatment options remain limited. The mitochondria is commonly considered to be the main target of doxorubicin and mitochondrial dysfunction is the hallmark of doxorubicin-induced cardiotoxicity. Here, we review the pathogenic mechanisms of doxorubicin-induced cardiotoxicity and present an update on cardioprotective strategies for this disorder. Specifically, we focus on strategies that can protect the mitochondria and cover different therapeutic modalities encompassing small molecules, post-transcriptional regulators, and mitochondrial transfer. We also discuss the shortcomings of existing models of doxorubicin-induced cardiotoxicity and explore advances in the use of human pluripotent stem cell derived cardiomyocytes as a platform to facilitate the identification of novel treatments against this disorder.

Remdesivir induces persistent mitochondrial and structural damage in human induced pluripotent stem cell-derived cardiomyocytes.

AIMS: Remdesivir is a prodrug of an adenosine triphosphate analogue and is currently the only drug formally approved for the treatment of hospitalized coronavirus disease of 2019 (COVID-19) patients. Nucleoside/nucleotide analogues have been shown to induce mitochondrial damage and cardiotoxicity, and this may be exacerbated by hypoxia, which frequently occurs in severe COVID-19 patients. Although there have been few reports of adverse cardiovascular events associated with remdesivir, clinical data are limited. Here, we investigated whether remdesivir induced cardiotoxicity using an in vitro human cardiac model. METHODS AND RESULTS: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to remdesivir under normoxic and hypoxic conditions to simulate mild and severe COVID-19, respectively. Remdesivir induced mitochondrial fragmentation, reduced redox potential, and suppressed mitochondrial respiration at levels below the estimated plasma concentration under both normoxic and hypoxic conditions. Non-mitochondrial damage such as electrophysiological alterations and sarcomere disarray were also observed. Importantly, some of these changes persisted after the cessation of treatment, culminating in increased cell death. Mechanistically, we found that inhibition of DRP1, a regulator of mitochondrial fission, ameliorated the cardiotoxic effects of remdesivir, showing that remdesivir-induced cardiotoxicity was preventable and excessive mitochondrial fission might contribute to this phenotype. CONCLUSIONS: Using an in vitro model, we demonstrated that remdesivir can induce cardiotoxicity in hiPSC-CMs at clinically relevant concentrations. These results reveal previously unknown potential side-effects of remdesivir and highlight the importance of further investigations with in vivo animal models and active clinical monitoring to prevent lasting cardiac damage to patients.

R4 RGS proteins suppress engraftment of human hematopoietic stem/progenitor cells by modulating SDF-1/CXCR4 signaling.

Homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM) microenvironment are tightly regulated by the chemokine stromal cell-derived factor-1 (SDF-1) and its G-protein-coupled receptor C-X-C motif chemokine receptor 4 (CXCR4), which on engagement with G-protein subunits, trigger downstream migratory signals. Regulators of G-protein signaling (RGS) are GTPase-accelerating protein of the Gα subunit and R4 subfamily members have been implicated in SDF-1-directed trafficking of mature hematopoietic cells, yet their expression and influence on HSPCs remain mostly unknown. Here, we demonstrated that human CD34+ cells expressed multiple R4 RGS genes, of which RGS1, RGS2, RGS13, and RGS16 were significantly upregulated by SDF-1 in a CXCR4-dependent fashion. Forced overexpression of RGS1, RGS13, or RGS16 in CD34+ cells not only inhibited SDF-1-directed migration, calcium mobilization, and phosphorylation of AKT, ERK, and STAT3 in vitro, but also markedly reduced BM engraftment in transplanted NOD/SCID mice. Genome-wide microarray analysis of RGS-overexpressing CD34+ cells detected downregulation of multiple effectors with established roles in stem cell trafficking/maintenance. Convincingly, gain-of-function of selected effectors or ex vivo priming with their ligands significantly enhanced HSPC engraftment. We also constructed an evidence-based network illustrating the overlapping mechanisms of RGS1, RGS13, and RGS16 downstream of SDF-1/CXCR4 and Gαi. This model shows that these RGS members mediate compromised kinase signaling and negative regulation of stem cell functions, complement activation, proteolysis, and cell migration. Collectively, this study uncovers an essential inhibitory role of specific R4 RGS proteins in stem cell engraftment, which could potentially be exploited to develop improved clinical HSPC transplantation protocols.

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.

Generation of genomic-integration-free human induced pluripotent stem cells and the derived cardiomyocytes of X-linked dilated cardiomyopathy from DMD gene mutation.

We derived an integration-free induced pluripotent stem cell (iPSC) line from the peripheral blood mononuclear cells (PBMCs) of a 23-year-old male patient. This patient carries a 5' splice site point mutation in intron 1 (c.31+1G>A) of the dystrophin gene, a mutation associated with X-linked dilated cardiomyopathy (XLDCM). Sendai virus was used to reprogram the PBMCs and deliver OCT3/4, SOX2, c-MYC, and KLF4 factors. The iPSC line (HKUi002-A) generated preserved the mutation, expressed common pluripotency markers, differentiated into three germ layers in vivo, and exhibited a normal karyotype. Further differentiation into cardiomyocytes enables the study of the disease mechanisms of XLDCM.

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.

Organic Electrochemical Transistor Arrays for In Vitro Electrophysiology Monitoring of 2D and 3D Cardiac Tissues.

Here, a multichannel organic electrochemical transistor (OECT) array is reported for electrophysiological monitoring and mapping of action potential propagation of a wide range of cardiac cells, including cell lines, primary cell lines, and human-sourced stem cell derivatives in 2D and 3D structures. The results suggest that the ability to exploit this OECT-based platform to map 2D action potential propagation provides a viable strategy to better characterize cardiac cells in response to various chronotropic drugs. The effects of chronotropic agents Isoproterenol and Verapamil on cardiac tissues validate the utility of OECT for drug screening capability, and a preliminary demonstration of a 64-channel OECT array to monitor the cardiac action potentials for better spatial resolution is presented. The study demonstrates that OECT will be a viable and versatile platform for applications in medical and pharmacological industries.

Integrated transcriptomic and regulatory network analyses identify microRNA-200c as a novel repressor of human pluripotent stem cell-derived cardiomyocyte differentiation and maturation.

Aims: MicroRNAs (miRNAs) are crucial for the post-transcriptional control of protein-encoding genes and together with transcription factors (TFs) regulate gene expression; however, the regulatory activities of miRNAs during cardiac development are only partially understood. In this study, we tested the hypothesis that integrative computational approaches could identify miRNAs that experimentally could be shown to regulate cardiomyogenesis. Methods and results: We integrated expression profiles with bioinformatics analyses of miRNA and TF regulatory programs to identify candidate miRNAs involved with cardiac development. Expression profiling showed that miR-200c, which is not normally detected in adult heart, is progressively down-regulated both during cardiac development and in vitro differentiation of human embryonic stem cells (hESCs) to cardiomyocytes (CMs). We employed computational methodologies to predict target genes of both miR-200c and five key cardiac TFs to identify co-regulated gene networks. The inferred cardiac networks revealed that the cooperative action of miR-200c with these five key TFs, including three (GATA4, SRF and TBX5) targeted by miR-200c, should modulate key processes and pathways necessary for CM development and function. Experimentally, over-expression (OE) of miR-200c in hESC-CMs reduced the mRNA levels of GATA4, SRF and TBX5. Cardiac expression of Ca2+, K+ and Na+ ion channel genes (CACNA1C, KCNJ2 and SCN5A) were also significantly altered by knockdown or OE of miR-200c. Luciferase reporter assays validated miR-200c binding sites on the 3' untranslated region of CACNA1C. In hESC-CMs, elevated miR-200c increased beating frequency, and repressed both Ca2+ influx, mediated by the L-type Ca2+ channel and Ca2+ transients. Conclusions: Our analyses demonstrate that miR-200c represses hESC-CM differentiation and maturation. The integrative computation and experimental approaches described here, when applied more broadly, will enhance our understanding of the interplays between miRNAs and TFs in controlling cardiac development and disease processes.

Polycystin-2 Plays an Essential Role in Glucose Starvation-Induced Autophagy in Human Embryonic Stem Cell-Derived Cardiomyocytes.

Autophagy is a process essential for cell survival under stress condition. The patients with autosomal dominant polycystic kidney disease, which is caused by polycystin-1 or polycystin-2 (PKD2) mutation, display cardiovascular abnormalities and dysregulation in autophagy. However, it is unclear whether PKD2 plays a role in autophagy. In the present study, we explored the functional role of PKD2 in autophagy and apoptosis in human embryonic stem cell-derived cardiomyocytes. HES2 hESC line-derived cardiomyocytes (HES2-CMs) were transduced with adenoviral-based PKD2-shRNAs (Ad-PKD2-shRNAs), and then cultured with normal or glucose-free medium for 3 hours. Autophagy was upregulated in HES2-CMs under glucose starvation, as indicated by increased microtubule-associated protein 1 light chain 3-II level in immunoblots and increased autophagosome and autolysosome formation. Knockdown of PKD2 reduced the autophagic flux and increased apoptosis under glucose starvation. In Ca2+ measurement, Ad-PKD2-shRNAs reduced caffeine-induced cytosolic Ca2+ rise. Co-immunoprecipitation and in situ proximity ligation assay demonstrated an increased physical interaction of PKD2 with ryanodine receptor 2 (RyR2) under glucose starvation condition. Furthermore, Ad-PKD2-shRNAs substantially attenuated the starvation-induced activation of AMP-activated protein kinase (AMPK) and inactivation of mammalian target of rapamycin (mTOR). The present study for the first time demonstrates that PKD2 functions to promote autophagy under glucose starvation, thereby protects cardiomyocytes from apoptotic cell death. The mechanism may involve PKD2 interaction with RyR2 to alter Ca2+ release from sarcoplasmic reticulum, consequently modulating the activity of AMPK and mTOR, resulting in alteration of autophagy and apoptosis. Stem Cells 2018;36:501-513.

Graphene Sheet-Induced Global Maturation of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) can proliferate infinitely. Their ability to differentiate into cardiomyocytes provides abundant sources for disease modeling, drug screening and regenerative medicine. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) display a low degree of maturation and fetal-like properties. Current in vitro differentiation methods do not mimic the structural, mechanical, or physiological properties of the cardiogenesis niche. Recently, we present an efficient cardiac maturation platform that combines hiPSCs monolayer cardiac differentiation with graphene substrate, which is a biocompatible and superconductive material. The hiPSCs lines were successfully maintained on the graphene sheets and were able to differentiate into functional cardiomyocytes. This strategy markedly increased the myofibril ultrastructural organization, elevated the conduction velocity, and enhanced both the Ca2+ handling and electrophysiological properties in the absence of electrical stimulation. On the graphene substrate, the expression of connexin 43 increased along with the conduction velocity. Interestingly, the bone morphogenetic proteins signaling was also significantly activated during early cardiogenesis, confirmed by RNA sequencing analysis. Here, we reasoned that graphene substrate as a conductive biomimetic surface could facilitate the intrinsic electrical propagation, mimicking the microenvironment of the native heart, to further promote the global maturation of hiPSC-CMs. Our findings highlight the capability of electrically active substrates to influence cardiomyocyte development. We believe that application of graphene sheets will be useful for simple, fast, and scalable maturation of regenerated cardiomyocytes.

Facile formation of a microporous chitosan hydrogel based on self-crosslinking.

A facile approach for the formation of microporous (chitosan) hydrogel scaffolds based on self-crosslinking is presented. It is simple and does not require any sacrificial porogen, toxic initiator/catalyst, harmful irradiation, or sophisticated equipment. The pore size, porosity, and mechanical properties of our hydrogels can be readily tuned.