Introduction:Idiopathic nephrotic syndrome (INS) is associated with important kidney and cardiovascular morbidities. We tested the hypothesis that serum syndecan-1, a marker of endothelial glycocalyx injury, can help identify patients at risk for unfavorable kidney and cardiovascular outcomes. Methods:We included 348 children and adults with INS (n = 35 unbiopsied, n = 141 minimal change disease [MCD] and n = 172 focal segmental glomerulosclerosis [FSGS]) from the Nephrotic Syndrome Study Network (NEPTUNE) cohort and 34 healthy participants (n = 22 adults, n = 12 children). We measured baseline serum syndecan-1 levels using an enzyme-linked immunosorbent assay and evaluated their relationship with kidney and cardiovascular outcomes. We performed in vitro studies to test whether INS sera and different therapeutics may alter syndecan-1 expression in human glomerular endothelial cells (GEnC). Results:Serum syndecan-1 was higher in patients with INS than in controls and was high in approximately one-third of patients without proteinuria or with subnephrotic proteinuria. Patients receiving steroids, regardless of disease activity, showed higher syndecan-1 than those off immunosuppression. Syndecan-1 was higher in proteinuric patients with MCD than in FSGS. At the time of serum collection, syndecan-1 modestly correlated with proteinuria but not with kidney function. In longitudinal analyses, serum syndecan-1 was associated with the composite of 40% decline in kidney function or kidney failure and with dyslipidemia. Compared with controls, INS sera in relapse increased syndecan-1 expression in cultured GEnC, and this was partially or fully mitigated when dexamethasone or a metalloprotease inhibitor were added, respectively, to culture media. Conclusion:Baseline serum syndecan-1 is associated with unfavorable kidney outcomes and cardiovascular risk factors in INS.

Extracellular vesicles (EVs) are associated with intercellular communications, immune responses, viral pathogenicity, cardiovascular diseases, neurological disorders, and cancer progression. EVs deliver proteins, metabolites, and nucleic acids into recipient cells to effectively alter their physiological and biological response. During their transportation from the donor to the recipient cell EVs face differential ionic concentrations, which can be detrimental to their integrity and impact their cargo content. EVs are known to possess ion channels and transporters in their membrane but neither the function nor the role of these channels in EVs is known. In this study, we discover a functional calcium-activated large-conductance potassium channel (BKCa) in the membrane of EVs. Furthermore, we establish that BKCa is essential for the structural and functional integrity of EVs. Together, these findings establish the critical role of ion channels such as BKCa in functioning as gatekeepers and maintaining EV-mediated signaling.

Rationale & Objective:Idiopathic nephrotic syndrome (INS) is viewed as a podocyte-specific disease. Recent reports indicate endothelial involvement, but its significance is unclear. Here, we investigated the relationship between the glomerular expression of selected genes relevant to endothelial health and clinical markers of disease severity. Study Design:A cross-sectional study. Setting & Participants:Patients with INS (n = 70 minimal change disease and n = 83 focal segmental glomerulosclerosis) from the Nephrotic Syndrome Study Network cohort study and 53 control participants. Validation studies, including animal and cell culture experiments, were performed. Exposure:Gene expression analysis from micro-dissected human glomeruli. The study is focused on 10 genes highly relevant for endothelial homeostasis and barrier integrity (nitric oxide synthase 3 [NOS3], endothelial cell adhesion molecule, and endothelial cell specific molecule 1 [ESM1]), endothelial glycocalyx remodeling (HPSE, HYAL1, MMP2, MMP9, and ADAMTS1), and endothelial activation (ICAM1 and CAV1). Outcomes:Kidney function, ultrastructural changes in podocytes and glomerular endothelium, interstitial fibrosis and tubular atrophy. Analytical Approach:One-way ANOVA and Tukey's multiple comparisons test, Pearson Correlation and Cohen's d statistics. Results:Transcriptomic analysis revealed that all genes of interest were highly expressed in glomeruli from INS patients compared with controls, except for ESM1 and MMP9, which were decreased. Expression of endothelial-specific genes correlated with those of glycocalyx injury and cell activation. HPSE, ADAMTS1, ICAM1, and CAV1 expression was inversely associated with kidney function, whereas ADAMTS1 showed a positive association with proteinuria. NOS3, HPSE, and ADAMTS1 were associated with podocyte foot process effacement, and ICAM1 with podocyte detachment. HPSE and MMP2 were associated with ultrastructural endothelial injury, whereas HPSE, MMP2, ICAM1, and CAV1 were associated with interstitial fibrosis and tubular atrophy. Several genes (ESM1, HPSE, HYAL1, MMP2, and ICAM1) were also dysregulated in experimental INS and validated in cultured glomerular endothelial cells (NOS3 and heparanase) following exposure to INS sera. Limitations:Observational study, selection bias, unmeasured confounders. Conclusions:INS involves dysregulation of genes relevant for endothelial health.

The molecular pathophysiology of nephrotic syndrome remains largely elusive in pediatric patients. While most children with minimal change disease (MCD) show favorable responses to immunosuppressive therapy, those with focal segmental glomerulosclerosis (FSGS) often exhibit poorer treatment responses, with many experiencing either partial remission or no remission of proteinuria. The need for reliable glomerular disease biomarkers to predict treatment response and understand molecular pathways governing responsiveness and resistance is a critical unmet need in pediatric nephrology. In this study, we sought to characterize urine proteomes in children with MCD and FSGS to identify biomarkers distinguishing disease activity and associated molecular pathways. Using quantitative proteomics, urine proteins from children with MCD and FSGS in the CureGN Study were identified and correlated with disease onset and activity. Unbiased cluster analyses of nephrotic urine proteomes demonstrated a cluster with relatively increased immune response and complement proteins, suggesting important distinctions in disease characteristics within the nephrotic subgroups. These analyses yielded patient subpopulations with proteinuria and distinct urine proteome differences associated with 116 proteins exerting cluster separation in the multivariate analyses. These findings highlight the potential of unsupervised clustering to identify disease subgroups and provide insights into the underlying molecular heterogeneity within nephrotic syndrome, paving the way for more tailored therapeutic strategies and improved patient management.

Background: Human induced pluripotent stem cells (hiPSCs) and their derived cardiomyocytes (hiPSC-CMs) have been used for cell-based therapies for myocardial regeneration. Underlying paracrine signals derived from these hiPSC-CMs have been shown to play a critical role in improving the function of the ischemic myocardium. However, characterization of the transplanted cell proteome in vivo has been challenging due to the lack of tools to distinguish cell-specific proteome in a complex multicellular microenvironment. Methods: To establish cell-specific proteome labelling, we adopted the biorthogonal non-canonical amino acid tagging (BONCAT) system in hiPSCs. We expressed the mutant mouse tRNA synthetase gene, “L274GmMetRS” in hiPSCs (L274G-hiPSCs) using lentiviral transduction to enable incorporation of the non-canonical amino acid azidonorleucine (Anl) in these cells. We assessed the pluripotency and trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo (teratoma formation assay). Furthermore, we validated the cell-specific Anl incorporation in the L274G-hiPSC-CMs both in vitro (co-culture) and in vivo (post-transplantation). Results: Our studies showed that the L274G-hiPSCs could efficiently differentiate into all the three germ lineages and can be used to track the cell-specific proteome in their differentiated progenies, including L274G-hiPSC-CMs. Furthermore, immunostaining and western blots showed cell-specific BONCAT in L274G-hiPSC-CMs both in co-cultures (in vitro) and post-transplantation (in vivo). Conclusion: The newly-established L274G-hiPSC line can be used to track cell-specific proteome of hiPSC-CMs in vitro and in vivo, to characterize cell-specific proteomic responses and delineate mechanisms underlying cell therapies.

BACKGROUND:Human induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of using hiPSCs has historically been to regenerate damaged tissue, emerging studies have shown a more potent effect of hiPSC-derived paracrine factors on tissue regeneration. However, the precise contents of the transplanted hiPSC-derived cell secretome are ambiguous. This is mainly due to the lack of tools to distinguish cell-specific secretome from host-derived proteins in a complex tissue microenvironment in vivo. METHODS:In this study, we present the generation and characterization of a novel hiPSC line, L274G-hiPSC, expressing the murine mutant methionyl-tRNA synthetase, L274GMmMetRS, which can be used for tracking the cell specific proteome via biorthogonal non-canonical amino acid tagging (BONCAT). We assessed the trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo. Furthermore, we assessed the cell-specific proteome labelling in the L274G-hiPSC derived cardiomyocytes (L274G-hiPSC-CMs) in vitro following co-culture with wild type human umbilical vein derived endothelial cells and in vivo post transplantation in murine hearts. RESULTS:We demonstrated that the L274G-hiPSCs exhibit typical hiPSC characteristics and that we can efficiently track the cell-specific proteome in their differentiated progenies belonging to the three germ lineages, including L274G-hiPSC-CMs. Finally, we demonstrated cell-specific BONCAT in transplanted L274G-hiPSC-CMs. CONCLUSION:The novel L274G-hiPSC line can be used to study the cell-specific proteome of hiPSCs in vitro and in vivo, to delineate mechanisms underlying hiPSC-based cell therapies for a variety of regenerative medicine applications.

IntroductionNephrotic syndrome (NS) occurs commonly in children with glomerular disease and glucocorticoids (GCs) are the mainstay treatment. Steroid resistant NS (SRNS) develops in 15% to 20% of children, increasing the risk of chronic kidney disease compared to steroid sensitive NS (SSNS). NS pathogenesis is unclear in most children, and no biomarkers exist that predict the development of pediatric SRNS.MethodsWe studied a unique patient cohort with plasma specimens collected before GC treatment, yielding a disease-only sample not confounded by steroid-induced gene expression changes (SSNS n = 8; SRNS n = 7). A novel "patient-specific" bioinformatic approach merged paired pretreatment and posttreatment proteomic and metabolomic data and identified candidate SRNS biomarkers and altered molecular pathways in SRNS versus SSNS.ResultsJoint pathway analyses revealed perturbations in nicotinate or nicotinamide and butanoate metabolic pathways in patients with SRNS. Patients with SSNS had perturbations of lysine degradation, mucin type O-glycan biosynthesis, and glycolysis or gluconeogenesis pathways. Molecular analyses revealed frequent alteration of molecules within these pathways that had not been observed by separate proteomic and metabolomic studies. We observed upregulation of NAMPT, NMNAT1, and SETMAR in patients with SRNS, in contrast to upregulation of ALDH1B1, ACAT1, AASS, ENPP1, and pyruvate in patients with SSNS. Pyruvate regulation was the change seen in our previous analysis; all other targets were novel. Immunoblotting confirmed increased NAMPT expression in SRNS and increased ALDH1B1 and ACAT1 expression in SSNS, following GC treatment.ConclusionThese studies confirmed that a novel "patient-specific" bioinformatic approach can integrate disparate omics datasets and identify candidate SRNS biomarkers not observed by separate proteomic or metabolomic analysis.

Idiopathic nephrotic syndrome (NS) is a common glomerular disease. Although glucocorticoids (GC) are the primary treatment, the PPARγ agonist pioglitazone (Pio) also reduces proteinuria in patients with NS and directly protects podocytes from injury. Because both drugs reduce proteinuria, we hypothesized these effects result from overlapping transcriptional patterns. Systems biology approaches compared glomerular transcriptomes from rats with PAN-induced NS treated with GC vs. Pio and identified 29 commonly regulated genes-of-interest, primarily involved in extracellular matrix (ECM) remodeling. Correlation with clinical idiopathic NS patient datasets confirmed glomerular ECM dysregulation as a potential mechanism of injury. Cellular deconvolution in silico revealed GC- and Pio-induced amelioration of altered genes primarily within podocytes and mesangial cells. While validation studies are indicated, these analyses identified molecular pathways involved in the early stages of NS (prior to scarring), suggesting that targeting glomerular ECM dysregulation may enable a future non-immunosuppressive approach for proteinuria reduction in idiopathic NS.

Extracellular vesicles specifically exosomes play an important role in transferring genetic information and mediating communication between cells. Exosomes get packaged inside the cell, excise out to the extracellular environment, and deliver the cargo to the target cells. However, the precise mechanism of how exosomes handle the differential ionic environment and the physiological role and identity of their ion channels is not determined. Given that potassium (K+) ions have the largest gradient, we focused on identifying the presence and physiological relevance of K+ channels in exosomes.

Masson’s Trichrome Staining (MTS) is a useful tool for analyzing fibrosis in a plethora of disease pathologies by differential staining of tissue components. It is used to identify collagen fibers in different tissues like heart, lung, skin, and muscles. Especially in cardiac fibrosis, MTS stains the collagen fibers (blue color), which helps in the distinction of scar area versus the healthy area (red color). However, there are several challenges to stain both paraffin-embedded sections and frozen (cryosections) using a single protocol. Therefore, the goal of this study was to develop a simple short protocol to assess cardiac fibrosis in both paraffin-embedded and cryo heart sections. MTS uses three different stains, i.e., Weigert’s Iron Hematoxylin, Biebrich scarlet-acid fuchsin, and aniline blue to detect nuclei, cytoplasm, and collagen, respectively. In this study, we developed a simple short protocol that can be adapted by any lab to easily assess cardiac fibrosis in paraffin and frozen heart sections. Furthermore, we have addressed the challenges that are commonly faced during the immunostaining process and troubleshooting techniques. Overall, we have successfully developed a simple one-step protocol to assess myocardial fibrosis in paraffin-embedded and frozen cryosections.

Extracellular vesicles (EVs) specifically exosomes are important in mediating intracellular communications, and are capable of transferring genetic information between cells. Exosomes are used as a drug delivery vehicle to carry cargo for targeted therapy and have emerged as one of the most promising candidate for treating cardiovascular diseases. Exosomes get packaged inside the cell, excise out to the extracellular environment, and deliver the cargo to the target cells. However, the precise mechanism of how exosomes handle the differential ionic environment and the physiological role of their ion channels is not determined. Given that potassium (K + ) ions has the largest gradient, we focused on identifying the presence and physiological relevance of K+ channels in exosomes. Using the in silico approach, several ion channel candidates were identified, the most prominent ion channel being large conductance Ca 2+ and voltage-activated potassium channel (BK). To record BK in exosomes, we incorporated planar bilayers and a novel electrophysiology approach called near field electrophysiology (NFE), as the canonical patch-clamp methods are not feasible due to the size of EVs. Our NFE indicates a presence of K + channels in intact exosomes and 45% of them are sensitive to IbTX. Since IbTX specifically blocks, BK channels, we estimated 2 functional channels (single-channel conductance of 300 pS with 50% open probability) in a single exosome. Plasma-derived exosomes from BK +/+ and BK -/- mice subjected to differential K + gradient indicated that functional BK channels exist in exosomes, and help in maintaining their structural integrity. Furthermore, plasma derived exosomes from BK +/+ mice showed cardioprotection from ischemia-reperfusion injury whereas exosomes from BK -/- mice did not. Thus, the presence of BK determines the packaging as well as cardioprotective function of exosomes. Overall, the study for the first time indicate a presence of functional ion channel (BK) in exosomes which plays a role in protecting cells and heart against ischemia and reperfusion injury.

Tumor angiogenesis is initiated and maintained by the tumor microenvironment through secretion of autocrine and paracrine factors, including extracellular vesicles (EVs). Although tumor-derived EVs (t-EVs) have been implicated in tumor angiogenesis, growth and metastasis, most studies on t-EVs are focused on proangiogenic miRNAs and growth factors. We have recently demonstrated that conditioned media from human lung tumor cells (A549) downregulate TRPV4 channels and transform normal endothelial cells to a tumor endothelial cell-like phenotype and induce abnormal angiogenesis in vitro, via t-EVs. However, the underlying molecular mechanism of t-EVs on endothelial cell phenotypic transition and abnormal angiogenesis in vivo remains unknown. Here, we demonstrate that t-EVs downregulate TRPV4 expression post-translationally and induce abnormal angiogenesis by activating Rho/Rho kinase/YAP/VEGFR2 pathways. Further, we demonstrate that t-EVs induce abnormal vessel formation in subcutaneously implanted Matrigel plugs in vivo (independent of tumors), which are characterized by increased VEGFR2 expression and reduced pericyte coverage. Taken together, our findings demonstrate that t-EVs induce abnormal angiogenesis via TRPV4 downregulation-mediated activation of Rho/Rho kinase/YAP/VEGFR2 pathways and suggest t-EVs and TRPV4 as novel targets for vascular normalization and cancer therapy.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are highly reactive molecules, with significant effects in human diseases including cancer and cardiovascular disease. The ability to accurately and precisely detect the formation of free radicals within cells and tissues is crucial to developing proper treatments for the problems caused by ROS/RNS. Fluorescent probes have become widely available reagents of detecting ROS/RNS within cells. Several commercially available kits have shown their specificity toward detecting the formation of ROS and RNS. In this chapter, we discuss the principle behind each kit and the benefits and shortcomings of these kits, namely dihydroethidium (DHE), dichlorohydrofluoresin diacetate (DCF-DA), 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM diacetate), and 10-acetyl-3,7-dihyrdroxyphenoxazine (Amplex red). DHE is used to specifically detect superoxide, while DCF-DA readily detects hydroxyl radicals. Amplex red is used to detect hydrogen peroxide, and DAF-FM is used for measuring nitric oxide. However, due to the nature of their reactivity, the probes are not absolutely specific for the noted ROS/RNS species, and will react with others. ROS measurement may need to be made in real time, and they are short-lived within the cell, especially superoxide and nitric oxide. This chapter explains the mechanism behind each chemical kit, the protocols used with the kit, and show typical results after imaging. Additionally, an assessment is made on the use of the kit, identifying the advantages and disadvantages of each probe.

Angiogenesis, the formation of new blood vessels from existing ones, is a normal physiological process. However, deregulation of angiogenesis can lead to pathological states such as cancer, that is characterized by hyper‐permeable and tortuous vessels. We have recently shown a significant decrease in functional expression of the mechanosensitive ion channel, transient potential receptor vanilloid 4 (TRPV4), in tumor endothelial cells (TEC). Further, pharmacological activation of TRPV4 induced normalization of tumor vasculature and improved cancer therapy. However, the molecular mechanisms by which TRPV4 is downregulated in TEC is not yet known. To determine this mechanism, we focused on extracellular vesicles (EVs) derived from tumor cells. We first collected conditioned media (TCM) from tumor cells with and without pre‐treatment of an exosome inhibitor, GW4869. We found that treatment of human normal endothelial cells (hNEC) with TCM transformed them into tumor‐endothelial like (hTEC) phenotype as revealed by expression of TEM8, VEGFR2 membrane translocation, and abnormal tube formation. However, TCM from exosome inhibitor‐treated cells, failed to induce endothelial transformation. Further, we found that EVs isolated from TCM induced hNEC transformation to hTEC. Mechanistically, we found that tumor derived EVs induced functional downregulation of TRPV4 in hNEC as assessed by calcium imaging. Taken together, our results suggest that tumor derived EVs transforms normal endothelial cells via downregulation of TRPV4 channels.Support or Funding InformationNational Institutes of Health R15CA202847 and R01HL119705

Studies on cardiac progenitor cells (CPCs) and their derived exosomes therapeutic potential have demonstrated only modest improvements in cardiac function. Therefore, there is an unmet need to improve the therapeutic efficacy of CPCs and their exosomes to attain clinically relevant improvement in cardiac function. The hypothesis of this project is to assess the therapeutic potential of exosomes derived from human CPCs (hCPCs) cultured under normoxia (21% O2), physoxia (5% O2) and hypoxia (1% O2) conditions. hCPCs were characterized by immunostaining of CPC-specific markers (NKX-2.5, GATA-4, and c-kit). Cell proliferation and cell death assay was not altered under physoxia. A gene expression qPCR array (84 genes) was performed to assess the modulation of hypoxic genes under three different oxygen conditions as mentioned above. Our results demonstrated that very few hypoxia-related genes were modulated under physoxia (5 genes upregulated, 4 genes down regulated). However, several genes were modulated under hypoxia (23 genes upregulated, 9 genes downregulated). Furthermore, nanoparticle tracking analysis of the exosomes isolated from hCPCs under physoxia had a 1.6-fold increase in exosome yield when compared to normoxia and hypoxia conditions. Furthermore, tube formation assay for angiogenesis indicated that exosomes derived from hCPCs cultured under physoxia significantly increased tube formation as compared to no-exosome control, 21% O2, and 1% O2 groups. Overall, our study demonstrated the therapeutic potential of physoxic oxygen microenvironment cultured hCPCs and their derived exosomes for myocardial repair.

Introduction: Recent studies have demonstrated the great potential of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for testing the efficacy of various cardiac drugs. Additionally, studies have shown that the hiPSC-CMs grown in a 3D environment express better physiological characteristics than 2D cultures. The convergence of polymeric cardiac patch technology with hiPSC-CMs has opened up innovative ways for generating biomimetic 3D cardiac tissues. Hypothesis: The central hypothesis of this study was to develop a 3D cardiac tissue model for pharmacological testing of various cardiac drugs on a 3D nanofibrous aligned co-axial cardiac patch. Methods: A co-axial (Co-A) PCL-gelatin aligned nanofibrous patch was fabricated using the electrospinning technique and its mechanical properties were assessed using Universal Test Machine. Then, the hiPSC-CMs were cultured on this Co-A patch for 2 weeks and the LDH assay was performed to determine the cell viability. The functionality of the cardiac patch was determined by an assessment of calcium cycling in hiPSC-CMs. Further, particle image velocimetry (PIV) and microelectrode array (MEA) was used to evaluate the physiological functionality of the cardiac patch in response to various cardiac drugs. Results: Our studies showed that the mean diameter and thickness of aligned Co-A nanofibrous patch was 578±184 nm and 115±11 μm respectively, while its tensile strength was 0.780 ± 0.098 MPa. Further, confocal imaging confirmed the core-shell structure of the Co-A patches with a core diameter of 2.21 ± 0.50 μm. Additionally, The hiPSC-CMs cultured on these aligned Co-A patches showed an aligned morphology and expressed Troponin-T, GATA4, α-sarcomeric actinin, and connexin-43. The hiPSC-CMs seeded on a 3D scaffold showed efficient calcium cycling properties, which were similar to the hiPSC-CMs cultured in 2D scaffold. Furthermore, PIV and MEA analysis showed that hiPSC-CMs cultured in 2D and 3D showed a similar response to various cardiac drugs, isoproterenol, verapamil and E4031. Conclusions: Overall, this study demonstrated a successful fabrication of aligned Co-A nanofibrous cardiac patch and its evaluation as a 3D cardiac tissue model in-vitro, which could be applied towards drug screening, toxicity studies and cardiac repair applications for ischemic heart disease.

Human-induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs) have been explored for cardiac regeneration and repair as well as for the development of in vitro 3D cardiac tissue models. Existing protocols for cardiac differentiation of hiPSCs utilize a 2D culture system. However, the efficiency of hiPSC differentiation to cardiomyocytes in 3D culture systems has not been extensively explored. In the present study, we investigated the efficiency of cardiac differentiation of hiPSCs to functional cardiomyocytes on 3D nanofibrous scaffolds. Coaxial polycaprolactone (PCL)-gelatin fibrous scaffolds were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and fourier transform infrared (FTIR) spectroscopy. hiPSCs were cultured and differentiated into functional cardiomyocytes on the nanofibrous scaffold and compared with 2D cultures. To assess the relative efficiencies of both the systems, SEM, immunofluorescence staining and gene expression analyses were performed. Contractions of differentiated cardiomyocytes were observed in 2D cultures after 2 weeks and in 3D cultures after 4 weeks. SEM analysis showed no significant differences in the morphology of cells differentiated on 2D versus 3D cultures. However, gene expression data showed significantly increased expression of cardiac progenitor genes (ISL-1, SIRPA) in 3D cultures and cardiomyocytes markers (TNNT, MHC6) in 2D cultures. In contrast, immunofluorescence staining showed no substantial differences in the expression of NKX-2.5 and α-sarcomeric actinin. Furthermore, uniform migration and distribution of the in situ differentiated cardiomyocytes was observed in the 3D fibrous scaffold. Overall, our study demonstrates that coaxial PCL-gelatin nanofibrous scaffolds can be used as a 3D culture platform for efficient differentiation of hiPSCs to functional cardiomyocytes.

Recent advances in cardiac tissue engineering have shown that human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in a three-dimensional (3D) micro-environment exhibit superior physiological characteristics compared with their two-dimensional (2D) counterparts. These 3D cultured hiPSC-CMs have been used for drug testing as well as cardiac repair applications. However, the fabrication of a cardiac scaffold with optimal biomechanical properties and high biocompatibility remains a challenge. In our study, we fabricated an aligned polycaprolactone (PCL)-Gelatin coaxial nanofiber patch using electrospinning. The structural, chemical, and mechanical properties of the patch were assessed by scanning electron microscopy (SEM), immunocytochemistry (ICC), Fourier-transform infrared spectroscopy (FTIR)-spectroscopy, and tensile testing. hiPSC-CMs were cultured on the aligned coaxial patch for 2 weeks and their viability [lactate dehydrogenase (LDH assay)], morphology (SEM, ICC), and functionality [calcium cycling, multielectrode array (MEA)] were assessed. Furthermore, particle image velocimetry (PIV) and MEA were used to evaluate the cardiotoxicity and physiological functionality of the cells in response to cardiac drugs. Nanofibers patches were comprised of highly aligned core-shell fibers with an average diameter of 578 ± 184 nm. Acellular coaxial patches were significantly stiffer than gelatin alone with an ultimate tensile strength of 0.780 ± 0.098 MPa, but exhibited gelatin-like biocompatibility. Furthermore, hiPSC-CMs cultured on the surface of these aligned coaxial patches (3D cultures) were elongated and rod-shaped with well-organized sarcomeres, as observed by the expression of cardiac troponin-T and α-sarcomeric actinin. Additionally, hiPSC-CMs cultured on these coaxial patches formed a functional syncytium evidenced by the expression of connexin-43 (Cx-43) and synchronous calcium transients. Moreover, MEA analysis showed that the hiPSC-CMs cultured on aligned patches showed an improved response to cardiac drugs like Isoproterenol (ISO), Verapamil (VER), and E4031, compared to the corresponding 2D cultures. Overall, our results demonstrated that an aligned, coaxial 3D cardiac patch can be used for culturing of hiPSC-CMs. These biomimetic cardiac patches could further be used as a potential 3D in vitro model for “clinical trials in a dish” and for in vivo cardiac repair applications for treating myocardial infarction.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been developed for cardiac cell transplantation studies more than a decade ago. In order to establish the hiPSC-CM-based platform as an autologous source for cardiac repair and drug toxicity, it is vital to understand the functionality of cardiomyocytes. Therefore, the goal of this study was to assess functional physiology, ultrastructural morphology, gene expression, and microRNA (miRNA) profiling at Wk-1, Wk-2 & Wk-4 in hiPSC-CMs in vitro. Functional assessment of hiPSC-CMs was determined by multielectrode array (MEA), Ca2+ cycling and particle image velocimetry (PIV). Results demonstrated that Wk-4 cardiomyocytes showed enhanced synchronization and maturation as compared to Wk-1 & Wk-2. Furthermore, ultrastructural morphology of Wk-4 cardiomyocytes closely mimicked the non-failing (NF) adult human heart. Additionally, modulation of cardiac genes, cell cycle genes, and pluripotency markers were analyzed by real-time PCR and compared with NF human heart. Increasing expression of fatty acid oxidation enzymes at Wk-4 supported the switching to lipid metabolism. Differential regulation of 12 miRNAs was observed in Wk-1 vs Wk-4 cardiomyocytes. Overall, this study demonstrated that Wk-4 hiPSC-CMs showed improved functional, metabolic and ultrastructural maturation, which could play a crucial role in optimizing timing for cell transplantation studies and drug screening.