Biology subjects

Kim, D.-H.

Publications and source records attributed to Kim, D.-H..

36 records · Page 2Linked to original sources

bioRxiv2023

Spatially resolved molecular analysis of host response to medical device implantation using the 3D OrbiSIMS

A key goal for implanted medical devices is that they do not elicit a detrimental immune response. Macrophages play critical roles in modulation of the host immune response and are the major cells responsible for persistent inflammatory reactions to implanted biomaterials. We investigate two novel immune-instructive polymers that stimulate pro- or anti-inflammatory responses from macrophages in vitro. These also modulate in vivo foreign body responses (FBR) when implanted subcutaneously in mice as coatings on biomedical grade silicone rubber. The tissue surrounding the implant is mechanically sectioned and imaged to assess the response of the polymers compared to silicone rubber. Immunofluorescent staining reveals responses consistent with pro- or anti-inflammatory responses previously described for these polymers. We apply 3D OrbiSIMS analysis to provide spatial analysis of the metabolite signature in the tissue surrounding the implant for the first time, providing molecular histology insight into the metabolite response in the host tissue. For the pro-inflammatory coating, monoacylglycerols (MG) and diacylglycerols (DG) are observed at increased intensity, while for the anti-inflammatory coating the number of phospholipid species detected decrease and pyridine and pyrimidine levels were elevated. These findings link to observations of small molecule signature from single cell studies of M2 macrophages in vitro where cell and tissue ion intensities were found to correlate suggesting potential for prediction. This illustrates the power of metabolite characterization by the 3D OrbiSIMS to gain insight into the mechanism of bio-instructive materials as medical devices and to inform on the FBR to biomaterials.

bioengineering↗

bioRxiv2022

Survivin Regulates Intracellular Stiffness and Extracellular Matrix Production in Vascular Smooth Muscle Cells

Vascular dysfunction is a common cause of cardiovascular diseases characterized by the narrowing and stiffening of arteries, such as atherosclerosis, restenosis, and hypertension. Arterial narrowing results from the aberrant proliferation of vascular smooth muscle cells (VSMCs) and their increased synthesis and deposition of extracellular matrix (ECM) proteins. These, in turn, are modulated by arterial stiffness, but the mechanism for this is not fully understood. We found that survivin (an inhibitor of apoptosis) is an important regulator of stiffness-mediated ECM synthesis and intracellular stiffness in VSMCs. Whole-transcriptome analysis and cell culture experiments showed that survivin expression is upregulated in injured femoral arteries in mice and in human VSMCs cultured on stiff fibronectin-coated hydrogels. Suppressed expression of survivin in human VSMCs and mouse embryonic fibroblasts decreased the stiffness-mediated expression of ECM components implicated in arterial stiffness, namely, collagen-I, fibronectin, and lysyl oxidase. By contrast, expression of these proteins was upregulated by the overexpression of survivin in human VSMCs cultured on soft hydrogels. Atomic force microscopy analysis showed that suppressed or enhanced expression of survivin decreases or increases intracellular stiffness, respectively. These findings suggest a novel mechanism by which survivin modulates arterial stiffness.

cell biology↗

bioRxiv2022

Perpendicular Shear Stresses Drive Transmural Helical Remodeling in Engineered Human Ventricular Models

Tissue engineering with human induced pluripotent stem cell-derived cardiomyocytes enables unique opportunities for creating physiological models of the heart in vitro. However, there are few approaches available that can recapitulate the complex structure-function relationships that govern cardiac function at the macroscopic organ level. Here, we report a down-scaled, conical human 3D ventricular model with controllable cellular organization using multilayered, patterned cardiac sheets. Tissue engineered ventricles whose cardiomyocytes were pre-aligned parallel or perpendicular to the long axis outperformed those whose cardiomyocytes were angled or randomly oriented. Notably, the inner layers of perpendicular cardiac sheets realigned over 4 days into a parallel orientation, creating a helical transmural architecture, whereas minimal remodeling occurred in the parallel or angled sheets. Finite element analysis of engineered ventricles demonstrated that circumferential alignment leads to maximal perpendicular shear stress at the inner layer, whereas longitudinal orientation leads to maximal parallel stress. We hypothesize that cellular remodeling occurs to reduce perpendicular shear stresses in myocardium. This advanced platform provides evidence that physical forces such as shear stress drive self-organization of cardiac architecture.

bioengineering↗

bioRxiv2022

Expression Microdissection for use in qPCR based analysis of miRNA in a single cell type

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of this data is obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) as a means to acquire in vivo estimates, directly from formalin fixed tissues, albeit with limited yield. Here we optimized each step of the xMD process including tissue retrieval, tissue transfer, film preparation, and RNA isolation to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, including the development of a non-crosslinked ethylene vinyl acetate (EVA) membrane, resulted in a 23-45 fold increase in miRNA yield, depending on cell type. By qPCR, miR-200a was increased 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143, relative to the matched non-dissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells.

genomics↗

bioRxiv2022

The lateral habenula mediates the association of a conditioned stimulus with the absence of an appetitive unconditioned stimulus

The lateral habenula (LHb) has been implicated in conditioned inhibition. Here, the modulating effects of LHb activity on the association of a conditioned stimulus (CS) with the nonoccurrence of an unconditioned stimulus (US) were assessed in vivo using chemogenetic methods. Animals initially received explicitly unpaired CS light and US food presentations. Animals subjected to a retardation-of-acquisition task subsequently underwent light and food pairings, whereas those subjected to a summation task underwent compound light-tone and food pairings. The inhibitory light strength was assessed based on retardation of the acquisition of food-cup conditioned responses (CRs) in light-food pairings and comparisons of food-cup CRs to each stimulus in a CS-alone test following compound training. Neurotoxic LHb lesions and chemogenetic LHb inhibition throughout unpaired training attenuated the inhibitory light strength. Furthermore, chemogenetic LHb activation accelerated the decline in CR induced by repeated light-alone presentations following light-food pairings. Therefore, the LHb critically contributes to conditioned inhibition.

neuroscience↗

bioRxiv2021

Early flowering in oilseed-type Brassica rapa plants results from nonsense-mediated mRNA decay (NMD) of BrFLC2

Many Brassica species require vernalization (long-term winter-like cooling) for transition to the reproductive stage. In the past several decades, scientific efforts have been made to discern the molecular mechanisms underlying vernalization in many species. Thus, to identify the key regulators required for vernalization in Brassica rapa L., we constructed a linkage map composed of 7,833 single nucleotide polymorphism (SNP) markers using the late-flowering Chinese cabbage (B. rapa L. ssp. pekinensis) inbred line Chiifu and the early-flowering yellow sarson (B. rapa L. ssp. trilocularis (Roxb.)) line LP08 and identified a single major QTL on the upper-arm of the chromosome A02. In addition, we compared the transcriptomes of the lines Chiifu and LP08 at five vernalization time points, including both non-vernalized and post-vernalization conditions. We observed that BrFLC2 was significantly downregulated in the early flowering LP08 and had two deletion sites around the BrFLC2 genomic region compared with the BrFLC2 genomic region in Chiifu. In the present study, we also demonstrate that early flowering in LP08 line is attributed to the low expression of BrFLC2, which is caused by nonsense-mediated mRNA decay (NMD). Therefore, this study provides a better understanding of the molecular mechanisms underlying floral transition in B. rapa. One sentence summaryNMD-mediated degradation of BrFLC2 mRNA transcripts is the main cause of rapid flowering of oilseed-type B. rapa LP08 plants.

plant biology↗

bioRxiv2020

Development of A Decellularized Meniscus Matrix-Based Nanofibrous Scaffold for Meniscus Tissue Engineering

The meniscus plays a critical role in knee mechanical function but is commonly injured given its central load bearing role. In the adult, meniscus repair is limited, given the low number of endogenous cells, the density of the matrix, and the limited vascularity. Menisci are fibrocartilaginous tissues composed of a micro-/nano-fibrous extracellular matrix (ECM) and a mixture of chondrocyte-like and fibroblast-like cells. Here, we developed a fibrous scaffold system that consists of bioactive components (decellularized meniscus ECM (dME) within a poly(e-caprolactone) material) fashioned into a biomimetic morphology (via electrospinning) to support and enhance meniscus cell function and matrix production. This work supports that the incorporation of dME into synthetic nanofibers increased hydrophilicity of the scaffold, leading to enhanced meniscus cell spreading, proliferation, and fibrochondrogenic gene expression. This work identifies a new biomimetic scaffold for therapeutic strategies to substitute or replace injured meniscus tissue. STATEMENT OF SIGNIFICANCEIn this study, we show that a scaffold electrospun from a combination of synthetic materials and bovine decellularized meniscus ECM provides appropriate signals and a suitable template for meniscus fibrochondrocyte spreading, proliferation, and secretion of collagen and proteoglycans. Material characterization and in vitro cell studies support that this new bioactive material is susceptible to enzymatic digestion and supports meniscus-like tissue formation.

bioengineering↗

bioRxiv2020

Metabolic profiles as putative predictors of relapse in pediatric brain tumour using direct surface and imaging mass spectrometry

We present here a novel surface mass spectrometry strategy to perform untargeted metabolite profiling of formalin-fixed paraffin-embedded (FFPE) pediatric ependymoma archives. Sequential Orbitrap secondary ion mass spectrometry (3D OrbiSIMS) and liquid extraction surface analysis-tandem MS (LESA-MS/MS) permitted the detection of 887 metabolites (163 chemical classes) from pediatric ependymoma tumor tissue microarrays (diameter <1 mm; thickness: 4 m). From these 163 classes, 60 classes were detected with both techniques, whilst LESA-MS/MS and 3D OrbiSIMS individually allowed the detection of another 83 and 20 unique metabolite classes, respectively. Through data fusion and multivariate analysis, we were able to identify key metabolites and corresponding pathways predictive of tumor relapse which were retrospectively confirmed using gene expression analysis with publicly available data. Altogether, this sequential mass spectrometry strategy has shown to be a versatile tool to perform high throughput metabolite profiling on sample-limited tissue archives. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/182071v3_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1f9c850org.highwire.dtl.DTLVardef@1ce0568org.highwire.dtl.DTLVardef@c5023borg.highwire.dtl.DTLVardef@15ad6e_HPS_FORMAT_FIGEXP M_FIG For Table of Contents Only C_FIG

cancer biology↗

bioRxiv2020

Quantitative super-resolution microscopy reveals distinct ULK1 oligomeric states and nanoscopic morphologies during autophagy initiation

Autophagy induction involves extensive molecular and membrane reorganization. Despite significant progress, the mechanism underlying autophagy initiation remains poorly understood. Here, we used quantitative Photoactivated Localization Microscopy with single-molecule sensitivity to analyze the nanoscopic distribution of endogenous ULK1, the kinase that triggers autophagy. Under amino acid starvation, ULK1 formed large clusters containing up to 161 molecules at the endoplasmic reticulum. Cross-correlation analysis revealed that ULK1 clusters engaging in autophagosome formation require 30 or more molecules. The ULK1 structures with more than the threshold number contained varying levels of Atg13, Atg14, Atg16, LC3B, GEC1 and WIPI2. We found that ULK1 activity is dispensable for the initial clustering of ULK1, but necessary for the subsequent expansion of the clusters, which involves interaction with Atg14, Atg16, and LC3B and relies on Vps34 activity. This quantitative analysis at the single molecule level has provided unprecedented insights into the behavior of ULK1 during autophagy initiation.

biophysics↗

bioRxiv2020

VIL1, a Polycomb-associated protein, modulates high ambient temperature response via H3K27me3 and H2A.Z in Arabidopsis thaliana

Adapting to the everchanging environment is key to a successful life for an organism. Eukaryotes reprogram their transcriptome in order to adapt to an unfavorable environment. To achieve this reprogramming, plants and animals employ multiple responses including epigenetic regulation. In the search for mutations compromised in high ambient temperature response, we found that VIL1, a PHD finger protein displays aberrant development at high temperature. RNA-seq analysis shows that vil1 fails to downregulate heat suppressed genes. H2A.Z ChIP-seq showed that unlike wild type, vil1 fails to evict H2A.Z from heat responsive genes. We also found that vil1 suppresses constitutive thermo-morphogenic phenotype of arp6. Supporting this phenotype, RNA-seq analysis revealed that constitutive heat responsive transcriptome of arp6 reverted back to the wild-type levels in arp6vil1. This observation suggests an antagonistic relationship between VIL1 and ARP6. We found that this antagonism can be explained in part by interaction between H3K27me3 and H2A.Z.

plant biology↗

bioRxiv2020

Assessing the impact of physicochemical parameters in the predictive capabilities of thermodynamics-based stoichiometric approaches under mesophilic and thermophilic conditions

Metabolic engineering in the post-genomic era is characterised by the development of new methods for metabolomics and fluxomics, supported by the integration of genetic engineering tools and mathematical modelling. Particularly, constraint-based stoichiometric models have been widely studied: (i) flux balance analysis (FBA) (in silico), and (ii) metabolic flux analysis (MFA) (in vivo). Recent studies have enabled the incorporation of thermodynamics and metabolomics data to improve the predictive capabilities of these approaches. However, an in-depth comparison and evaluation of these methods is lacking. This study presents a thorough analysis of two different in silico methods tested against experimental data (metabolomics and 13C-MFA) for the mesophile Escherichia coli. In particular, a modified version of the recently published matTFA toolbox was created, providing a broader range of physicochemical parameters. Validating against experimental data allowed the determination of the best physicochemical parameters to perform the TFA (Thermodynamics-based Flux Analysis). An analysis of flux pattern changes in the central carbon metabolism between 13C-MFA and TFA highlighted the limited capabilities of both approaches for elucidating the anaplerotic fluxes. In addition, a method based on centrality measures was suggested to identify important metabolites that (if quantified) would allow to further constrain the TFA. Finally, this study emphasised the need for standardisation in the fluxomics community: novel approaches are frequently released but a thorough comparison with currently accepted methods is not always performed. Author summaryBiotechnology has benefitted from the development of high throughput methods characterising living systems at different levels (e.g. concerning genes or proteins), allowing the industrial production of chemical commodities. Recently, focus has been placed on determining reaction rates (or metabolic fluxes) in the metabolic network of certain microorganisms, in order to identify bottlenecks hindering their exploitation. Two main approaches are commonly used, termed metabolic flux analysis (MFA) and flux balance analysis (FBA), based on measuring and estimating fluxes, respectively. While the influence of thermodynamics in living systems was accepted several decades ago, its application to study biochemical networks has only recently been enabled. In this sense, a multitude of different approaches constraining well-established modelling methods with thermodynamics has been suggested. However, physicochemical parameters are generally not properly adjusted to the experimental conditions, which might affect their predictive capabilities. In this study, we have explored the reliability of currently available tools by investigating the impact of varying said parameters in the simulation of metabolic fluxes and metabolite concentration values. Additionally, our in-depth analysis allowed us to highlight limitations and potential solutions that should be considered in future studies.

systems biology↗

bioRxiv2019

Functional Maturation of Human iPSC-based Cardiac Microphysiological Systems with Tunable Electroconductive Decellularized Extracellular Matrices

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer tremendous potential for use in engineering human tissues for regenerative therapy and drug screening. However, differentiated cardiomyocytes are phenotypically immature, reducing assay reliability when translating in vitro results to clinical studies and precluding hiPSC-derived cardiac tissues from therapeutic use in vivo. To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cellular and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces at 14 days of culture and had increased the expression of genes that regulate contractile function. Similar improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial. We also demonstrate that dECM-rGO hydrogels can be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced maturation of these tissues corresponded well with a capability to produce physiologically relevant drug responses.

bioengineering↗

bioRxiv2019

High-throughput 16S rRNA gene sequencing reveals gut microbial changes in 6-hydroxydopamine-induced Parkinsons disease mice

Recently, there has been a rapid increase in studies on the relationship between brain diseases and gut microbiota, and clinical evidence on gut microbial changes in Parkinsons disease (PD) has accumulated. 6-hydroxydopamine (6-OHDA) is a widely used neurotoxin that leads to PD pathogenesis, but whether the alterations of gut microbial community in 6-OHDA-treated mice has not been investigated. Here we performed the 16S rRNA gene sequencing to analyze changes in gut microbial community of mice. We found that there were no significant changes in species richness and its diversity in the 6-OHDA-lesioned mice. The relative abundance of Lactobacillus gasseri and L. reuteri probiotic species in feces of 6-OHDA-lesioned mice was significantly decreased compared with those of sham-operated mice, while the commensal bacterium Bacteroides acidifaciens in 6-OHDA-treated mice was remarkably higher than sham-operated mice. These results provides a baseline for understanding the microbial communities of 6-OHDA-induced PD model to investigate the role of gut microbiota in the pathogenesis of PD.

neuroscience↗

bioRxiv2019

A NOVEL MECHANISM FOR NF-κB-ACTIVATION VIA IκB-AGGREGATION: IMPLICATIONS FOR HEPATIC MALLORY-DENK-BODY INDUCED INFLAMMATION

Background & AimsMallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-{kappa}B activation. However, the precise mechanism that links protein aggregation to NF{kappa}B-activation and inflammatory response remains unclear.\n\nMethodsHerein, we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin, protoporphyrin IX (PPIX), or ZnPPIX) elicited an I{kappa}B-loss with consequent NF-{kappa}B activation. We characterized the underlying mechanism in detail using hepatocytes from various knockout mice and MEF cell lines and multiple approaches including immunoblotting, EMSA, RT-PCR, confocal immunofluorescence microscopy, affinity immunoprecipitation, and protein solubility assays. Additionally, we performed rigorous proteomic analyses to identify the proteins aggregating upon PPIX treatment and/or co-aggregating with I{kappa}B.\n\nResultsFour known mechanisms of I{kappa}B-loss were probed and excluded. Immunofluorescence analyses of ZnPPIX-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this I{kappa}B-loss was due to its sequestration along with I{kappa}B{beta} into insoluble aggregates. Through proteomic analyses we identified 47 aggregation-prone proteins that co-aggregate with I{kappa}B through direct interaction or proximity. Of these ZnPPIX-aggregation targets, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as likely mediators of I{kappa}B-nuclear import.\n\nConclusionWe discovered a novel mechanism of inflammatory NF-{kappa}B activation through I{kappa}B-sequestration into insoluble aggregates along with interacting aggregation-prone proteins. This mechanism may account for the protein aggregate-induced inflammation observed in MDB-associated liver diseases, thereby identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.\n\nLay SummaryMallory-Denk-bodies (MDBs) are hepatic protein aggregates commonly featured in many liver diseases. MDB-presence is associated with the induction of inflammatory responses both clinically and in all MDB-inducing models. Similar protein aggregation in neurodegenerative diseases is also known to trigger inflammation and NF{kappa}B pathway activation via an as yet to be characterized non-canonical mechanism. Herein using a MDB-inducing cell model, we uncovered a novel mechanism for NF{kappa}B activation via cytosolic I{kappa}B-sequestration into insoluble aggregates. Furthermore, using a proteomic approach, we identified 47 aggregation-prone proteins that interact and co-aggregate with I{kappa}B. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thereby identifying novel targets for therapeutic intervention.

cell biology↗

bioRxiv2019

Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy

Pathogenic mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses (Hi-C) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared to gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape inactivation during cardiogenesis. These regions exhibit upregulation of multiple non-cardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. On the other hand, A/B compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. We conclude that global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency. SummaryBertero et al. observe that lamin A/C haploinsufficiency in human cardiomyocytes markedly alters electrophysiology, contractility, gene expression, and chromosomal topology. Contrary to expectations, however, changes in chromatin compartments involve just few regions, and most dysregulated genes lie outside these hotspots. Condensed titleGenomic effects of lamin A/C haploinsufficiency

genomics↗

bioRxiv2018

Clathrin-coated Pits are Shredded Organelles Presorting Receptors in the Plasma Membrane

A myriad of receptor types crowds the plasma membrane of cells. Here, we revealed that clathrin-coated pits (CCPs) presort receptors to modulate receptor activation on the plasma membrane. We visualized individual molecules of receptors, including epidermal growth factor receptor (EGFR), ErbB2, transferrin receptor (TfR), and beta2-adrenergic receptors ({beta}2-AR), inside a single CCP in a living cell using single-molecule diffusivity-based colocalization analysis. The spatially distinct subsets of CCPs selectively allocated for these receptors were observed in a resting state. The EGFR pre-allocated CCP subset was partially shared with that for ErbB2, whereas the EGFR and TfR pre-allocated CCP subsets were mutually exclusive. PICALM was necessary for the pre-allocation of CCPs for EGFR. Furthermore, EGFR dimerization was markedly elevated inside the pre-allocated CCP subset after dynamin recruitment. The pre-sorting function of CCPs provides an efficient mechanism to control competition and cooperation between receptors on the crowded plasma membrane.\n\nOne Sentence SummaryClathrin-coated pits are small-sized organelles pre-sorting receptor kinds to modulate receptor activation on the crowded plasma membrane.

cell biology↗

bioRxiv2018

Engineered developmental niche enables predictive phenotypic screening in human dystrophic cardiomyopathy

Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes typically produces cells with structural, functional, and biochemical properties that most closely resemble those present in the fetal heart. Here we establish an in vitro engineered developmental cardiac niche to produce matured hPSC-derived cardiomyocytes (hPSC-CMs) with enhanced sarcomere development, electrophysiology, contractile function, mitochondrial capacity, and a more mature transcriptome. When this developmental cardiac niche was applied to dystrophin mutant hPSC-CMs, a robust disease phenotype emerged, which was not observed in non-matured diseased hPSC-CMs. Matured dystrophin mutant hPSC-CMs exhibited a greater propensity for arrhythmia as measured via beat rate variability, most likely due to higher resting cytosolic calcium content. Using a custom nanopatterned microelectrode array platform to screen functional output in hPSC-CMs exposed to our engineered developmental cardiac niche, we identified calcium channel blocker, nitrendipine, mitigated hPSC-CM arrhythmogenic behavior and correctly identified sildenafil as a false positive. Taken together, we demonstrate our developmental cardiac niche platform enables robust hPSC-CM maturation allowing for more accurate disease modeling and predictive drug screening.

bioengineering↗

bioRxiv2018

NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

Somatic cells derived from human pluripotent stem cell (hPSC) sources hold significant potential as a means to improve current in vitro screening assays. However, their inconsistent ability to recapitulate the structural and functional characteristics of native cells has raised questions regarding their ability to accurately predict the functional behavior of human tissues when exposed to chemical or pathological insults. In addition, the lack of cytoskeletal organization within conventional culture platforms prevents analysis of how structural changes in human tissues affect functional performance. Using cation-permeable hydrogels, we describe the production of multiwell nanotopographically-patterned microelectrode arrays (nanoMEAs) for studying the effect of structural organization on hPSC-derived cardiomyocyte and neuronal function in vitro. We demonstrate that nanoscale topographic substrate cues promote the development of more ordered cardiac and neuronal monolayers while simultaneously enhancing cytoskeletal organization, protein expression patterns, and electrophysiological function in these cells. We then show that these phenotypic improvements act to alter the sensitivity of hPSC-derived cardiomyocytes to treatment with arrhythmogenic and conduction-blocking compounds that target structural features of the cardiomyocyte. Similarly, we demonstrate that neuron sensitivity to synaptic blockers is increased when cells are maintained on nanotopographically-patterned Nafion surfaces. The improved structural and functional capacity of hPSC-derived cardiomyocyte and neuronal populations maintained on nanoMEAs may have important implications for improving the predictive capabilities of cell-based electrophysiological assays used in preclinical screening applications.

bioengineering↗