Biology subjects

Huebsch, N.

Publications and source records attributed to Huebsch, N..

16 recordsLinked to original sources

bioRxiv2026

Tunable Magnetorheological Elastomer Substrates for Studying Mechano-Genetic Interactions in Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. While sarcomeric genomic variants drive HCM, a role for mechanical stress in disease pathogenesis is increasingly appreciated. However, existing in vitro models of HCM rely on model systems (e.g., hydrogels) with static mechanical properties, precluding investigation of how dynamic mechanical loading interacts with genetic susceptibility during disease initiation. Here, we leveraged a dynamically tunable magnetorheological elastomer (MRE) platform to model progressive mechanical stiffening in order to study mechano-genetic interactions in human induced pluripotent stem cell (hiPSC) derived cardiomyocytes harboring an HCM-associated MYBPC3 frameshift (MYBPC3fs) loss-of-function variant. Dynamic stiffening selectively unmasked pathological structural remodeling in MYBPC3fs cardiomyocytes while producing only modest changes in isogenic controls, suggesting that MYBPC3fs confers heightened mechanosensitivity. In MYBPC3fs cardiomyocytes, mechanical stiffening drove hypertrophic remodeling, sarcomere disorganization, nuclear remodeling, dysregulated NFAT3 signaling, and aberrant cell-cycle activity. MRE stiffening also drove fibroblast-to-myofibroblast transition in primary cardiac fibroblasts, recapitulating early fibrotic remodeling. Together, these findings position dynamically tunable MRE substrates as a human-relevant platform for dissecting mechano-genetic disease mechanisms and evaluating therapies targeting mechanotransduction pathways in HCM.

bioengineering↗

bioRxiv2026

Mechanical history shapes phenotypic plasticity and fibroblast-autonomous 3D remodeling in iPSC-derived cardiac fibroblasts

Human induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-cFBs) offer a renewable, genetically defined stromal cell source for studying cardiac fibrosis and cardiomyocyte-fibroblast crosstalk during development and disease. Because cardiac fibroblasts are proliferative, expansion of iPSC-cFBs in culture is attractive for applications requiring large cell numbers, including drug screening and tissue engineering. However, phenotypic drift during expansion, particularly premature fibroblast-to-myofibroblast transition (FMT), remains a concern. Here, we investigated how differentiation protocol, mechanical culture history, and serial expansion shape iPSC-cFB phenotype, transforming growth factor beta 1 (TGF-beta1) responsiveness, and fibroblast-autonomous remodeling in three-dimensional (3D) tissues. Initial studies of iPSC-cFBs expanded on conventional tissue-culture polystyrene (TCP) revealed passage-associated increases in projected cell area and alpha-smooth muscle actin (alpha-SMA) expression, two markers associated with FMT. These findings prompted us to examine how the stiffness of poly(dimethylsiloxane) (PDMS) substrates (15 kPa or 2 MPa) used during iPSC-cFB derivation and expansion influences basal phenotype and response to TGF-beta1. Across two independent differentiation protocols, mechanical culture history shaped passage-dependent basal phenotypes and responses to TGF-beta1. Notably, its effects on collective TGF-beta1 responses in three-dimensional cultures differed from those observed after replating cells onto two-dimensional substrates. Together, these findings demonstrate that mechanical culture history modulates iPSC-cFB responsiveness and tissue-remodeling behavior without irreversibly determining fibroblast state. Subsequent passage and microenvironmental conditions can attenuate or re-express these history-dependent phenotypes. This work supports the use of iPSC-cFBs in basic biological discovery and future tissue engineering applications.

bioengineering↗

bioRxiv2026

A PDMS-based microfluidic platform enabling dual biochemical and electric-field stimulation for modeling sensory neuron-intervertebral disc crosstalk

Inflammation-driven increases in nociception are prominent in pain pathologies associated with intervertebral disc (IVD) degeneration yet are difficult to model in vitro. Since neurons are exposed to multi-modal stimuli in vivo, it is critical for these exposures to be conserved in an in vitro test system. We developed a polydimethylsiloxane (PDMS)-based microfluidic platform to interrogate peripheral sensory neurons (SNs) in the presence of conditioned media from nucleus pulposus cells from the degenerated IVD, to model a potential impact of IVD cells secretome on pain sensing. Our platform enables controlled perfusion of cell-derived biochemical cues alongside a defined homogeneous electric field (EF) and supports real-time optical analysis. Computational modeling, fluid perfusion experiments, and conductivity measurements confirmed stable fluid transport and tunable homogeneous EF generation within the device. As proof of concept for neuronal stimulation, neuroblastoma (N2a) cells loaded with a fluorescent Ca2+ indicator exhibited a 56% increase in Ca2+ transient activity when exposed to media from degenerated IVDs, concomitant with increased IVD-derived IL-1{beta} production. Importantly, EF-stimulated Ca2+ transients increased in SNs derived from human induced pluripotent stem cells when exposed to conditioned media from primary human IVD cells, demonstrating the translation of this model system to human cells. Together, these results establish a versatile platform that enables controlled and simultaneous exposure to biochemical and electrical stimuli to quantify inflammation-driven peripheral neuronal hyperexcitability in tissue-neuron crosstalk.

bioengineering↗

bioRxiv2026

Xeno-Free Peptide-Functionalized Hydrogels Support hiPSC Encapsulation and In Situ Differentiation into Structurally Mature Cardiomyocytes

While defined synthetic substrates can replace Matrigel for human induced pluripotent stem cell (hiPSC) culture and hiPSC-derived cardiomyocyte (hiPSC-CM) production, existing approaches culture cells on two-dimensional surfaces and yield structurally immature cardiomyocytes, limiting their use in disease modeling and regenerative medicine. Here, we developed a xeno-free, fully-defined cyclic RGD (cRGD)-functionalized alginate platform in which we encapsulated hiPSCs to support their expansion and in situ cardiac differentiation. cRGD functionalization was essential for hiPSC survival and pluripotency, with maximal support achieved at a low ligand density (25 {micro}M). In the presence of cRGD, hiPSC encapsulation into softer gels made from lower molecular weight alginates led to enhanced hiPSC expansion and improved cardiogenesis. Strikingly, differentiation in situ with 3D gels led to hiPSC-CM with higher structural maturity, including a markedly increased proportion of Desmin-positive cardiomyocytes. Finally, after enzymatic retrieval from hydrogels, cardiomyocytes derived from softer gels formed tissue-engineered myocardium with superior contractile force compared to tissue fashioned from hiPSC-CM derived from more rigid gels. Together, these results demonstrate the promise of this defined, tunable platform for biomanufacturing of structurally mature cardiomyocytes from hiPSC.

bioengineering↗

bioRxiv2026

Stress Granule Coarsening Is a Pathological Inflection Point for Cardiac Electrophysiological Dysfunction

Arrhythmia risk rises early in many forms of cardiac stress, often before contractile failure is evident. Stressed cardiomyocytes accumulate biomolecular condensates, known as stress granules (SGs), whose contribution to this electrical vulnerability has been unclear. We mapped where SGs reside and followed their life cycle under acute and chronic oxidative stress across complementary model systems and assessed electrophysiological consequences with pharmacological tools targeting granule assembly, microtubule integrity, and calcium channel function. Under both stress regimes, SGs localized preferentially to z-lines and intercalated discs, marking these mechanically critical sites as hubs of condensate assembly. Merging of granules, referred to as coarsening, rather than initial formation, emerged as a pathological connection. Early granules were broadly cytoprotective, whereas progressive coarsening was accompanied by disruption of alpha-actinin and L-type calcium channel (Cav1.2) nanodomains and by shortening of action potential (AP) duration. Coarsened granules disorganized Cav1.2 nanodomains through a microtubule-dependent mechanism, and arresting coarsening with nocodazole preserved nanodomain integrity and restored AP morphology. The transition from nascent to coarsened SGs therefore represents a targetable inflection point, and limiting coarsening may prevent proarrhythmic remodeling during cardiac oxidative stress.

bioengineering↗

bioRxiv2026

Molecular and Cellular Determinants of Human Iron Overload Cardiomyopathy

Iron overload cardiomyopathy (IOC) is a serious heart condition that is caused by elevated levels of systemic iron. IOC is characterized by both systolic and diastolic dysfunction as well as arrhythmias. It has been challenging to isolate the cardiac-specific cellular and molecular mechanisms driving IOC because the disease affects multiple interconnected organ systems. Here, we leverage stem cell technologies, cardiac tissue engineering, and protein reconstitution assays to model key aspects of human IOC in vitro and to probe the cellular and molecular mechanisms driving cardiac dysfunction. We demonstrate that human engineered heart tissues consisting of both cardiomyocytes and cardiac fibroblasts faithfully recapitulate key aspects of the human disease, including reduced systolic function, impaired diastolic function, and increased prevalence of arrhythmogenic events. We demonstrate that while both cardiomyocytes and cardiac fibroblasts show increased intracellular iron levels, leading to reduced viability, cardiomyocytes show higher levels of iron accumulation and higher levels of reactive oxygen species production. Moreover, we show that in a tissue, iron overload has little effect on the action potential kinetics; however, it directly impacts the amplitude and kinetics of the calcium transient, potentially driving arrhythmogenesis. Finally, we demonstrate that iron overload decreases force production, in part, through oxidative damage of sarcomeric proteins and direct iron-based inhibition of myosin. In summary, our results reveal new insights into the cellular and molecular mechanisms of human IOC pathogenesis, and they establish new in vitro models that can be harnessed to faithfully recapitulate key aspects of the human disease phenotype. HighlightsO_LIContractile aspects of iron overload cardiomyopathy have been difficult to study in vitro. C_LIO_LIWe developed engineered heart tissues to model key aspects of the human disease. C_LIO_LIIn vitro iron overload reduces contractility and induces arrhythmogenesis. C_LIO_LIIron differentially affects cardiomyocytes and cardiac fibroblasts. C_LIO_LIIron overload directly impacts the actomyosin contractile apparatus. C_LI

biophysics↗

bioRxiv2025

A Micro-Engineered Heart Tissue Model of Desmin-related Cardiomyopathy Caused by Mutant αB Crystalin

Protein quality control (PQC) is essential for maintaining sarcomere integrity in cardiomyocytes. Crystallin B chain (CRYAB) R120G mutation disrupts CRYABs chaperone activity, leading to aggregation of CRYAB and its client proteins (including Desmin), leading to Desmin-related cardiomyopathy (DRM). Prior experimental systems for modeling DRM linked to CRYAB require massive overexpression of CRYAB mutant isoforms, raising questions about translational relevance. Here, we establish the first model of CRYAB-linked DRM that uses genome-edited hiPSC together with isogenic controls, allowing us to study the impact of mutant CRYAB expressed at near endogenous levels. Within micro-engineered heart tissues (HT), CRYAB-R120G mutant hiPSC-derived cardiomyocytes recapitulated key DRM hallmarks, including Desmin and CRYAB aggregation, contractile dysfunction, and increased vulnerability to PQC pathway inhibition. CRYAB-R120G mutant HT also exhibited dysfunctional calcium-contraction coupling, which exacerbated contractile deficits at higher pacing frequencies. JAK1 inhibition with Itacitinib partially restored contractile function at higher pacing frequencies, suggesting JAK1 inhibition as a viable therapeutic strategy. By preserving human-specific structural and functional features, our {micro}HT platform enables mechanistic characterization of proteotoxic cardiomyopathies and offers a scalable system for targeted drug screening.

bioengineering↗

bioRxiv2025

3D-printing-assisted, microfabricated devices reveal hierarchical and temporal mechanosensing in high-density fibroblast culture

Understanding how cells integrate mechanical forces across multiple directions, length scales, and timescales remains a fundamental challenge in mechanobiology. Deciphering how cells integrate this information is particularly important in the context of wound healing, where the timing and duration of the fibroblast-to-myofibroblast transition can determine healing outcomes. Here, we discovered that fibroblasts in engineered tissues respond to directional anisotropy in stress through a hierarchical temporal cascade, with individual cell elongation (24 hr) preceding collective alignment (40 hr), which then drives -smooth muscle actin expression and myofibroblast transition (96h). To enable this discovery, we developed a modified hydrogel-assisted stereolithographic elastomer (HASTE) prototyping platform to incorporate a detergent that improves wettability of template agar hydrogels by poly(dimethylsiloxane) elastomer. HASTE allowed rapid prototyping of intricate 3D micropost arrays that provides isotropic (8-post) versus anisotropic (4-post) boundary conditions. Fibroblasts sensed and responded to stress directionality before bulk tissue reorganization occurs. Computational modeling predicted steady-state activation patterns based on initial stress anisotropy rather than magnitude, and our experiments reveal that reaching this state requires sequential mechanosensitive processes operating across distinct timescales. This temporal hierarchy persists even when extensive cell-cell contacts might be expected to mask matrix-mediated mechanical signals. Our findings demonstrate that fibroblast mechanosensing involves adaptive responses encoded through progressive cell and tissue reorganization. Results provide insight into how nanoscale mechanosensing scales up to direct tissue-level organization, with implications for understanding wound healing, understanding fibrosis, and engineering functional tissue replacements.

bioengineering↗

bioRxiv2025

Rapid thermoforming of polycarbonate cell culture accessories from 3D printed molds

Bespoke cell culture devices are essential for tissue engineering applications. Traditional manufacturing methods for cell culture accessories involve injection molding and machining, which are too costly and time-consuming to implement for producing custom designs in small batches, and/or while testing the usefulness of a new design before mass producing it. Materials typically used for rapid design iteration, like poly(dimethylsiloxane) (PDMS) elastomers, surmount this issue but present new challenges of affinity for hydrophobic small molecules and sub-optimal interactions with sensitive cell types. Here, we propose polycarbonate (PC) thermoforming as a solution for creating customized transparent and autoclavable accessories. We demonstrate that optimized preheating of PC overcomes issues with bubbling during thermoforming. The use of high heat deflection temperature (HDT) resins allows these PC devices to be thermoformed off molds created by Digital Light Processing (DLP) 3D prints, enabling rapid prototyping of the PC. Using this approach, we fabricated custom PC well plate inserts. These inserts combine many advantages of tissue culture polystyrene (negligible absorption of hydrophobic molecules, transparency, rigidity) and elastomers (ease of creating bespoke devices, ability to be sterilized by autoclaving) and are compatible with a variety of cell biology applications, including human induced pluripotent stem cells (iPSC) culture. PC inserts also supported iPSC differentiation into cardiomyocytes (iPS-CM) and micro-patterning of iPS-CM into cardiospheres. This low cost, customizable approach holds promise for a variety of bioengineering applications.

bioengineering↗

bioRxiv2024

Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

BackgroundProtein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiac myocytes, and mutations in PQC pathway proteins, such as CRYAB (arginine to glycine at position 120, R120G) and BAG3 (proline to lysine at position 209, P209L) induce protein aggregate pathology with cardiomyopathy in humans. Novel observations in yeast and mammalian cells demonstrate mitochondrial uptake of cytosolic protein aggregates. We hypothesized that mitochondrial uptake of cytosolic protein aggregates and their removal by mitophagy, a lysosomal degradative pathway essential for myocardial homeostasis, facilitates cytosolic protein quality control in cardiac myocytes. MethodsMice with inducible cardiac myocyte specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assessed for protein aggregates with biochemical fractionation and super-resolution imaging in comparison to floxed controls. Induced pluripotent stem cell (iPSC)-derived cardiac myocytes with R120G knock-in to the CRYAB locus were assessed for localization of the CRYAB protein. Transgenic mice expressing R120G CRYAB protein (R120G-TG) were subjected to both TRAF2 gain-of-function (with AAV9-cardiac Troponin T promoter-driven TRAF2 transduction) and TRAF2 loss-of-function (with tamoxifen-inducible ablation of one Traf2 allele) in cardiac myocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology. ResultsCardiomyocyte-specific ablation of TRAF2 results accumulation of mitochondrial and cytosolic protein aggregates and DESMIN mis-localization to protein aggregates. Isolated mitochondria take up cardiomyopathy-associated aggregate-prone cytosolic chaperone proteins, namely arginine to glycine (R120G) CRYAB mutant and proline to lysine (P209L) BAG3 mutant. R120G-CRYAB mutant protein increasingly localizes to mitochondria in human and mouse cardiomyocytes. R120G-TG mice demonstrate upregulation of TRAF2 in the mitochondrial fraction with increased mitophagy as compared with wild type. Adult-onset inducible haplo-insufficiency of TRAF2 resulted in accelerated mortality, impaired left ventricular systolic function and increased protein aggregates in R120G-TG mice as compared with controls. Conversely, AAV9-mediated TRAF2 transduction in R120G-TG mice reduced mortality and attenuated left ventricular systolic dysfunction, with reduced protein aggregates and restoration of normal localization of DESMIN, a cytosolic scaffolding protein chaperoned by CRYAB, as compared with control AAV9-GFP group. ConclusionsTRAF2-mediated mitophagy in cardiac myocytes facilitates removal of cytosolic protein aggregates and can be stimulated to ameliorate proteotoxic cardiomyopathy.

cell biology↗

bioRxiv2024

IGF-1 Peptide Mimetic-functionalized Hydrogels Enhance MSC Survival and Immunomodulatory Activity

Human mesenchymal stem cells (MSCs) have demonstrated promise when delivered to damaged tissue or tissue defects for their cytokine secretion and inflammation modulation behaviors that can promote repair. Insulin-like growth factor 1 (IGF-1) has been shown to augment MSCs viability and survival and promote their secretion of cytokines that signal to endogenous cells, in the treatment of myocardial infarction, wound healing, and age-related diseases. Biomaterial cell carriers can be functionalized with growth factor-mimetic peptides (i.e. IGF-1 mimicking peptides) to enhance MSC function while promoting cell retention and minimizing off-target effects seen with direct administration of soluble growth factors. Here, we functionalized alginate hydrogels with three distinct IGF-1 peptide mimetics and the integrin-binding peptide, cyclic RGD. One IGF-1 peptide mimetic (IGM-3) in combination with integrin ligand was found to activate Akt and ERK1/2 signaling and support survival of serum-deprived MSCs. MSCs encapsulated in alginate hydrogels that presented both IGM-3 and cRGD showed a significant reduction in pro-inflammatory cytokine secretion when challenged with interleukin-1{beta}. Finally, MSCs cultured within the cRGD/IGM-3 hydrogels were able to blunt pro-inflammatory gene expression of human primary cells from degenerated intervertebral discs. These studies indicate the potential to leverage cell adhesive and IGF-1 growth factor peptide mimetics together to control therapeutic secretory behavior of MSCs. Significance StatementInsulin-like growth factor 1 (IGF-1) plays a multifaceted role in stem cell biology and may promote proliferation, survival, migration, and immunomodulation for MSCs. In this study, we functionalized alginate hydrogels with integrin-binding and IGF-1 peptide mimetics to investigate their impact on MSC function. Encapsulating MSCs in the dual peptide (cRGD/IGM-3) hydrogels enhanced their ability to reduce inflammatory cytokine production and promote anti-inflammatory gene expression in cells from degenerative human intervertebral discs exposed to proteins secreted by the MSC. This approach suggests a new way to retain and augment MSC functionality using IGF-1 peptide mimetics, offering an alternative to co-delivery of cells and high dose soluble growth factors for tissue repair and immune-system modulation.

bioengineering↗

bioRxiv2023

Mechanical Resistance to Micro-Heart Tissue Contractility unveils early Structural and Functional Pathology in iPSC Models of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy is the most common cause of sudden death in the young. Because the disease exhibits variable penetrance, there are likely nongenetic factors that contribute to the manifestation of the disease phenotype. Clinically, hypertension is a major cause of morbidity and mortality in patients with HCM, suggesting a potential synergistic role for the sarcomeric mutations associated with HCM and mechanical stress on the heart. We developed an in vitro physiological model to investigate how the afterload that the heart muscle works against during contraction acts together with HCM-linked MYBPC3 mutations to trigger a disease phenotype. Micro-heart muscle arrays (HM) were engineered from iPSC-derived cardiomyocytes bearing MYBPC3 loss-of-function mutations and challenged to contract against mechanical resistance with substrates stiffnesses ranging from the of embryonic hearts (0.4 kPa) up to the stiffness of fibrotic adult hearts (114 kPa). Whereas MYBPC3+/- iPSC-cardiomyocytes showed little signs of disease pathology in standard 2D culture, HMs that included components of afterload revealed several hallmarks of HCM, including cellular hypertrophy, impaired contractile energetics, and maladaptive calcium handling. Remarkably, we discovered changes in troponin C and T localization in the MYBPC3+/- HM that were entirely absent in 2D culture. Pharmacologic studies suggested that excessive Ca2+ intake through membrane-embedded channels, rather than sarcoplasmic reticulum Ca2+ ATPase (SERCA) dysfunction or Ca2+ buffering at myofilaments underlie the observed electrophysiological abnormalities. These results illustrate the power of physiologically relevant engineered tissue models to study inherited disease mechanisms with iPSC technology.

bioengineering↗

bioRxiv2022

Hydrogel Assisted Double Molding of 3D-Print Enables Prestress Regulation of Micro-Heart Muscle Physiology

Engineered heart tissues have been created to study cardiac biology and disease in a setting that more closely mimics in-vivo heart muscle than 2D monolayer culture. Previously published studies suggest that geometrically anisotropic micro-environments are crucial for inducing "in vivo-like" physiology from immature cardiomyocytes. We hypothesized that such anisotropic tissue geometries regulate tissue prestress, and that in turn this prestress is a major factor regulating cardiomyocytes electrophysiological development. Thus, we studied the effects of tissue geometry on electrophysiology of micro-heart muscle arrays (HM) engineered from human induced pluripotent stem cells (iPSC). Geometries predicted to increase tissue prestress not only affected cardiomyocyte structure, but also had profound effects on electrophysiology. Elongated geometries led to adaptations that yielded increased calcium intake during each contraction cycle. Strikingly, pharmacologic studies revealed a prestress threshold is required for sodium channel function, whereas L-type calcium and rapidly-rectifying potassium channels were largely insensitive. Analysis of RNA and protein levels suggest sodium channel activity changes were related to post-transcriptional, and potentially post-translational, changes. HM formed from Plakophilin 2 (PKP2) knockout iPSC had a cellular structure similar to isogenic controls. However, these tissues exhibited no functional sodium current and an overall lesser degree of electrical remodeling in response to prestress. These results suggest that PKP2, a key component of the nascent desmosome, is crucial to transducing tissue prestress into physiologically beneficial electrical remodeling via activation of sodium channels.

bioengineering↗

bioRxiv2020

Integrated hiPSC-based liver and heart microphysiological systems predict unsafe drug-drug interaction

Microphysiological systems (MPSs) mimicking human organ function in vitro are an emerging alternative to conventional cell culture and animal models for drug development. Human induced pluripotent stem cells (hiPSCs) have the potential to capture the diversity of human genetics and provide an unlimited supply of cells. Combining hiPSCs with microfluidics technology in MPSs offers new perspectives for drug development. Here, the integration of a newly developed liver MPS with a cardiac MPS--both built with the same hiPSC line--to study drug-drug interaction (DDI) is reported. As a prominent example of clinically relevant DDI, the interaction of the arrhythmogenic gastroprokinetic cisapride with the fungicide ketoconazole was investigated. As seen in patients, metabolic conversion of cisapride to non-arrhythmogenic norcisapride in the liver MPS by the cytochrome P450 enzyme CYP3A4 was inhibited by ketoconazole, leading to arrhythmia in the cardiac MPS. These results establish functional integration of isogenic hiPSC-based liver and cardiac MPSs, which allows screening for DDI, and thus drug efficacy and toxicity, in the same genetic background.

bioengineering↗

bioRxiv2018

Metabolically-Driven Maturation of hiPSC-Cell Derived Heart-on-a-Chip

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are a promising in vitro tool for drug development and disease modeling, but their immature electrophysiology limits diagnostic utility. Tissue engineering approaches involving aligned 3D cultures enhance hiPSC-CM structural maturation but are insufficient to induce mature electrophysiology. We hypothesized that mimicking post-natal switching of the hearts primary ATP source from glycolysis to fatty acid oxidation could enhance electrophysiological maturation of hiPSC-CM. We combined hiPSC-CM with microfabricated culture chambers to form 3D cardiac microphysiological systems (MPS) that enhanced immediate microtissue alignment and tissue specific extracellular matrix (ECM) production. Using Robust Experimental design, we identified a maturation media that improved calcium handling in MPS derived from two genetically distinct hiPSC sources. Although calcium handling and metabolic maturation were improved in both genotypes, there was a divergent effect on action potential duration (APD): MPS that started with abnormally prolonged APD exhibited shorter APD in response to maturation media, whereas the same media prolonged the APD in MPS that started with aberrantly short APD. Importantly, the APD of both genotypes was brought near the range of 270-300ms observed in human left ventricular cardiomyocytes. Mathematical modeling explained these divergent phenotypes, and further predicted the response of matured MPS to drugs with known pro-arrhythmic effects. These results suggest that systematic combination of biophysical stimuli and metabolic cues can enhance the electrophysiological maturation of hiPSC-derived cardiomyocytes. However, they also reveal that maturation-inducing cues can have differential effects on electrophysiology depending on the baseline phenotype of hiPSC-CM. In silico models provide a valuable tool for predicting how changes in cellular maturation will manifest in drug responsiveness.

bioengineering↗

bioRxiv2018

Inversion and computational maturation of drug response using human stem cell derived cardiomyocytes in microphysiological systems

While cardiomyocytes differentiated from human induced pluripotent stems cells (hiPSCs) hold great promise for drug screening, the electrophys-iological properties of these cells can be variable and immature, producing results that are significantly different from their human adult counterparts. Here, we describe a computational framework to address this limitation, and show how in silico methods, applied to measurements on immature cardiomyocytes, can be used to both identify drug action and to predict its effect in mature cells. Our synthetic and experimental results indicate that optically obtained waveforms of voltage and calcium from microphysiological systems can be inverted into information on drug ion channel blockage, and then, through assuming functional invariance of proteins during maturation, this data can be used to predict drug induced changes in mature ventricular cells. Together, this pipeline of measurements and computational analysis could significantly improve the ability of hiPSC derived cardiomycocytes to predict dangerous drug side effects.

biophysics↗