Biology subjects

Anseth, K. S.

Publications and source records attributed to Anseth, K. S..

18 recordsLinked to original sources

bioRxiv2026

hMSCs for Osteocyte-like Cell Networks within Strain-Stiffening Bottlebrush Polymer Hydrogels

Bone formation and remodeling depend on dynamic biochemical and biomechanical signaling from the collagen-rich osteoid that precedes mineralization, yet the role of osteoid nonlinear mechanics in regulating osteocyte-like network formation remains poorly understood. Here, we engineered a synthetic bottlebrush polymer hydrogel (BB) that mimics key mechanical features of osteoid and compared it to collagen type-I (Col1) matrices with matched shear modulus (~70 Pa) and strain-stiffening behavior. Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) were cultured for 28 days in growth (GM) or osteogenic media (OM) to examine network formation, and functional connectivity using live cell fluorescence recovery after photobleaching. hMSCs cultured in BB networks and OM showed upregulation of early osteocyte markers compared to Col1. We find that strain-stiffening materials with minimal stress relaxation promote osteocyte-like cell differentiation with functional connectivity, establishing osteoid-mimetic BB hydrogels as a promising matrix to study human osteocytogenesis and osteocyte mechanotransduction in vitro.

bioengineering↗

bioRxiv2026

Redundant YAP and TAZ functions are essential for skeletal muscle regeneration

Muscle stem cells orchestrate skeletal muscle regeneration through complex fate decisions. The transcriptional co-activators Yes-associated protein 1 (YAP) and WW domain-containing transcription regulator 1 (TAZ) contribute to multiple stages of myogenesis, yet their individual contributions to regeneration remain unclear due to substantial functional overlap. We genetically titrated YAP and TAZ expression in MuSCs with double knockout and single allele mutants by crossing Pax7CreERT mice with TAZflox/flox;YAPflox/flox mice. Conditional deletion of both YAP and TAZ in muscle stem cells severely disrupted muscle regeneration with dramatically increased fibrosis and impaired myofiber formation following injury. In contrast, a single allele of either YAP or TAZ was sufficient to rescue injured muscle weight and myofiber cross-sectional area. Similarly, the reduced proliferation of double knockout muscle stem cells on isolated myofibers was restored by a single allele of either YAP or TAZ. In addition, disrupted actin cytoskeleton organization and reduced focal adhesion formation drove double knockout muscle stem cell migration defects, negatively impacting muscle stem cell congregation prior to fusion. Thus, YAP and TAZ function redundantly as critical transcriptional co-activators to regulate progenitor proliferation and migration during muscle regeneration. By challenging myogenesis with double knockout of both YAP and TAZ, we unmasked regenerative requirements previously undetected in single-gene loss models, highlighting genetic redundancy as a key principle buffering regenerative robustness.

cell biology↗

bioRxiv2026

Fibroblasts impair muscle stem cell self-renewal via excessive fibronectin deposition in viscoelastic hydrogel co-cultures

Muscle satellite cells (SCs) regenerate skeletal muscle, but their regenerative capacity declines with age, in part due to extracellular matrix (ECM) remodeling and aberrant fibroblast activation within the SC niche. In regenerating young mouse muscle, fibronectin remodeling is transient, whereas in aged mouse muscle, fibronectin remodeling is prolonged and disorganized. Fibroblasts in aged mice are activated, increasing fibronectin deposition and expressing elevated -smooth muscle actin (SMA), which negatively influence SC fate. We develop a viscoelastic hydrogel co-encapsulation system, enabling three-dimensional co-culture of intact myofibers with primary fibroblasts. Using this 3D co-culture system, we show that fibroblasts from young mice support SC quiescence and self-renewal, whereas fibroblasts from aged mice aberrantly activate SCs and promote their differentiation on myofibers isolated from either young or aged mice. Knocking down fibronectin (Fn1) in fibroblasts from aged mice partially restores SC function, promoting quiescence and limiting differentiation. Using a novel 3D hydrogel co-culture system, we demonstrate that fibroblast-deposited fibronectin is a key age-associated regulator negatively affecting SC fate within the SC niche of aged mice.

bioengineering↗

bioRxiv2026

The Muscle Tissue Environment Limits Muscle Stem Cells in Aged Mice

Frailty arising from loss of muscle function and mass is a significant health concern impacting quality of life and dramatically increasing health care costs as our population ages. Ameliorating frailty derived from reduced muscle function is thus a critical research priority to improve health span. Cell intrinsic defects in muscle stem cells (MuSC), or satellite cells, occur as skeletal muscle ages, reducing the capacity of MuSCs to maintain and repair skeletal muscle and are accompanied by cell nonautonomous changes. Although rejuvenating stem cells in aged tissues or organs has potential to improve muscle aging phenotypes, we found that the extracellular environment in aged mice abrogates rejuvenated muscle stem cell potential. MuSCs from young mice were unable to grow on extracellular matrix derived from aged mice that contains elevated collagen protein levels, establishing a critical role for the environment in contributing to muscle phenotypes in aging. Combining an inducible FGF receptor 1 (FGFR1) to rescue MuSC intrinsic aging defects with a drug to reduce fibrosis partially rescued muscle mass loss in aged mice. We conclude that aging affects tissues, and particularly skeletal muscle tissue, via complex multifactorial processes requiring multifaceted interventions to improve aging phenotypes.

cell biology↗

bioRxiv2026

Activating FGFR1 restores Integrin-β1--mediated fibronectin sensing in satellite cells of aged mice

Muscle satellite cells (SCs), essential for skeletal muscle regeneration, decline in number and function with age, contributing to sarcopenia. A fully defined viscoelastic hydrogel that preserves SC-myofiber interactions and supports tunable densities of fibronectin-derived RGD ligands was used to investigate age-related defects in extracellular matrix sensing by SCs. Elevating RGD density increased the number of activating and proliferating SCs on myofibers from young mice, whereas SCs from aged mice were unresponsive. Loss of FGF receptor 1 signaling in SCs from aged mice abrogated the coordinated Syndecan-4 and Integrin-{beta}1 matrix response observed in SCs from young mice. Activating Integrin-{beta}1 promoted asymmetric division and self-renewal in SCs from young mice whereas combined FGFR1 and Integrin-{beta}1 signaling drove symmetric expansion. In SCs from aged mice, FGFR1 dysfunction disrupted this balance, impairing asymmetric division, but constitutive FGFR1 activation restored receptor co-localization, self-renewal, and fibronectin responsiveness. Therefore, FGFR1 integrates matrix and growth factor signals, suggesting that targeting the FGFR1-Integrin-{beta}1 axis may enhance SC regenerative potential in aging organisms.

cell biology↗

bioRxiv2026

Integration and harmonization of cell shape images for generative modeling

As cell imaging grows in scale, precision, and complexity, data integration and harmonization become increasingly important for studying cell-material interactions. Quantitative understanding of how cells respond to mechanical cues, such as substrate stiffness and topography, is often limited by differences in experimental conditions and imaging formats. This study presents a framework that combines compact, interpretable cell shape models with generative artificial intelligence to harmonize 2D and 3D immunofluorescent datasets within defined experimental contexts. By efficiently capturing morphology and associated biological features, the approach enables generation of realistic synthetic cells, including rare or intermediate phenotypes, to augment machine-learning analyses and support scalable in silico studies. This work advances data-driven investigation of cellular responses to biomaterial-derived mechanical cues.

bioengineering↗

bioRxiv2026

Multi-omics Insight into Cardiac Myofibril Remodeling in Post-Prandial Burmese Pythons

Burmese pythons exhibit rapid cardiac remodeling in response to a dramatic increase in metabolic rate during digestion. Here, we performed single-myofibril mechanics measurements and myosin heavy chain metabolic assays to evaluate the impact of feeding on the cardiomyocyte sarcomere - the fundamental molecular unit of muscle contraction - using two experimental paradigms: normal feeding (one meal per month) and frequent feeding (eight meals per month). Myofibril tension and rate of relaxation increased during digestion in both paradigms, while frequent feeding was further associated with slower myofibril activation kinetics and faster myosin heavy chain ATP turnover. To identify molecular changes at the sarcomere and gain potential mechanistic insight, we performed multi-omics analyses. RNA sequencing identified increased expression of some sarcomere genes during digestion; however, proteomics analysis suggested a delay in sarcomere protein synthesis at the peak of remodeling, as expression of many sarcomere proteins decreased. Analysis of post-translational modifications (ubiquitinomics, phospho-proteomics, acetylomics) identified hundreds of significantly regulated sites on sarcomere proteins during digestion, including many on the tension-regulating titin and myosin heavy chain proteins. Our results detail the molecular underpinnings of cardiac remodeling in digesting Burmese pythons and suggest that natures solution for rapidly increasing cardiac contractility is a post-translational sarcomere tuning program.

molecular biology↗

bioRxiv2025

Nuclei sense complex tissue shape and direct intestinal stem cell fate

Tissue architecture and function are influenced by mechanical cues. Yet, how cell nuclei sense forces within 3D tissues and dictate differentiation remains unknown as prior studies focused on isolated mesenchymal cells, which fail to fully predict tissue-level mechanical properties. We fill this knowledge gap utilizing live reporters and material-based organoid models. We posit the nucleus as an active mechanosensor of tissue shape, with levels of the nuclear scaffolding protein lamin-A varying across intestinal stem cell differentiation trajectory. Elevated forces on differentiated Paneth cell nuclei, in both organoids and tissue explants, increase lamin-A and nuclear wrinkling. Enhancing nuclear mechanotransduction primes cell differentiation, in otherwise stem promoting conditions, revealing that nuclear mechanics can direct stem cell fate. By engineering spatiotemporally controlled de novo tissue curvature with photo-degradable hydrogels, we direct spatially patterned lamin-A levels across mouse and human organoids of healthy and diseased origin, uncovering conserved nuclear mechanosensing pathway in epithelial tissues.

bioengineering↗

bioRxiv2024

Fully synthetic hydrogels promote robust crypt formation in intestinal organoids

Initial landmark studies in the design of synthetic hydrogels for intestinal organoid culture identified precise matrix requirements for differentiation, namely decompression of matrix-imposed forces and supplementation of laminin. But beyond stating the necessity of laminin, organoid-laminin interactions have gone largely unstudied, as this ubiquitous requirement of exogenous laminin hinders investigation. In this work, we exploit a fast stress relaxing, boronate ester based synthetic hydrogel for the culture of intestinal organoids, and fortuitously discover that unlike all other synthetic hydrogels to date, laminin does not need to be supplemented for crypt formation. This highly defined material provides a unique opportunity to investigate laminin-organoid interactions and how it influences crypt evolution and organoid function. Via fluorescent labeling of non-canonical amino acids, we further show that adaptable boronate ester bonds increase deposition of nascent proteins, including laminin. Collectively, these results advance the understanding of how mechanical and matricellular signaling influence intestinal organoid development.

bioengineering↗

bioRxiv2024

PTEN Regulates Myofibroblast Activation in Valvular Interstitials Cells based on Subcellular Localization

Aortic valve stenosis (AVS) is characterized by altered mechanics of the valve leaflets, which disrupts blood flow through the aorta and can cause left ventricle hypotrophy. These changes in the valve tissue result in activation of resident valvular interstitial cells (VICs) into myofibroblasts, which have increased levels of SMA in their stress fibers. The persistence of VIC myofibroblast activation is a hallmark of AVS. In recent years, the tumor suppressor gene phosphatase and tensin homolog (PTEN) has emerged as an important player in the regulation of fibrosis in various tissues (e.g., lung, skin), which motivated us to investigate PTEN as a potential protective factor against matrix-induced myofibroblast activation in VICs. In aortic valve samples from humans, we found high levels of PTEN in healthy tissue and low levels of PTEN in diseased tissue. Then, using pharmacological inducers to treat VIC cultures, we observed PTEN overexpression prevented stiffness-induced myofibroblast activation, whereas genetic and pharmacological inhibition of PTEN further activated myofibroblasts. We also observed increased nuclear PTEN localization in VICs cultured on stiff matrices, and nuclear PTEN also correlated with smaller nuclei, altered expression of histones and a quiescent fibroblast phenotype. Together, these results suggest that PTEN not only suppresses VIC activation, but functions to promote quiescence, and could serve as a potential pharmacological target for the treatment of AVS.

cell biology↗

bioRxiv2024

Automated Prediction of Fibroblast Phenotypes Using Mathematical Descriptors of Cellular Features

Fibrosis is caused by pathological activation of resident fibroblasts to myofibroblasts that leads to aberrant tissue stiffening and diminished function of affected organs with limited pharmacological interventions. Despite the prevalence of myofibroblasts in fibrotic tissue, existing methods to grade fibroblast phenotypes are typically subjective and qualitative, yet important for screening of new therapeutics. Here, we develop mathematical descriptors of cell morphology and intracellular structures to identify quantitative and interpretable cell features that capture the fibroblast-to-myofibroblast phenotypic transition in immunostained images. We train and validate models on features extracted from over 2,500 primary heart valve interstitial cells (VICs) and test their predictive performance on VICs treated with the small molecule drug 5-azacytidine, which inhibited myofibroblast activation. Collectively, this work introduces an analytical framework that unveils key features associated with distinct fibroblast phenotypes via quantitative image analysis and is broadly applicable for high-throughput screening assays of candidate treatments for fibrotic diseases.

bioengineering↗

bioRxiv2024

Y chromosome linked UTY modulates sex differences in valvular fibroblast methylation in response to nanoscale extracellular matrix cues

Aortic valve stenosis (AVS) is a progressive disease wherein males more often develop valve calcification relative to females that develop valve fibrosis. Valvular interstitial cells (VICs) aberrantly activate to myofibroblasts during AVS, driving the fibrotic valve phenotype in females. Myofibroblasts further differentiate into osteoblast-like cells and produce calcium nanoparticles, driving valve calcification in males. We hypothesized the lysine demethylase UTY (ubiquitously transcribed tetratricopeptide repeat containing, Y-linked) decreases methylation uniquely in male VICs responding to nanoscale extracellular matrix cues to promote an osteoblast-like cell phenotype. Here, we describe a hydrogel biomaterial cell culture platform to interrogate how nanoscale cues modulate sex-specific methylation states in VICs activating to myofibroblasts and osteoblast-like cells. We found UTY modulates the osteoblast-like cell phenotype in response to nanoscale cues uniquely in male VICs. Overall, we reveal a novel role of UTY in the regulation of calcification processes in males during AVS progression.

bioengineering↗

bioRxiv2024

Nonlinear Elastic Bottlebrush Polymer Hydrogels Modulate Actomyosin Mediated Protrusion Formation in Mesenchymal Stromal Cells

The nonlinear elasticity of many tissue-specific extracellular matrices is difficult to recapitulate without the use of fibrous architectures, which couple strain-stiffening with stress relaxation. Herein, bottlebrush polymers are synthesized and crosslinked to form poly(ethylene glycol)-based hydrogels and used to study how strain-stiffening behavior affects human mesenchymal stromal cells (hMSCs). By tailoring the bottlebrush polymer length, the critical stress associated with the onset of network stiffening is systematically varied, and a unique protrusion-rich hMSC morphology emerges only at critical stresses within a biologically accessible stress regime. Local cell-matrix interactions are quantified using 3D traction force microscopy and small molecule inhibitors are used to identify cellular machinery that plays a critical role in hMSC mechanosensing of the engineered, strain-stiffening microenvironment. Collectively, this study demonstrates how covalently crosslinked bottlebrush polymer hydrogels can recapitulate strain-stiffening biomechanical cues at biologically relevant stresses and be used to probe how nonlinear elastic matrix properties regulate cellular processes.

bioengineering↗

bioRxiv2023

Fibrillar adhesion dynamics govern the timescales of nuclear mechano-response via the vimentin cytoskeleton

The cell nucleus is continuously exposed to external signals, of both chemical and mechanical nature. To ensure proper cellular response, cells need to regulate not only the transmission of these signals, but also their timing and duration. Such timescale regulation is well described for fluctuating chemical signals, but if and how it applies to mechanical signals reaching the nucleus is still unknown. Here we demonstrate that the formation of fibrillar adhesions locks the nucleus in a mechanically deformed conformation, setting the mechanical response timescale to that of fibrillar adhesion remodelling ([~]1 hour). This process encompasses both mechanical deformation and associated mechanotransduction (such as via YAP), in response to both increased and decreased mechanical stimulation. The underlying mechanism is the anchoring of the vimentin cytoskeleton to fibrillar adhesions and the extracellular matrix through plectin 1f, which maintains nuclear deformation. Our results reveal a mechanism to regulate the timescale of mechanical adaptation, effectively setting a low pass filter to mechanotransduction.

biophysics↗

bioRxiv2023

Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment

In skeletal muscle tissue, injury-related changes in stiffness activate muscle stem cells through mechanosensitive signaling pathways. Functional muscle tissue regeneration also requires the effective coordination of myoblast proliferation, migration, polarization, differentiation, and fusion across multiple length scales. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topographic features that could confer contact guidance, C2C12 myoblasts collectively polarize in the stiffest direction of the substrate. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of same chemical formulation. These findings reveal a role for stiffness anisotropy in coordinating emergent collective cellular dynamics, with implications for understanding skeletal muscle tissue development and regeneration.

bioengineering↗

bioRxiv2021

Network Modeling Predicts Personalized Gene Expression and Drug Responses in Valve Myofibroblasts Cultured with Patient Sera

Aortic valve stenosis (AVS) patients experience pathogenic valve leaflet stiffening due to excessive extracellular matrix (ECM) remodeling. Numerous microenvironmental cues influence pathogenic expression of ECM remodeling genes in tissue-resident valvular myofibroblasts, and the regulation of complex myofibroblast signaling networks depends on patient-specific extracellular factors. Here, we combined a manually curated myofibroblast signaling network with a data-driven transcription factor network to predict patient-specific myofibroblast gene expression signatures and drug responses. Using transcriptomic data from myofibroblasts cultured with AVS patient sera, we produced a large-scale, logic-gated differential equation model in which 11 biochemical and biomechanical signals are transduced via a network of 334 signaling and transcription reactions to accurately predict the expression of 27 fibrosis-related genes. Correlations were found between personalized model-predicted gene expression and AVS patient echocardiography data, suggesting links between fibrosis-related signaling and patient-specific AVS severity. Further, global network perturbation analyses revealed signaling molecules with the most influence over network-wide activity including endothelin 1 (ET1), interleukin 6 (IL6), and transforming growth factor {beta} (TGF{beta}) along with downstream mediators c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription (STAT), and reactive oxygen species (ROS). Lastly, we performed virtual drug screening to identify patient-specific drug responses, which were experimentally validated via fibrotic gene expression measurements in VICs cultured with AVS patient sera and treated with or without bosentan - a clinically approved ET1 receptor inhibitor. In sum, our work advances the ability of computational approaches to provide a mechanistic basis for clinical decisions including patient stratification and personalized drug screening.

systems biology↗

bioRxiv2020

Engineering Functional Pseudo-Islets of Defined Sizes from Primary Murine Cells Using PEG Microwell Devices

A major limitation of islet transplantation as a therapy for treating Type 1 Diabetes is eventual graft failure, which can be partially attributed to islet cell death. When cultured in vitro, cells in the center of large islets show increased necrosis and exhibit decreased viability and insulin secretion compared to smaller islets. Given the necessity of {beta}-cell-to-{beta}-cell coupling for the physiological response to glucose, a technique to re-aggregate primary islet cells or cells derived from progenitor cells into small clusters of defined sizes may prove advantageous for promoting function upon transplantation. Here, hydrogel microwell arrays were utilized to generate 3-dimensional pseudo-islets from primary murine islets. Pseudo-islets ranged from 50 to 100 m in diameter as controlled through the microwell dimensions, and contained {beta}-, -, and {delta}-cells with ratios similar to those in whole murine islets. Over two weeks in culture, pseudo-islets remained highly viable and responsive to glucose. Intracellular calcium flux showed more robust and coordinated dynamics at high glucose and decreased activity at low glucose compared to age-matched wild-type islets. Therefore, microwell devices can control the aggregation of cells isolated from primary islets to produce islet-like clusters that are functionally similar to freshly isolated islets, and may provide a technique to create improved cellular therapies for Type 1 Diabetes.

bioengineering↗

bioRxiv2019

Cardiac myocytes respond differentially and synergistically to matrix stiffness and topography

During cardiac disease progression, myocytes undergo molecular, functional and structural changes, including increases in cell size and shape, decreased myocyte alignment and contractility. The heart often increases extracellular matrix production and stiffness, which affect myocytes. The order and hierarchy of these events remain unclear as available in vitro cell culture systems do not adequately model both physiologic and pathologic environments. Traditional cell culture substrates are 5-6 orders of magnitude stiffer than even diseased native cardiac tissue. Studies that do account for substrate stiffness often do not consider intercellular alignment and vice versa. We developed a cardiac myocyte culture platform that better recapitulates native tissue stiffness while simultaneously introducing topographical cues that promote cellular alignment. We show that stiffness and topography impact myocyte molecular and functional properties. We used a spatiotemporally-tunable, photolabile hydrogel platform to generate a range of stiffness and micron-scale topographical patterns to guide neonatal rat ventricular myocyte morphology. Importantly, these substrate patterns were of subcellular dimensions to test whether cells would spontaneously respond to topographical cues rather than an imposed geometry. Cellular contractility was highest and the gene expression profile was most physiologic on gels with healthy cardiac tissue stiffness. Surprisingly, while elongated patterns in stiff gels yielded the greatest cellular alignment, the cells actually had more pathologic functional and molecular profiles. These results highlight that morphological measurements alone are not a surrogate for overall cellular health as many studies assume. In general, substrate stiffness and micropatterning synergistically affect cardiac myocyte phenotype to recreate physiologic and pathologic microenvironments.\n\nSignificance StatementHeart disease is accompanied by organ- and cellular-level remodeling, and deconvoluting their interplay is complex. Cellular-level change is best studied in vitro due to greater control and uniformity of cell types compared to animals. One common metric is degree of cellular alignment as misalignment of myocytes is a hallmark of disease. However, most studies utilize featureless culture surfaces that are orders of magnitude stiffer than, and do not mimic the scaffolding of, the heart. We developed a hydrogel platform with tunable stiffness and patterns providing topographical alignment cues. We cultured heart cells on and characterized multifactorial responses to these dynamic surfaces. Interestingly, conditions that yielded greatest alignment did not yield the healthiest functional and molecular state. Thus, morphology alone is not an indicator of overall cellular health.

cell biology↗