Biology subjects

Herberg, S.

Publications and source records attributed to Herberg, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Transcriptomic profiling of normal and glaucomatous trabecular meshwork cells in a tissue-like soft ECM environment

Purpose. Trabecular meshwork (TM) dysfunction contributes to the elevated intraocular pressure in primary open-angle glaucoma. To better understand the molecular signature distinguishing glaucomatous from normal TM cells, we interrogated their genome-wide transcriptomic differences in a tissue-like soft extracellular matrix (ECM) microenvironment. Methods. Primary human glaucomatous and normal TM cell strains (N = 5 each) from three independent laboratories were plated on bioengineered collagen type I/hyaluronic acid/elastin-like polypeptide ECM hydrogels in 10% FBS media for 4-5 days, then serum-free media for 2 days. Cell-laden hydrogels were homogenized in TRIzol for RNA isolation, quality assessment, and library preparation, followed by paired-end 100-bp Illumina sequencing. Differential expression was assessed using DESeq2, including glaucoma status, originating laboratory, and donor age/sex in the model. Differentially expressed genes (DEGs) were defined as p < 0.05 and |fold change| [≥] 2, followed by Gene Ontology (GO) enrichment and QIAGEN Ingenuity Pathway Analysis (IPA). Results. In total, 588 DEGs distinguished the glaucomatous from the normal TM transcriptome (393 up-regulated in glaucomatous TM; 195 up-regulated in normal TM, i.e., down-regulated in glaucomatous TM). GO and IPA causal analyses revealed three coordinated disease programs: (i) TGF-{beta}/Wnt-driven ECM remodeling and fibrosis, (ii) NF-{kappa}B-associated inflammation, and (iii) a hypoxia/metabolic-stress program linked to p300/CREBBP chromatin co-activation. Conclusions. These data define a transcriptomic signature of glaucomatous TM cells when cultured in a soft physiologic ECM microenvironment, independent of the originating laboratory, with three mechanistic axes (ECM/fibrosis, inflammation, hypoxia/metabolism), and nominate p300 (encoded by EP300) as a candidate upstream node integrating these programs.

cell biology↗

bioRxiv2026

Multiomic analysis of non-glaucomatous human trabecular meshwork cells.

Glaucoma is an irreversible blinding disease that affects millions of individuals worldwide. Elevated intraocular pressure (IOP), regulated by the trabecular meshwork (TM) in the anterior eye, is the only modifiable risk factor. Human TM cells can be cultured from donor eyes, providing a precious resource for studying factors that induce or prevent glaucoma. The goal of this study was to produce datasets that define the molecular profile of human non-glaucomatous TM cells. Using 18 human TM cell strains cultured from non-glaucomatous individuals deposited from seven laboratories in the USA and UK, this study used transcriptomic, proteomic, lipidomic, and metabolomic analyses to characterize the molecular content of TM cells. The data herein provides the most comprehensive multiomic analyses of human TM cells to date and will be a useful resource for researchers and clinicians in the TM and glaucoma fields.

cell biology↗

bioRxiv2025

Decreased substrate stiffness leads to mitochondrial dysfunctions and Endothelial to Mesenchymal transition through Focal Adhesion Kinase activity in corneal endothelial cells

PurposeFuchs Endothelial Corneal Dystrophy (FECD), a degenerative corneal disorder, is marked by the thickening of Descemets membrane and a progressive loss of corneal endothelial cells, ultimately leading to vision loss. A feature associated with the disease is the reduced stiffness of Descemets membrane. However, the effects of this change in Descemets membrane, on corneal endothelial cell health are not well understood. To explore this, we used in vitro, in vivo, and ex vivo studies to investigate how changes in substrate stiffness and the signaling pathways associated with these changes influence corneal endothelial functions. MethodsFor in-vitro studies, we cultured bovine corneal endothelial cells for 96 hours on stiff (32 kPa) and soft (8 kPa) substrate CytoSoft plates. By using Jess immunoassay and traditional western blotting, we evaluated changes in integrin signaling components, endothelial-to-mesenchymal transition, apoptosis, autophagy, and ubiquitin-proteasome pathway markers. Mitochondrial health and mitochondrial superoxide levels were assessed using commercial kits. We assessed the protein levels of the above-mentioned markers in the Col8a2Q455K/Q455K FECD mouse model. To evaluate whether FAK signaling contributes to the FECD pathogenesis, we injected the Col8a2Q455K/Q455K mice with an FAK inhibitor and assessed the corneal phenotypes. ResultsWe observed increased levels of phosphorylated FAK, integrins 4 and 5 in bovine corneal endothelial cells cultured on soft substrate. We also found upregulated endothelial-to-mesenchymal transition (EndMT) markers, mitochondrial dysfunction, and apoptosis in cells grown on soft substrate. In the Col8a2Q455K/Q455K mouse model of FECD, there was increased pFAK Y397 levels coincident with the onset of phenotypes. Intraperitoneal injections of a pFAK inhibitor improved antioxidant protein expression and decreased EndMT; however, it did not improve FECD-associated disease progression. ConclusionIn this study, we explored how changes in the physical characteristics of the Descemets membrane impact corneal endothelial cell health. While we discovered activation of Focal adhesion kinase as a result of stiffness changes, its inhibition alone was insufficient to improve cell health in an FECD mouse model.

molecular biology↗

bioRxiv2025

Harnessing cell-encapsulated hydrogels to study astrocyte mechanoresponse in 4D

In glaucoma, the optic nerve head (ONH) is exposed to increased biomechanical strain, impacting the resident astrocytes that maintain neural homeostasis. During disease progression astrocytes exhibit morphologic and metabolic shifts; however, the specific impact of glaucoma-related biomechanical strains on astrocyte behavior remains poorly understood. To address this, we used our previously established 3D cell-encapsulated extracellular matrix (ECM) hydrogel to investigate ONH astrocyte cellular and transcriptomic responses to varying biomechanical strain levels over time. Murine ONH astrocyte were encapsulated within an ECM hydrogel made from photocrosslinkable collagen type I and hyaluronic acid, and subjected to 0, 3, or 10% cyclic compression for 4h and 24h. We found significant restructuring of cytoskeletal morphology, metabolic dysregulation, and astrocyte-mediated ECM modulation that were strain-, duration- and hydrogel subregion-dependent. These phenotypic alterations were associated with diverse transcriptional changes in genes related to cell cycle and morphology, inflammation, metabolism and matrix remodeling that were driven by compressive strain intensity and duration. Our work reveals the direct role of compressive strain in eliciting a complex astrocyte response, supports targeting mechanosensation to prevent these pathologic astrocyte responses, and establishes ECM-based hydrogels as a platform to test mechanisms driving astrocyte mechanodysfunction. Altogether, our study offers new insights into astrocyte responses to biomechanical insult and demonstrates the use of a tunable 3D ECM hydrogel for future mechanistic studies of neurodegeneration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/660800v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@f355fborg.highwire.dtl.DTLVardef@191e096org.highwire.dtl.DTLVardef@2fae32org.highwire.dtl.DTLVardef@55913c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

The risk for developing primary open-angle glaucoma (POAG) correlates with the magnitude of ocular hypertension (OHT) and the concentration of transforming growth factor-{beta}2 (TGF{beta}2) in the aqueous humor. Effective treatment of POAG requires detailed understanding of interaction between pressure sensing mechanisms in the trabecular meshwork (TM) and biochemical risk factors. Here, we employed molecular, optical, electrophysiological and tonometric strategies to establish the role of TGF{beta}2 in transcription and functional expression of mechanosensitive channel isoforms alongside studies of TM contractility in biomimetic hydrogels, and intraocular pressure (IOP) regulation in a mouse model of TGF{beta}2 -induced OHT. TGF{beta}2 upregulated expression of TRPV4 and PIEZO1 transcripts and time-dependently augmented functional TRPV4 activation. TRPV4 agonists induced contractility of TM-seeded hydrogels whereas pharmacological inhibition suppressed TGF{beta}2-induced hypercontractility and abrogated OHT in eyes overexpressing TGF{beta}2. Trpv4-deficient mice resisted TGF{beta}2-driven increases in IOP, but nocturnal OHT was not additive to TGF{beta}-evoked OHT. Our study establishes the fundamental role of TGF{beta} as a modulator of mechanosensing in nonexcitable cells, identifies the TRPV4 channel as the final common mechanism for TM contractility and circadian and pathological OHT, and offers insights for future treatments that can lower IOP in the sizeable cohort of hypertensive glaucoma patients that resist current treatments.

physiology↗

bioRxiv2023

Targeting YAP mechanosignaling to ameliorate stiffness-induced Schlemm's canal cell pathobiology

Pathologic alterations in the biomechanical properties of the Schlemms canal (SC) inner wall endothelium and its immediate vicinity are strongly associated with ocular hypertension in glaucoma due to decreased outflow facility. Specifically, the underlying trabecular meshwork is substantially stiffer in glaucomatous eyes compared to that from normal eyes. This raises the possibility of a critical involvement of mechanotransduction processes in driving SC cell dysfunction. Yes-associated protein (YAP) has emerged as a key contributor to glaucoma pathogenesis. However, the molecular underpinnings of SC cell YAP mechanosignaling in response to glaucomatous extracellular matrix (ECM) stiffening are not well understood. Using a novel biopolymer hydrogel that facilitates dynamic and reversible stiffness tuning, we investigated how ECM stiffening modulates YAP activity in primary human SC cells, and whether disruption of YAP mechanosignaling attenuates SC cell pathobiology and increases ex vivo outflow facility. We demonstrated that ECM stiffening drives pathologic YAP activation and cytoskeletal reorganization in SC cells, which was fully reversible by matrix softening in a distinct time-dependent manner. Furthermore, we showed that pharmacologic or genetic disruption of YAP mechanosignaling abrogates stiffness-induced SC cell dysfunction involving altered cytoskeletal and ECM remodeling. Lastly, we found that perfusion of the clinically-used, small molecule YAP inhibitor verteporfin (without light activation) increases ex vivo outflow facility in normal mouse eyes. Collectively, our data provide new evidence for a pathologic role of aberrant YAP mechanosignaling in SC cell dysfunction and suggest that YAP inhibition has therapeutic value for treating ocular hypertension in glaucoma.

cell biology↗

bioRxiv2022

YAP/TAZ inactivation with simvastatin attenuates glucocorticoid-induced human trabecular meshwork cell dysfunction

PurposeImpairment of the trabecular meshwork (TM) is the principal cause of increased outflow resistance in the glaucomatous eye. Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) are emerging as potential mediators of TM cell/tissue dysfunction. Furthermore, YAP/TAZ activity was recently found to be controlled by the mevalonate pathway in non-ocular cells. Clinically-used statins block the mevalonate cascade and were shown to improve TM cell pathobiology; yet, the link to YAP/TAZ signaling was not investigated. In this study, we hypothesized that YAP/TAZ inactivation with simvastatin attenuates glucocorticoid-induced human TM (HTM) cell dysfunction. MethodsPrimary HTM cells were seeded atop or encapsulated within bioengineered extracellular matrix (ECM) hydrogels. Dexamethasone was used to induce a pathologic phenotype in HTM cells in the absence or presence of simvastatin. Changes in YAP/TAZ activity, actin cytoskeletal organization, phospho-myosin light chain levels, hydrogel contraction/stiffness, and fibronectin deposition were assessed. ResultsSimvastatin potently blocked pathologic YAP/TAZ nuclear localization/activity, actin stress fiber formation, and myosin light chain phosphorylation in HTM cells. Importantly, simvastatin co-treatment significantly attenuated dexamethasone-induced ECM contraction/stiffening and extracellular fibronectin deposition. Sequential treatment was similarly effective but did not match clinically-used Rho kinase inhibition. ConclusionsYAP/TAZ inactivation with simvastatin attenuates HTM cell pathobiology in a tissue-mimetic ECM microenvironment. Our data may help explain the association of statin use with a reduced risk of developing glaucoma via indirect YAP/TAZ inhibition as a proposed regulatory mechanism.

cell biology↗

bioRxiv2022

YAP/TAZ mediate TGFβ2-induced Schlemm's canal cell dysfunction

PurposeElevated transforming growth factor beta2 (TGF{beta}2) levels in the aqueous humor have been linked to glaucomatous outflow tissue dysfunction. Potential mediators of dysfunction are the transcriptional co-activators, Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ). However, the molecular underpinnings of YAP/TAZ modulation in Schlemms Canal (SC) cells under glaucomatous conditions are not well understood. Here, we investigate how TGF{beta}2 regulates YAP/TAZ activity in human SC (HSC) cells using biomimetic extracellular matrix (ECM) hydrogels, and examine whether pharmacologic YAP/TAZ inhibition would attenuate TGF{beta}2-induced HSC cell dysfunction. MethodsPrimary HSC cells were seeded atop photocrosslinked ECM hydrogels, made of collagen type I, elastin-like polypeptide and hyaluronic acid, or encapsulated within the hydrogels. HSC cells were induced with TGF{beta}2 in the absence or presence of concurrent actin destabilization or pharmacologic YAP/TAZ inhibition. Changes in actin cytoskeletal organization, YAP/TAZ activity, ECM production, phospho-myosin light chain levels, and hydrogel contraction were assessed. ResultsTGF{beta}2 significantly increased YAP/TAZ nuclear localization in HSC cells, which was prevented by either filamentous (F)-actin relaxation or depolymerization. Pharmacologic YAP/TAZ inhibition using verteporfin without light stimulation decreased fibronectin expression and reduced actomyosin cytoskeletal rearrangement in HSC cells induced by TGF{beta}2. Similarly, verteporfin significantly attenuated TGF{beta}2-induced HSC cell-encapsulated hydrogel contraction. ConclusionsOur data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in HSC cells under simulated glaucomatous conditions, and suggest that pharmacologic YAP/TAZ inhibition has promising potential to improve outflow tissue dysfunction.

cell biology↗

bioRxiv2022

Effects of netarsudil-family Rho kinase inhibitors on human trabecular meshwork cell contractility and actin remodeling using a bioengineered ECM hydrogel

Interactions between trabecular meshwork (TM) cells and their extracellular matrix (ECM) are critical for normal outflow function in the healthy eye. Multifactorial dysregulation of the TM is the principal cause of elevated intraocular pressure that is strongly associated with glaucomatous vision loss. Key characteristics of the diseased TM are pathologic contraction and actin stress fiber assembly, contributing to overall tissue stiffening. Among first-line glaucoma medications, the Rho-associated kinase inhibitor (ROCKi) netarsudil is known to directly target the stiffened TM to improve outflow function via tissue relaxation involving focal adhesion and actin stress fiber disassembly. Yet, no in vitro studies have explored the effect of netarsudil on human TM (HTM) cell contractility and actin remodeling in a 3D ECM environment. Here, we use our bioengineered HTM cell-encapsulated ECM hydrogel to investigate the efficacy of different netarsudil-family ROCKi compounds on reversing pathologic contraction and actin stress fibers. Netarsudil and all related experimental ROCKi compounds exhibited significant ROCK1/2 inhibitory and focal adhesion disruption activities. Furthermore, all ROCKi compounds displayed potent contraction-reversing effects on HTM hydrogels upon glaucomatous induction in a dose-dependent manner, relatively consistent with their biochemical/cellular inhibitory activities. At their tailored EC50 levels, netarsudil-family ROCKi compounds exhibited distinct effect signatures of reversing pathologic HTM hydrogel contraction and actin stress fibers, independent of the cell strain used. Netarsudil outperformed the experimental ROCKi compounds in support of its clinical status. In contrast, at uniform EC50-levels using netarsudil as reference, all ROCKi compounds performed similarly. Collectively, our data suggest that netarsudil exhibits high potency to rescue HTM cell pathobiology in a tissue-mimetic 3D ECM microenvironment, solidifying the utility of our bioengineered hydrogel model as a viable screening platform to further our understanding of TM pathophysiology in glaucoma.

cell biology↗

bioRxiv2022

Engineering a 3D hydrogel system to study optic nerve head astrocyte morphology and behavior in response to glaucomatous insult

In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGF{beta}2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGF{beta}2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405-500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGF{beta}2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHA retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with longer process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGF{beta}2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONHA-encapsulated hydrogel to investigate ONHA behavior in response to glaucomatous insult.

bioengineering↗

bioRxiv2021

Extracellular matrix stiffness and TGFβ2 regulate YAP/TAZ activity in human trabecular meshwork cells

Primary open-angle glaucoma progression is associated with increased human trabecular meshwork (HTM) stiffness and elevated transforming growth factor beta 2 (TGF{beta}2) levels in aqueous humor. Increased transcriptional activity of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), central players in mechanotransduction, are implicated in glaucomatous HTM (GTM) cell dysfunction. Yet, the detailed mechanisms underlying YAP/TAZ modulation in HTM cells in response to alterations in extracellular matrix (ECM) stiffness and TGF{beta}2 levels are not well understood. Using biomimetic ECM hydrogels with tunable stiffness, here we show that increased ECM stiffness elevates YAP/TAZ transcriptional activity potentially through modulating focal adhesions and cytoskeletal rearrangement. Furthermore, TGF{beta}2 increased YAP/TAZ nuclear localization in both normal and glaucomatous HTM cells, which was prevented by inhibiting extracellular-signal-regulated kinase and Rho-associated kinase signaling pathways. Filamentous (F)-actin depolymerization reversed TGF{beta}2-induced YAP/TAZ nuclear localization. YAP/TAZ depletion using siRNA or verteporfin decreased focal adhesions, ECM remodeling and cell contractile properties. Similarly, YAP/TAZ inactivation with verteporfin partially blocked TGF{beta}2-induced HTM/GTM hydrogel contraction and stiffening. Collectively, our data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in glaucomatous HTM cell dysfunction, and may help inform strategies for the development of novel multifactorial approaches to prevent progressive ocular hypertension in glaucoma.

cell biology↗

bioRxiv2021

Engineering a thixotropic and biochemically tunable hyaluronan and collagen bioink for biofabrication of multiple tissue construct types

The field of three-dimensional (3D) bioprinting has advanced rapidly in recent years. Significant reduction in the costs associated with obtaining functional 3D bioprinting hardware platforms is both a cause and a result of these advances. As such, there are more laboratories than ever integrating bioprinting methodologies into their research. However, there is a lack of standards in the field of biofabrication governing any requirements or characteristics to support cross-compatibility with biomaterial bioinks, hardware, and different tissue types. Here we describe a modular extracellular matrix (ECM) inspired bioink comprised of collagen and hyaluronic acid base components that: 1) employ reversible internal hydrogen bonding forces to generate thixotropic materials that dynamically reduce their elastic moduli in response to increased shear stress, thus enabling increased compatibility with printing hardware; and 2) modular addons in the form of chemically-modified fibronectin and laminin that when covalently bound within the bioink support a variety of tissue types, including liver, neural, muscle, pancreatic islet, and adipose tissue. These features aim to accelerate the deployment of such bioinks for tissue engineering of functional constructs in the hands of various end users.

bioengineering↗

bioRxiv2021

Mechanosensitive channel inhibition attenuates TGFβ2-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes.

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGF{beta}2) levels within astrocytes have been described in glaucoma, and TGF{beta} signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGF{beta}2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGF{beta}2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGF{beta}2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining for GFAP. Levels of fibronectin deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (Iba1) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGF{beta}2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGF{beta}2 treatment and the presence of f-actin stress fibers. TGF{beta}2 treatment significantly increased GFAP and fibronectin fluorescence intensity, which were decreased with GsMTx4 treatment. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

cell biology↗

bioRxiv2021

The sperm protein SPACA4 is required for efficient fertilization in mice

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in non-mammalian species do not have obvious mammalian homologs. We have recently identified the Ly6/uPAR protein Bouncer as a new, essential fertilization factor in zebrafish (Herberg et al., 2018). Here, we show that Bouncers homolog in mammals, SPACA4, is also required for efficient fertilization in mice. In contrast to fish, where Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely sub-fertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

developmental biology↗

bioRxiv2020

A tissue-engineered human trabecular meshwork hydrogel for advanced glaucoma disease modeling

PurposeAbnormal human trabecular meshwork (HTM) cell function and extracellular matrix (ECM) remodeling contribute to HTM stiffening in primary open-angle glaucoma (POAG). Most current cellular HTM model systems do not sufficiently replicate the complex native three dimensional (3D) cell-ECM interface, which makes them less than ideal to investigate POAG pathology. Tissue-engineered protein-based hydrogels are ideally positioned to overcome shortcomings of current models. Here, we report a novel biomimetic HTM hydrogel and test its utility as a POAG disease model. MethodsHTM hydrogels were engineered by mixing normal donor-derived HTM cells with collagen type I, elastin-like polypeptide and hyaluronic acid, each containing photoactive functional groups, followed by UV light-activated free-radical crosslinking. Glaucomatous conditions were induced with dexamethasone (DEX), and therapeutic effects of the Rho-associated kinase (ROCK) inhibitor Y27632 on cytoskeletal organization and tissue-level function, contingent on HTM cell-ECM interactions, were assessed. ResultsDEX exposure increased HTM hydrogel contractility, f-actin and alpha smooth muscle actin abundance and rearrangement, ECM remodeling, and fibronectin and collagen type IV deposition, all contributing to HTM hydrogel condensation and stiffening consistent with recent data from normal vs. glaucomatous HTM tissue. Y27632 treatment produced precisely the opposite effects and attenuated the DEX-induced pathologic changes, resulting in HTM hydrogel relaxation and softening. ConclusionsWe have developed a biomimetic HTM hydrogel model for detailed investigation of 3D cell-ECM interactions under normal and simulated glaucomatous conditions. Its bidirectional responsiveness to pharmaceutical challenge and rescue suggests promising potential to serve as screening platform for new POAG treatments with focus on HTM biomechanics.

bioengineering↗

bioRxiv2019

Simulating the human tumor microenvironment in colorectal cancer organoids in vitro and in vivo

The tumor microenvironment (TME) plays a significant role in cancer growth and metastasis. Bioengineered models of the TME will advance our understanding of cancer progression and facilitate identification of novel anti-cancer therapeutics that target TME components such as extracellular matrix (ECM) and stromal cells. However, most current in vitro models fail to recapitulate the extensive features of the human tumor stroma, especially ECM architecture, and are not exposed to intact body physiology. On the other hand, in vivo animal models do not accurately capture human tumor architecture. Using the features of biopsied colorectal cancer (CRC) tissue as a guide, we address these deficiencies by creating human organoids containing a colonic stromal ECM layer and CRC spheroids. Organoids were studied in vitro and upon implantation in mice for 28 days. We show that the stromal ECM micro-architecture, generated in vitro, was maintained in vivo for at least 28 days. Furthermore, comparisons with biopsied CRC tumors revealed that organoids with orderly structured TMEs induce an epithelial phenotype in CRC cells, similar to low-grade tumors, compared to a mesenchymal phenotype observed in disordered TMEs, similar to high-grade tumors. Altogether, these results are the first demonstration of replicating the human tumor ECM architecture in biofabricated tumor organoids under ex vivo and in vivo conditions.

cancer biology↗

bioRxiv2019

Scaffold-free human mesenchymal stem cell construct geometry regulates long bone regeneration

Scaffold-based bone tissue engineering approaches frequently induce repair processes dissimilar to normal developmental programs. In contrast, biomimetic strategies aim to recapitulate aspects of development through cellular self-organization, morphogenetic pathway activation, and mechanical cues. This may improve regenerative outcome in large long bone defects that cannot heal on their own; however, no study to date has investigated the role of scaffold-free construct geometry, in this case tubes mimicking long bone diaphyses, on bone regeneration. We hypothesized that microparticle-mediated in situ presentation of transforming growth factor-{beta}1 (TGF-{beta}1) and bone morphogenetic protein-2 (BMP-2) to engineered human mesenchymal stem cell (hMSC) tubes induces the endochondral cascade, and that TGF-{beta}1 + BMP-2-presenting hMSC tubes facilitate enhanced endochondral healing of critical-sized femoral segmental defects under delayed in vivo mechanical loading conditions compared to loosely-packed hMSC sheets. Here, localized morphogen presentation imparted early chondrogenic lineage priming, and stimulated robust endochondral differentiation of hMSC tubes in vitro. In an ectopic environment, hMSC tubes formed a cartilage template that was actively remodeled into trabecular bone through endochondral ossification without lengthy predifferentiation. Similarly, hMSC tubes stimulated in vivo cartilage and bone formation and more robust healing in femoral defects compared to hMSC sheets. New bone was formed through endochondral ossification in both groups; however, only hMSC tubes induced regenerate tissue partially resembling normal growth plate architecture. Together, this study demonstrates the interaction between mesenchymal cell condensation geometry, bioavailability of multiple morphogens, and defined in vivo mechanical environment to recapitulate developmental programs for biomimetic bone tissue engineering.\n\nSignificance StatementEngineered bone constructs must be capable of withstanding and adapting to harsh conditions in a defect site upon implantation, and can be designed to facilitate repair processes that resemble normal developmental programs. Self-assembled tubular human mesenchymal stem cell constructs were engineered to resemble the geometry of long bone diaphyses. By mimicking the cellular, biochemical, and mechanical environment of the endochondral ossification process during embryonic development, successful healing of large femoral segmental defects upon implantation was achieved and the extent was construct geometry dependent. Importantly, results were obtained without a supporting scaffold or lengthy predifferentiation of the tubular constructs. This indicates that adult stem/progenitor cells retain features of embryonic mesenchyme, and supports the concept of developmental engineering for bone regeneration approaches.

bioengineering↗

bioRxiv2019

Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair

Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation and is directed by local morphogen signals and mechanical cues. Here, we aimed to mimic these developmental conditions for regeneration of large bone defects. We hypothesized that engineered human mesenchymal stem cell (hMSC) condensations with in situ presentation of transforming growth factor-{beta}1 (TGF-{beta}1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles would promote endochondral regeneration of critical-sized rat femoral bone defects in a manner dependent on the in vivo mechanical environment. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with dual BMP-2 + TGF-{beta}1 fully restoring mechanical bone function by week 12. In vivo ambulatory mechanical loading, initiated at week 4 by delayed unlocking of compliant fixation plates, significantly enhanced the bone formation rate in the four weeks after load initiation in the dual morphogen group. In vitro, local presentation of either BMP-2 alone or BMP-2 + TGF-{beta}1 initiated endochondral lineage commitment of mesenchymal condensations, inducing both chondrogenic and osteogenic gene expression through SMAD3 and SMAD5 signaling. In vivo, however, endochondral cartilage formation was evident only in the BMP-2 + TGF-{beta}1 group and was enhanced by mechanical loading. The degree of bone formation was comparable to BMP-2 soaked on collagen but without the ectopic bone formation that limits the clinical efficacy of BMP-2/collagen. In contrast, mechanical loading had no effect on autograft-mediated repair. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering. One Sentence SummaryMimicking aspects of the cellular, biochemical, and mechanical environment during early limb development, chondrogenically-primed human mesenchymal stem cell condensations promoted functional healing of critical-sized femoral defects via endochondral ossification, and healing rate and extent was a function of the in vivo mechanical environment.

bioengineering↗