Publications by authors named "Ryan S Stowers"

9 Publications

  • Page 1 of 1

Identification of cell context-dependent YAP-associated proteins reveals β and β integrin mediate YAP translocation independently of cell spreading.

Sci Rep 2019 11 20;9(1):17188. Epub 2019 Nov 20.

Department of Mechanical Engineering, Stanford University, Stanford, CA, 94305, USA.

Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin stress fiber mediated nuclear pore opening, however the protein mediators of YAP translocation remain unclear. Here, we show that YAP co-localizes with F-actin during activating conditions, such as sparse plating and culturing on stiff 2D substrates. To identify proteins mediating YAP translocation, we performed co-immunoprecipitation followed by mass spectrometry (co-IP/MS) for proteins that differentially associated with YAP under activating conditions. Interestingly, YAP preferentially associates with β integrin under activating conditions, and β integrin under inactivating conditions. In activating conditions, CRISPR/Cas9 knockout (KO) of β integrin (ΔITGB1) resulted in decreased cell area, which correlated with decreased YAP nuclear localization. ΔITGB1 did not significantly affect the slope of the correlation between YAP nuclear localization with area, but did decrease overall nuclear YAP independently of cell spreading. In contrast, β integrin KO (ΔITGB4) cells showed no change in cell area and similarly decreased nuclear YAP. These results reveal proteins that differentially associate with YAP during activation, which may aid in regulating YAP nuclear translocation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-53659-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868278PMC
November 2019

Matrix stiffness induces a tumorigenic phenotype in mammary epithelium through changes in chromatin accessibility.

Nat Biomed Eng 2019 12 8;3(12):1009-1019. Epub 2019 Jul 8.

Department of Mechanical Engineering, Stanford University, Stanford, CA, USA.

In breast cancer, the increased stiffness of the extracellular matrix is a key driver of malignancy. Yet little is known about the epigenomic changes that underlie the tumorigenic impact of extracellular matrix mechanics. Here, we show in a three-dimensional culture model of breast cancer that stiff extracellular matrix induces a tumorigenic phenotype through changes in chromatin state. We found that increased stiffness yielded cells with more wrinkled nuclei and with increased lamina-associated chromatin, that cells cultured in stiff matrices displayed more accessible chromatin sites, which exhibited footprints of Sp1 binding, and that this transcription factor acts along with the histone deacetylases 3 and 8 to regulate the induction of stiffness-mediated tumorigenicity. Just as cell culture on soft environments or in them rather than on tissue-culture plastic better recapitulates the acinar morphology observed in mammary epithelium in vivo, mammary epithelial cells cultured on soft microenvironments or in them also more closely replicate the in vivo chromatin state. Our results emphasize the importance of culture conditions for epigenomic studies, and reveal that chromatin state is a critical mediator of mechanotransduction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41551-019-0420-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899165PMC
December 2019

Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling.

Nat Mater 2017 12 30;16(12):1233-1242. Epub 2017 Oct 30.

Department of Materials Science & Engineering, Stanford University, Stanford, California 94305, USA.

Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from ∼0.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell-cell contact and promote β-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmat5020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708569PMC
December 2017

Extracellular Matrix Stiffening Induces a Malignant Phenotypic Transition in Breast Epithelial Cells.

Cell Mol Bioeng 2017 Feb 19;10(1):114-123. Epub 2016 Oct 19.

1Department of Biomedical Engineering, The University of Texas at Austin, Austin, USA.

Tumors are much stiffer than healthy tissue, and progressively stiffen as the cancer develops. Tumor stiffening is largely the result of extracellular matrix (ECM) remodeling, for example, deposition and crosslinking of collagen I. Well established models have demonstrated the influence of the microenvironment in regulating tissue homeostasis, with matrix stiffness being a particularly influential mediator. Non-malignant MCF10A mammary epithelial cells (MECs) lose their epithelial characteristics and become invasive when cultured in stiff microenvironments, leading to the hypothesis that tumor stiffening could contribute directly to disease progression. However, previous studies demonstrating MCF10A invasion have been performed in gels with constant mechanical properties, unlike the dynamically stiffening tumor microenvironment. Here, we employ a temporally stiffening hydrogel platform to demonstrate that matrix stiffening induces invasion from and proliferation in MCF10A mammary acini. After allowing MCF10A acini to form in soft hydrogels for 14 days, the gels were stiffened to the level of a malignant tumor, giving rise to a proliferative and invasive phenotype. Cells were observed to collectively migrate away from mammary acini while maintaining cell-cell contacts. Small molecule inhibition of PI3K and Rac1 pathways was sufficient to significantly reduce the number and size of invasive acini after stiffening. Our results demonstrate that temporal matrix stiffening can induce invasion from mammary acini and supports the notion that tumor stiffening could be implicated in disease progression and metastasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12195-016-0468-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816676PMC
February 2017

Fibrin-based stem cell containing scaffold improves the dynamics of burn wound healing.

Wound Repair Regen 2016 Sep 13;24(5):810-819. Epub 2016 Sep 13.

The University of Texas at Austin, Austin, Texas.

For severe burn injuries, successful medical intervention is accomplished by rapidly and safely providing physical barriers that can cover damaged skin tissues, thereby preventing critical danger of extensive bleeding and infection. Despite availability of a large assortment of wound coverage options, the etiology of wound healing is rather complex leading to significant defects in skin repair. The use of cell-mediated treatment approaches in combination with bioengineered wound coverage constructs may provide the missing tool to improve wound healing outcomes. In this study, we have used an engineered 3D PEGylated fibrin (P-fibrin) gel as a scaffold for adipose derived stem cells (ASCs) delivery into the burn injury model. We were able to confirm the presence of ASCs in the wound site two weeks after the initial injury. Delivery of ASCs-containing gels was associated with improved vascularization of the injured area at early time points accompanied by an increased abundance of mannose receptor expressing cells. Moreover, the application of P-fibrin biomaterial exhibited positive effects on early mononuclear cell recruitment and granulation tissue formation without negatively affecting wound closure kinetics or extent of connective tissue deposition. Collectively, our data support the feasibility of using P-fibrin gels in wound dressing applications requiring controlled delivery of viable cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/wrr.12459DOI Listing
September 2016

Dynamic phototuning of 3D hydrogel stiffness.

Proc Natl Acad Sci U S A 2015 Feb 2;112(7):1953-8. Epub 2015 Feb 2.

Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712

Hydrogels are widely used as in vitro culture models to mimic 3D cellular microenvironments. The stiffness of the extracellular matrix is known to influence cell phenotype, inspiring work toward unraveling the role of stiffness on cell behavior using hydrogels. However, in many biological processes such as embryonic development, wound healing, and tumorigenesis, the microenvironment is highly dynamic, leading to changes in matrix stiffness over a broad range of timescales. To recapitulate dynamic microenvironments, a hydrogel with temporally tunable stiffness is needed. Here, we present a system in which alginate gel stiffness can be temporally modulated by light-triggered release of calcium or a chelator from liposomes. Others have shown softening via photodegradation or stiffening via secondary cross-linking; however, our system is capable of both dynamic stiffening and softening. Dynamic modulation of stiffness can be induced at least 14 d after gelation and can be spatially controlled to produce gradients and patterns. We use this system to investigate the regulation of fibroblast morphology by stiffness in both nondegradable gels and gels with degradable elements. Interestingly, stiffening inhibits fibroblast spreading through either mesenchymal or amoeboid migration modes. We demonstrate this technology can be translated in vivo by using deeply penetrating near-infrared light for transdermal stiffness modulation, enabling external control of gel stiffness. Temporal modulation of hydrogel stiffness is a powerful tool that will enable investigation of the role that dynamic microenvironments play in biological processes both in vitro and in well-controlled in vivo experiments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1421897112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343123PMC
February 2015

Mesenchymal stem cell response to TGF-β1 in both 2D and 3D environments.

Biomater Sci 2013 Aug 21;1(8):860-869. Epub 2013 May 21.

Laboratory for Cardiovascular Tissue Engineering, Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA.

Smooth muscle cells (SMC) are critical in stabilizing developing vascular networks, and transforming growth factor β1 (TGF-β1) has been shown to promote SMC differentiation from stem cells. Previously, our lab has developed a chemically modified fibrin-based hydrogel that induces endothelial cell (EC) phenotype and network formation from human mesenchymal stem cells (hMSCs) without exogenous cytokines. Additionally, we have shown that this hydrogel system is capable of releasing growth factors in a controlled manner. In the present work, the effects of TGF-β1 on hMSCs in both monolayer and fibrin-based gel culture systems were demonstrated. The objective was to enhance SMC properties through TGF-β1 signaling for vessel stability while maintaining EC gene expression and morphology. Proliferation was decreased with higher TGF-β1 concentration in both monolayer and 3D gel cultures. EC genes were predominantly downregulated in the presence of TGF-β1 in monolayer cultures, while SMC genes were generally upregulated. In fibrin-based gels, several SMC genes were significantly upregulated at high concentrations of TGF-β1. Even at elevated TGF-β1 concentrations, no significant differences were seen in EC genes for hMSCs in gels compared to controls. Network formation and growth occurred in PEGylated fibrin gels loaded with TGF-β1 and were not significantly different from gels without loaded growth factor. Additionally, production of smooth muscle α-actin (SMA) was significantly increased in gels loaded with TGF-β1. These results demonstrate a simultaneous response of hMSCs to both the 3D biomatrix and cytokine signaling cues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c3bm60057bDOI Listing
August 2013

Multifunctional nanoscale strategies for enhancing and monitoring blood vessel regeneration.

Nano Today 2012 Dec 17;7(6):514-531. Epub 2012 Nov 17.

Department of Biomedical Engineering, The University of Texas at Austin, 1 University Station, C0800, Austin, TX 78712-0238, USA.

Nanomedicine has great potential in biomedical applications, and specifically in regenerative medicine and vascular tissue engineering. Designing nanometer-sized therapeutic and diagnostic devices for tissue engineering applications is critical because cells experience and respond to stimuli on this spatial scale. For example, nanoscaffolds, including nanoscalestructured or nanoscale surface-modified vascular scaffolds, can influence cell alignment, adhesion, and differentiation to promote better endothelization. Furthermore, nanoscale contrast agents can be extended to the field of biomedical imaging to monitor and track stem cells to better understand the process of neovascularization. In addition, nanoscale systems capable of delivering biomolecules ( peptides and angiogenic genes/proteins) can influence cell behavior, function, and phenotype to promote blood vessel regeneration. This review will focus on nanomedicine and nanoscale strategies applied to vascular tissue engineering. In particular, some of the latest research and potential applications pertaining to nanoscaffolds, biomedical imaging and cell tracking using nanoscale contrast agents, and nanodelivery systems of bioactive molecules applied to blood vessel regeneration will be discussed. In addition, the overlap between these three areas and their synergistic effects will be examined as related to vascular tissue engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nantod.2012.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630157PMC
December 2012

A PEGylated fibrin-based wound dressing with antimicrobial and angiogenic activity.

Acta Biomater 2011 Jul 13;7(7):2787-96. Epub 2011 Apr 13.

United States Army Institute of Surgical Research, Fort Sam Houston, TX 78234, USA.

Wounds sustained under battlefield conditions are considered to be contaminated and their initial treatment should focus on decreasing this contamination and thus reducing the possibility of infection. The early and aggressive administration of antimicrobial treatment starting with intervention on the battlefield has resulted in improved patient outcomes and is considered the standard of care. Chitosan microspheres (CSM) loaded with silver sulfadiazine (SSD) were developed via a novel water-in-oil emulsion technique to address this problem. The SSD-loaded spheres were porous with needle-like structures (attributed to SSD) that were evenly distributed over the spheres. The average particle size of the SSD-CSM was 125-180 μm with 76.50 ± 2.8% drug entrapment. As a potential new wound dressing with angiogenic activity SSD-CSM particles were impregnated in polyethylene glycol (PEGylated) fibrin gels. In vitro drug release studies showed that a burst release of 27.02% in 6h was achieved, with controlled release for 72 h, with an equilibrium concentration of 27.7% (70 μg). SSD-CSM-PEGylated fibrin gels were able to exhibit microbicidal activity at 125 and 100 μg ml(-1) against Staphylococcus aureus and Pseudomonas aeruginosa, respectively. The in vitro vasculogenic activity of this composite dressing was shown by seeding adipose-derived stem cells (ASC) in SSD-CSM-PEGylated fibrin gels. The ASC spontaneously formed microvascular tube-like structures without the addition of any exogenous factors. This provides a method for the extended release of an antimicrobial drug in a matrix that may provide an excellent cellular environment for revascularization of infected wounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2011.04.003DOI Listing
July 2011