Microenvironmental determinants of organized iPSC-cardiomyocyte tissues on synthetic fibrous matrices.
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ABSTRACT:
SUBMITTER: DePalma SJ
PROVIDER: S-EPMC7971708 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mechanical interactions between fibroblasts and their surrounding extracellular matrix (ECM) guide fundamental behaviors such as spreading, migration, and proliferation that underlie disease pathogenesis. The challenges of studying ECM mechanics in vivo have motivated the development of in vitro models of the fibrous ECM in which fibroblasts reside. Natural materials such as collagen hydrogels bear structural and biochemical resemblance to stromal ECM, but mechanistic studies in these settings are often confounded by cell-mediated material degradation and the lack of structural and mechanical tunability. Here, we established a new material system composed of electrospun dextran vinyl sulfone (DexVS) polymeric fibers. These fibrous matrices exhibit mechanical tunability at both the single fiber (80-340 MPa) and bulk matrix (0.77-11.03 kPa) level, as well as long-term stability in mechanical properties over a two-week period. Cell adhesion to these matrices can be either user-defined by functionalizing synthetic fibers with thiolated adhesive peptides or methacrylated heparin to sequester cell-derived ECM proteins. We utilized DexVS fibrous matrices to investigate the role of matrix mechanics on the activation of fibroblasts into myofibroblasts, a key step of the fibrotic progression. In contrast to previous findings with non-fibrous hydrogel substrates, we find that fibroblasts in soft and deformable matrices exhibit increased spreading, focal adhesion formation, proliferation, and myofibroblast activation as compared to cells on stiffer matrices with equivalent starting architecture. STATEMENT OF SIGNIFICANCE: Cellular mechanosensing of fibrillar extracellular matrices plays a critical role in homeostasis and disease progression in stromal connective tissue. Here, we established a new material system composed of electrospun dextran vinyl sulfone polymeric fibers. These matrices exhibit architectural, mechanical, and biochemical tunability to accurately model diverse tissue microenvironments found in the body. In contrast to previous observations with non-fibrous hydrogels, we find that fibroblasts in soft and deformable fibrous matrices exhibit increased spreading and focal adhesion formation as compared to those in stiffer matrices with equivalent architecture. We also investigated the role of matrix stiffness on myofibroblast activation, a critical step in the fibrotic cascade, and find that low stiffness matrices promote increased myofibroblast activation.
| S-EPMC7369643 | biostudies-literature
Project description:IntroductionConnective tissue repair and mechanosensing are tightly entwined in vivo and occur within a complex three-dimensional (3D), fibrous extracellular matrix (ECM). Typically driven by activated fibroblasts, wound repair involves well-defined steps of cell spreading, migration, proliferation, and fibrous ECM deposition. While the role of Rho GTPases in regulating these processes has been explored extensively in two-dimensional cell culture models, much less is known about their role in more physiologic, 3D environments.MethodsWe employed a 3D, fibrous and protease-sensitive hydrogel model of interstitial ECM to study the interplay between Rho GTPases and fibrous matrix cues in fibroblasts during wound healing.ResultsModulating fiber density within protease-sensitive hydrogels, we confirmed previous findings that heightened fiber density promotes fibroblast spreading and proliferation. The presence of matrix fibers furthermore corresponded to increased cell migration speeds and macroscopic hydrogel contraction arising from fibroblast generated forces. During fibroblast spreading, Rac1 and RhoA GTPase activity proved crucial for fiber-mediated cell spreading and contact guidance along matrix fibers, while Cdc42 was dispensable. In contrast, interplay between RhoA, Rac1, and Cdc42 contributed to fiber-mediated myofibroblast differentiation and matrix contraction over longer time scales.ConclusionThese observations may provide insights into tissue repair processes in vivo and motivate the incorporation of cell-adhesive fibers within synthetic hydrogels for material-guided wound repair strategies.Supplementary informationThe online version contains supplementary material available at 10.1007/s12195-021-00698-5.
| S-EPMC8548490 | biostudies-literature
Project description:Cellular structure and function are interdependent. To understand this relationship in beating heart cells, individual neonatal rat ventricular myocytes (NRVMs) were analyzed one and 3 days after plating when cultured on different stiffness (100, 400 kPa) and surface structures (flat or [Formula: see text] high, [Formula: see text] diameter, microposts spaced [Formula: see text] apart) manufactured from polydimethylsiloxane. Myofibril structure seen by immunohistochemistry was organized in three dimensions when NRVMs were attached to microposts. On day three, paxillin distribution near the post serving as cellular anchorage was quantified on both soft posts (12.04 % of total voxel count) and stiff posts (8.16 %). Living NRVMs were analyzed using line scans for sarcomeric shortening and shortening velocity, and traction force microscopy for surface stress and surface tension. One day after plating, NRVMs shortened more on soft posts ([Formula: see text] at [Formula: see text]) compared to either soft flat ([Formula: see text] at [Formula: see text]), stiff posts ([Formula: see text] at [Formula: see text]) or stiff flat ([Formula: see text] at [Formula: see text]). NRVMs have decreased shortening and shortening velocity on soft posts ([Formula: see text] at [Formula: see text]) compared to soft flat ([Formula: see text] at [Formula: see text]) substrates. The surface stress and surface tension increased over time for both soft post ([Formula: see text] and [Formula: see text] to [Formula: see text] and [Formula: see text]) and flat ([Formula: see text] and [Formula: see text] to [Formula: see text] and [Formula: see text]) substrates. Paxillin displacement during contraction on day three was significantly greater in NRVMs attached to soft posts [Formula: see text] compared to flat [Formula: see text] substrates. The volume and time creating four-dimensional data, interpreted by structural engineering theory, demonstrate subdomain structure is maintained by the counterbalance between the external load acting upon and the internal forces generated by the cardiomyocyte. These findings provide further insight into localized regulation of cellular mechanical function.
| S-EPMC4383742 | biostudies-literature
Project description:Although epithelial morphogenesis is tightly controlled by intrinsic genetic programs, the microenvironment in which epithelial cells proliferate and differentiate also contributes to the morphogenetic process. The roles of the physical microenvironment in epithelial morphogenesis, however, have not been well dissected. In this study, we assessed the impact of the microenvironment on epithelial cyst formation, which often marks the beginning or end step of morphogenesis of epithelial tissues and the pathological characteristic of some diseases. Previous studies have demonstrated that Madin-Darby canine kidney (MDCK) epithelial cells form cysts when grown in a three-dimensional (3D) extracellullar matrix (ECM) environment. We have now further demonstrated that the presence of ECM in the 3D scaffold is required for the formation of properly polarized cysts. Also, we have found that the full interface of epithelial cells with the ECM environment (in-3D) is not essential for cyst formation, since partial contact (on-3D) is sufficient to induce cystogenesis. In addition, we have defined the minimal ECM environment or the physical threshold for cystogenesis under the on-3D condition. Only above the threshold can the morphological cues from the ECM environment induce cyst formation. Moreover, cyst formation under the on-3D condition described in this study defines a novel and more feasible model to analyze in vitro morphogenesis. Finally, we have found that, during cystogenesis, MDCK cells generate basal microprotrusions and produce vesicle-like structures to the basal extracellular space, which are specific to and correlated with cyst formation. For the first time, we have systematically and quantitatively elucidated the microenvironmental determinants for epithelial cystogenesis.
| S-EPMC4141498 | biostudies-literature
Project description:Cell therapy is a potential novel treatment for cardiac regeneration and numerous studies have attempted to transplant cells to regenerate the myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported. This is likely to be the result of low cell retention and survival following transplantation. This study aimed to improve the delivery and engraftment of viable cells by using an injectable microcarrier that provides an implantable, biodegradable substrate for attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC). We describe the fabrication and characterisation of Thermally Induced Phase Separation (TIPS) microcarriers and their surface modification to enable iPSC-derived cardiomyocyte attachment in xeno-free conditions is described. The selected formulation resulted in iPSC attachment, expansion, and retention of pluripotent phenotype. Differentiation of iPSC into cardiomyocytes on the microcarriers is investigated in comparison with culture on 2D tissue culture plastic surfaces. Microcarrier culture is shown to support culture of a mature cardiomyocyte phenotype, be compatible with injectable delivery, and reduce anoikis. The findings from this study demonstrate that TIPS microcarriers provide a supporting matrix for culturing iPSC and iPSC-derived cardiomyocytes in vitro and are suitable as an injectable cell-substrate for cardiac regeneration.
| S-EPMC11348074 | biostudies-literature
Project description:Since the first work by Laurencin and colleagues on the development of polymeric electrospinning for biomedical purposes, the use of electrospinning technology has found broad applications in such areas of tissue regeneration and drug delivery. More recently, coaxial electrospinning has emerged as an important technique to develop scaffolds for regenerative engineering incorporated with drug(s). However, the addition of a softer core layer leads to a reduction in mechanical properties. Here, novel robust tripolymeric triaxially electrospun fibrous scaffolds were developed with a polycaprolactone (PCL) (core layer), a 50:50 poly (lactic-co-glycolic acid) (PLGA) (sheath layer) and a gelatin (intermediate layer) with a dual drug delivery capability was developed through modified electrospinning. A sharp increase in elastic modulus after the incorporation of PCL in the core of the triaxial fibers in comparison with uniaxial PLGA (50:50) and coaxial PLGA (50:50) (sheath)-gelatin (core) fibers was observed. Thermal analysis of the fibrous scaffolds revealed an interaction between the core-intermediate and sheath-intermediate layers of the triaxial fibers contributing to the higher tensile modulus. A simultaneous dual release of model small molecule Rhodamine B (RhB) and model protein Fluorescein isothiocynate (FITC) Bovine Serum Albumin (BSA) conjugate incorporated in the sheath and intermediate layers of triaxial fibers was achieved. The tripolymeric, triaxial electrospun systems were seen to be ideal for the support of mesenchymal stem cell growth, as shrinkage of fibers normally found with conventional electrospun systems was minimized. These tripolymeric triaxial electrospun fibers that are biomechanically competent, biocompatible, and capable of dual drug release are designed for regenerative engineering and drug delivery applications.
| S-EPMC6969175 | biostudies-literature
Project description:Most tissues in multicellular organisms are maintained by continuous cell renewal processes. However, high turnover of many cells implies a large number of error-prone cell divisions. Hierarchical organized tissue structures with stem cell driven cell differentiation provide one way to prevent the accumulation of mutations, because only few stem cells are long lived. We investigate the deterministic dynamics of cells in such a hierarchical multi compartment model, where each compartment represents a certain stage of cell differentiation. The dynamics of the interacting system is described by ordinary differential equations coupled across compartments. We present analytical solutions for these equations, calculate the corresponding extinction times and compare our results to individual based stochastic simulations. Our general compartment structure can be applied to different tissues, as for example hematopoiesis, the epidermis, or colonic crypts. The solutions provide a description of the average time development of stem cell and non stem cell driven mutants and can be used to illustrate general and specific features of the dynamics of mutant cells in such hierarchically structured populations. We illustrate one possible application of this approach by discussing the origin and dynamics of PIG-A mutant clones that are found in the bloodstream of virtually every healthy adult human. From this it is apparent, that not only the occurrence of a mutant but also the compartment of origin is of importance.
| S-EPMC3228763 | biostudies-literature
Project description:Metastasis causes more than 90% of cancer-related deaths and most prostate cancer (PCa) patients also die from metastasis. The 'metastatic cascade' is a complex biological process that encompasses tumor cell dissociation (from the primary tumor), local invasion, intravasation, transport in circulation, extravasation, colonization, and overt growth in end organs. It has become clear that successful metastasis not only involves many tumor cell-intrinsic properties but also depends on productive interactions between cancer cells and the tumor microenvironment. In this Review, we begin with a general summary on cancer metastasis and a specific discussion on PCa metastasis. We then discuss recent advances in our knowledge of the cellular determinants of PCa metastasis and the importance of tumor microenvironment, especially an immunosuppressive tumor microenvironment, in shaping metastatic propensities. We conclude with a presentation of current and future therapeutic options for patients with PCa metastasis, emphasizing the development of novel, mechanism-based combinatorial strategies for treating metastatic and castration-resistant PCa.
| S-EPMC5491097 | biostudies-literature
Project description:The extracellular matrix (ECM) represents a highly charged and hydrated network in which different cells in vertebrate tissues are embedded. Hydrogels as minimal ECM mimetics with a controlled chemistry offer the opportunity to vary material properties by varying the negative network charge. In this paper, a synthetic biology model of the ECM based on natural and highly negatively charged polyelectrolyte hyaluronic acid (HA) is characterized with specific emphasis on its charge-related bioactivity. Therefore, the thiol-Michael addition click reaction is used to produce HA hydrogels with defined network structure and charge density. The presented hydrogels show enzymatic degradability and cell attachment. These properties depend on both covalent and electrostatic interactions within the hydrogel network. Furthermore, no unspecific or specific attachment of proteins to the presented hydrogels is observed. In addition, these fundamental insights into charge-related ECM behavior and the influence of electrostatic properties could also lead to innovations in existing biomedical products.
| S-EPMC6017843 | biostudies-other
Project description:IntroductionIn the myocardium, rapid propagation of action potentials and subsequent calcium waves is critical for synchronizing the contraction of cardiac myocytes and maximizing cardiac output. In many pathological settings, diverse remodeling of the tissue microenvironment is correlated with arrhythmias and decreased cardiac output, but the precise impact of tissue remodeling on propagation is not completely understood. Our objective was to delineate how multiple features within the cardiac tissue microenvironment modulate propagation velocity.MethodsTo recapitulate diverse myocardial tissue microenvironments, we engineered substrates with tunable elasticity, patterning, composition, and topography using two formulations of polydimethylsiloxane (PDMS) micropatterned with fibronectin and gelatin hydrogels with flat or micromolded features. We cultured neonatal rat ventricular myocytes on these substrates and quantified cell density, tissue alignment, and cell shape. We used a fluorescent calcium indicator, high-speed microscopy, and newly-developed analysis software to record and quantify calcium wave propagation velocity (CPV).ResultsFor all substrates, tissue alignment and cell aspect ratio were higher in aligned compared to isotropic tissues. Isotropic CPV and longitudinal CPV were similar across conditions, but transverse CPV was lower on micromolded gelatin hydrogels compared to micropatterned soft and stiff PDMS. In aligned tissues, the anisotropy ratio of CPV (longitudinal CPV/transverse CPV) was lower on micropatterned soft PDMS compared to micropatterned stiff PDMS and micromolded gelatin hydrogels.ConclusionPropagation velocity in engineered cardiac tissues is sensitive to features in the tissue microenvironment, such as alignment, matrix elasticity, and matrix topography, which may underlie arrhythmias in conditions with pathological tissue remodeling.
| S-EPMC6816715 | biostudies-literature