Cell-derived Extracellular Matrices Research Articles

Cornea-Specific Human Adipose Stem Cell-Derived Extracellular Matrix for Corneal Stroma Tissue Engineering.

Utilizing tissue-specific extracellular matrices (ECMs) is vital for replicating the composition of native tissues and developing biologically relevant biomaterials. Human- or animal-derived donor tissues and organs are the current gold standard for the source of these ECMs. To overcome the several limitations related to these ECM sources, including the highly limited availability of donor tissues, cell-derived ECM offers an alternative approach for engineering tissue-specific biomaterials, such as bioinks for three-dimensional (3D) bioprinting. 3D bioprinting is a state-of-the-art biofabrication technology that addresses the global need for donor tissues and organs. In fact, there is a vast global demand for human donor corneas that are used for treating corneal blindness, often resulting from damage in the corneal stromal microstructure. Human adipose tissue is one of the most abundant tissues and easy to access, and adipose tissue-derived stem cells (hASCs) are a highly advantageous cell type for tissue engineering. Furthermore, hASCs have already been studied in clinical trials for treating corneal stromal pathologies. In this study, a corneal stroma-specific ECM was engineered without the need for donor corneas by differentiating hASCs toward corneal stromal keratocytes (hASC-CSKs). Furthermore, this ECM was utilized as a component for corneal stroma-specific bioink where hASC-CSKs were printed to produce corneal stroma structures. This cost-effective approach combined with a clinically relevant cell type provides valuable information on developing more sustainable tissue-specific solutions and advances the field of corneal tissue engineering.

Improving Biological Functions of Three-Dimensional Printed Ti2448 Scaffolds by Decoration with Polydopamine and Extracellular Matrices.

Extracellular matrices (ECMs) provide important cues for cell proliferation and differentiation in the complex environment, which show a significant influence on cell functions. Herein, cell-derived ECMs were deposited on the polydopamine (PDA)-decorated porous Ti-24Nb-4Zr-8Sn (Ti2448) scaffolds fabricated by the electron beam melting method in order to improve biological functions. The influence of PDA-ECM coatings on cell functions was further investigated. The results demonstrated that the PDA-ECM coating facilitated adhesion, proliferation, and migration of MC3T3-E1 cells on Ti2448 scaffolds. Moreover, Ti2448-PDA-ECM scaffolds promoted osteogenesis differentiation of cells indicated by greater alkaline phosphatase activity and further mineralization, compared to the plain Ti2448 group. Meanwhile, Ti2448-PDA-ECM scaffolds enhanced bone growth after implantation for one month in rabbit femoral bone defects. Our findings suggest that the bioinspired PDA-ECM coating can be implemented on the porous Ti2448 scaffolds, which significantly improve the biological functions of orthopedic implants.

Cell-Derived Extracellular Matrix for Tissue Engineering and Regenerative Medicine.

Cell-derived extracellular matrices (CD-ECMs) captured increasing attention since the first studies in the 1980s. The biological resemblance of CD-ECMs to their in vivo counterparts and natural complexity provide them with a prevailing bioactivity. CD-ECMs offer the opportunity to produce microenvironments with costumizable biological and biophysical properties in a controlled setting. As a result, CD-ECMs can improve cellular functions such as stemness or be employed as a platform to study cellular niches in health and disease. Either on their own or integrated with other materials, CD-ECMs can also be utilized as biomaterials to engineer tissues de novo or facilitate endogenous healing and regeneration. This review provides a brief overview over the methodologies used to facilitate CD-ECM deposition and manufacturing. It explores the versatile uses of CD-ECM in fundamental research and therapeutic approaches, while highlighting innovative strategies. Furthermore, current challenges are identified and it is accentuated that advancements in methodologies, as well as innovative interdisciplinary approaches are needed to take CD-ECM-based research to the next level.

Osteogenic-differentiated mesenchymal stem cell-secreted extracellular matrix as a bone morphogenetic protein-2 delivery system for ectopic bone formation

While human bone morphogenetic protein-2 (BMP-2) is a promising growth factor for bone regeneration, a major challenge in biomedical applications is finding an optimal carrier for its delivery at the site of injury. Because of their natural affinities for growth factors (including BMP-2) as well as their role in instructing cell function, cultured cell-derived extracellular matrices (ECM) are of special interest. We hereby hypothesized that a “bony matrix” containing mineralized, osteogenic ECM is a potential efficacious carrier of BMP-2 for promoting bone formation and, therefore, compared the efficacy of the decellularized ECM derived from osteogenic-differentiated human mesenchymal stem cells (hMSCs) to the one obtained from ECM from undifferentiated hMSCs. Our results provided evidence that both ECMs can bind BMP-2 and promote bone formation when implanted ectopically in mice. The osteoinductive potential of BMP-2, however, was greater when loaded within an osteogenic MSC-derived ECM; this outcome was correlated with higher sequestration capacity of BMP-2 over time in vivo. Interestingly, although the BMP-2 mainly bound onto the mineral crystals contained within the osteogenic MSC derived-ECM, these mineral components were not involved in the observed higher osteoinductivity, suggesting that the organic components were the critical components for the matrix efficacy as BMP-2 carrier.

Synergistic Effect of Cell-Derived Extracellular Matrices and Topography on Osteogenesis of Mesenchymal Stem Cells.

Cell-derived matrices (CDMs) are an interesting alternative to conventional sources of extracellular matrices (ECMs) as CDMs mimic the natural ECM composition better and are therefore attractive as a scaffolding material for regulating the functions of stem cells. Previous research on stem cell differentiation has demonstrated that both surface topography and CDMs have a significant influence. However, not much focus has been devoted to elucidating possible synergistic effects of CDMs and topography on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). In this study, polydimethylsiloxane (PDMS)-based anisotropic topographies (wrinkles) with various topography dimensions were prepared and subsequently combined with native ECMs produced by human fibroblasts that remained on the surface topography after decellularization. The synergistic effect of CDMs combined with topography on osteogenic differentiation of hBM-MSCs was investigated. The results showed that substrates with specific topography dimensions, coated with aligned CDMs, dramatically enhanced the capacity of osteogenesis as investigated using immunofluorescence staining for identifying osteopontin (OPN) and mineralization. Furthermore, the hBM-MSCs on the substrates decorated with CDMs exhibited a higher percentage of (Yes-associated protein) YAP inside the nucleus, stronger cell contractility, and greater formation of focal adhesions, illustrating that enhanced osteogenesis is partly mediated by cellular tension and mechanotransduction following the YAP pathway. Taken together, our findings highlight the importance of ECMs mediating the osteogenic differentiation of stem cells, and the combination of CDMs and topography will be a powerful approach for material-driven osteogenesis.

Engineering Stem Cell-Derived Extracellular Matrices: Decellularization, Characterization, and Biological Function.

Stem cells, including mesenchymal stem cells and pluripotent stem cells, have attracted considerable attention in tissue engineering and regenerative medicine primarily because of their unique ability in self-renewal and multilineage differentiation. However, stem cells also have important secretory functions that form a specialized in vivo microenvironment and direct tissue development and regeneration. Extracellular matrices (ECMs) derived from stem cells retain the functional properties of their native environment and exhibit unique signaling that mediates stem cell self-renewal and lineage commitment. Stem cell-derived ECMs (scECMs) also have tunable properties corresponding to their developmental stages, suggesting that their lineage- and developmental specificity can be engineered for a wide range of applications. Hence, there is a growing interest in reconstructing stem cell microenvironment through decellularization and obtaining decellularized matrices that exhibit unique biological properties. This article summarizes recent advances in the use and understanding of scECMs. Moreover, future directions to extend the spectrum of applications of stem-derived ECMs in tissue engineering by comprehensively elucidating and engineering their regulatory function is highlighted. Impact statement Stem cells bear unique potency for multilineage differentiation as well as the capacity to secrete a vast amount of regulatory molecules. At different developmental stages, the extracellular matrices (ECMs) secreted by stem cells regulate their microenvironment and direct tissue development. The decellularization of stem cells effectively preserves ECM functional properties and can provide suitable templates to regulate stem cell fate decision, which can hardly be reproduced using single ECM proteins or synthetic scaffolds. This review highlights the unique regulatory functions of stem cell-derived ECMs, which can serve as novel sources of highly bioactive materials for tissue engineering and cell therapy.

Decellularized hASCs-derived matrices as biomaterials for 3D in vitro approaches.

Tissue regeneration strategies have been greatly evolving in the last years due to the use of more realistic approaches. These approaches rely in the use of biomaterials for the development of three dimensional (3D) structures that emulate the in vivo microenvironment of different tissues. Recently, extracellular matrices (ECM) secreted by cells have been caught a great deal of attention as an attractive biomaterial for the development 3D structures. In fact, different cells and/or different cellular culture conditions gave rise to different ECM's compositions, which can be used for the development of more physiologically relevant 3D structures. Nevertheless, the recovery of cell-derived ECM requires the use of a proper decellularization protocol. Herein, we report a decellularization protocol to recover the ECM produced by human adipose derived stem cells. This protocol comprises multiple steps (chemical, physical or enzymatic) which are described here in more detail. Furthermore, it is describe the methods that have been used to evaluate the effectiveness of this decellularization protocol. Overall, this protocol enables the production of hASCs-derived matrices that can be further used for the production of more physiologically relevant 3D in vitro models for tissue regeneration strategies.

Engineering clinically-relevant human fibroblastic cell-derived extracellular matrices.

Three-dimensional (3D) culturing models, replicating in vivo tissue microenvironments that incorporate native extracellular matrix (ECM), have revolutionized the cell biology field. Fibroblastic cells generate lattices of interstitial ECM proteins. Cell interactions with ECMs and with molecules sequestered/stored within these are crucial for tissue development and homeostasis maintenance. Hence, ECMs provide cells with biochemical and biomechanical cues to support and locally control cell function. Further, dynamic changes in ECMs, and in cell-ECM interactions, partake in growth, development, and temporary occurrences such as acute wound healing. Notably, dysregulation in ECMs and fibroblasts could be important triggers and modulators of pathological events such as developmental defects, and diseases associated with fibrosis and chronic inflammation such as cancer. Studying the type of fibroblastic cells producing these matrices and how alterations to these cells enable changes in ECMs are of paramount importance. This chapter provides a step-by-step method for producing multilayered (e.g., 3D) fibroblastic cell-derived matrices (fCDM). Methods also include means to assess ECM topography and other cellular traits, indicative of fibroblastic functional statuses, like naïve/normal vs. inflammatory and/or myofibroblastic. For these, protocols include indications for isolating normal and diseased fibroblasts (i.e., cancer-associated fibroblasts known as CAFs). Protocols also include means for conducting microscopy assessments, querying whether fibroblasts present with fCDM-dependent normal or CAF phenotypes. These are supported by discrete semi-quantitative digital imaging analyses, providing some imaging processing advice. Additionally, protocols include descriptions for effective fCDM decellularization, which renders cellular debris-free patho/physiological in vivo-like scaffolds, suitable as 3D substrates for subsequent cell culturing.

Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods.

Azide-bearing cell-derived extracellular matrices (“clickECMs”) have emerged as a highly exciting new class of biomaterials. They conserve substantial characteristics of the natural extracellular matrix (ECM) and offer simultaneously small abiotic functional groups that enable bioorthogonal bioconjugation reactions. Despite their attractiveness, investigation of their biomolecular composition is very challenging due to the insoluble and highly complex nature of cell-derived matrices (CDMs). Yet, thorough qualitative and quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specific biomolecules is essential for consistent advancement of CDMs and the understanding of the prospective functions of the developed biomaterial. In this study, we evaluated frequently used methods for the analysis of complex CDMs. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and (immune)histochemical staining methods in combination with several microscopic techniques were found to be highly eligible. Commercially available colorimetric protein assays turned out to deliver inaccurate information on CDMs. In contrast, we determined the nitrogen content of CDMs by elementary analysis and converted it into total protein content using conversion factors which were calculated from matching amino acid compositions. The amount of insoluble collagens was assessed based on the hydroxyproline content. The Sircol™ assay was identified as a suitable method to quantify soluble collagens while the Blyscan™ assay was found to be well-suited for the quantification of sulphated glycosaminoglycans (sGAGs). Eventually, we propose a series of suitable methods to reliably characterise the biomolecular composition of fibroblast-derived clickECM.

Glycosaminoglycan disaccharide compositional analysis of cell-derived extracellular matrices using liquid chromatography-tandem mass spectrometry.

Cell-derived extracellular matrices have emerged as promising scaffolds for tissue engineering (TE) strategies due to their ability to create a biomimetic microenvironment providing biochemical and physical cues to cells, without the limitations of availability and potential pathogen transmission associated with tissue-derived extracellular matrix (ECM) scaffolds. Glycosaminoglycans (GAGs) are important components of ECM with a crucial role in the maintenance of the mechanical properties of the tissue and as signaling regulators of several cellular processes, such as cell adhesion, growth and differentiation. However, despite their relevance to the field of TE, little information is available on the GAG composition of cell-derived ECM, mainly due to the lack of appropriate quantitative tools to determine different GAG and disaccharide subtypes in complex biological samples. In this chapter, we describe a highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to characterize decellularized cell-derived ECM generated in vitro in terms of their GAG and disaccharide composition.

Extracellular matrix from decellularized mesenchymal stem cells improves cardiac gene expressions and oxidative resistance in cardiac C-kit cells.

ObjectiveMyocardial infarction remains the number one killer disease worldwide. Cellular therapy using cardiac c-kit cells (CCs) are capable of regenerating injured heart. Previous studies showed mesenchymal stem cell-derived (MSC) extracellular matrices can provide structural support and are capable of regulating stem cell functions and differentiation. This study aimed to evaluate the effects of human MSC-derived matrices for CC growth and differentiation.MethodsHuman Wharton's Jelly-derived MSCs were cultured in ascorbic acid supplemented medium for 14 days prior to decellularisation using two methods. 1% SDS/Triton X-100 (ST) or 20 mM ammonia/Triton X-100 (AT). CCs isolated from 4-week-old C57/BL6N mice were cultured on the decellularised MSC matrices, and induced to differentiate into cardiomyocytes in cardiogenic medium for 21 days. Cardiac differentiation was assessed by immunocytochemistry and qPCR. All data were analysed using ANOVA.ResultsIn vitro decellularisation using ST method caused matrix delamination from the wells. In contrast, decellularisation using AT improved the matrix retention up to 30% (p < 0.05). This effect was further enhanced when MSCs were cultured in cardiogenic medium, with a matrix retention rate up to 90%. CCs cultured on cardiogenic MSC matrix (ECMcardio), however, did not significantly improve its proliferation after 3 days (p < 0.05), but the viability of CCs was augmented to 67.2 ± 0.7% after 24-h exposure to H2O2 stress as compared to 42.9 ± 0.5% in control CCs (p < 0.05). Furthermore, CCs cultured on cardiogenic MSC matrices showed 1.7-fold up-regulation in cardiac troponin I (cTnI) gene expression after 21 days (p < 0.05).ConclusionHighest matrix retention can be obtained by decellularization using Ammonia/Triton-100 in 2-D culture. ECMcardio could rescue CCs from exogenous hydrogen peroxide and further upregulated the cardiac gene expressions, offering an alternate in vitro priming strategy to precondition CCs which could potentially enhance its survival and function after in vivo transplantation.

Compositional and structural analysis of glycosaminoglycans in cell-derived extracellular matrices.

The extracellular matrix (ECM) is a highly dynamic and complex meshwork of proteins and glycosaminoglycans (GAGs) with a crucial role in tissue homeostasis and organization not only by defining tissue architecture and mechanical properties, but also by providing chemical cues that regulate major biological processes. GAGs are associated with important physiological functions, acting as modulators of signaling pathways regulating several cellular processes such as cell growth and differentiation. Recently, in vitro fabricated cell-derived ECM have emerged as promising materials for regenerative medicine due to their ability of better recapitulate the native ECM-like composition and structure, without the limitations of availability and pathogen transfer risks of tissue-derived ECM scaffolds. However, little is known about the molecular and more specifically, GAG composition of these cell-derived ECM. In this study, three different cell-derived ECM were produced in vitro and characterized in terms of their GAG content, composition and sulfation patterns using a highly sensitive liquid chromatography-tandem mass spectrometry technique. Distinct GAG compositions and disaccharide sulfation patterns were verified for the different cell-derived ECM. Additionally, the effect of decellularization method on the GAG and disaccharide relative composition was also assessed. In summary, the method presented here offers a novel approach to determine the GAG composition of cell-derived ECM, which we believe is critical for a better understanding of ECM role in directing cellular responses and has the potential for generating important knowledge to use in the development of novel ECM-like biomaterials for tissue engineering applications.

Rigidity controls human desmoplastic matrix anisotropy to enable pancreatic cancer cell spread via extracellular signal-regulated kinase 2

It is predicted that pancreatic ductal adenocarcinoma (PDAC) will become the second most lethal cancer in the US by 2030. PDAC includes a fibrous-like stroma, desmoplasia, encompassing most of the tumor mass, which is produced by cancer-associated fibroblasts (CAFs) and includes their cell-derived extracellular matrices (CDMs). Since elimination of desmoplasia has proven detrimental to patients, CDM reprogramming, as opposed to stromal ablation, is therapeutically desirable. Hence, efforts are being made to harness desmoplasia's anti-tumor functions. We conducted biomechanical manipulations, using variations of pathological and physiological substrates in vitro, to culture patient-harvested CAFs and generate CDMs that restrict PDAC growth and spread. We posited that extrinsic modulation of the environment, via substrate rigidity, influences CAF's cell-intrinsic forces affecting CDM production. Substrates used were polyacrylamide gels of physiological (~1.5 kPa) or pathological (~7 kPa) stiffnesses. Results showed that physiological substrates influenced CAFs to generate CDMs similar to normal/control fibroblasts. We found CDMs to be softer than the corresponding underlying substrates, and CDM fiber anisotropy (i.e., alignment) to be biphasic and informed via substrate-imparted morphological CAF aspect ratios. The biphasic nature of CDM fiber anisotropy was mathematically modeled and proposed a correlation between CAF aspect ratios and CDM alignment; regulated by extrinsic and intrinsic forces to conserve minimal free energy. Biomechanical manipulation of CDMs, generated on physiologically soft substrates, leads to reduction in nuclear translocation of pERK1/2 in KRAS mutated pancreatic cells. ERK2 was found essential for CDM-regulated tumor cell spread. In vitro findings correlated with in vivo observations; nuclear pERK1/2 is significantly high in human PDAC samples. The study suggests that altering underlying substrates enable CAFs to remodel CDMs and restrict pancreatic cancer cell spread in an ERK2 dependent manner.

Enhancement of collagen deposition and cross-linking by coupling lysyl oxidase with bone morphogenetic protein-1 and its application in tissue engineering

Cultured cell-derived extracellular matrices (ECM)-based biomaterials exploit the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, standard cell culture conditions are far from ideal given the fact that the diluted microenvironment does not favor the production of ECM components, a circumstance particularly relevant for collagen. An incomplete conversion of procollagen by C-proteinase/bone morphogenetic protein 1 (BMP1) has been proposed to severely limit in vitro collagen deposition. BMP1 also catalyzes the proteolytic activation of the precursor of the collagen cross-linking enzyme, lysyl oxidase (LOX) to yield the active form, suggesting a deficit in cross-linking activity under standard conditions. We hypothesized that the implementation of fibroblast cultures with LOX and BMP1 may be an effective way to increase collagen deposition. To test it, we have generated stable cell lines overexpressing LOX and BMP1 and studied the effect of supernatants enriched in LOX and BMP1 on collagen synthesis and deposition from fibroblasts. Herein, we demonstrate that the supplementation with LOX and BMP1 strongly increased the deposition of collagen onto the insoluble matrix at the expense of the soluble fraction in the extracellular medium. Using decellularization protocols, we also show that fibroblast-derived matrices regulate adipogenic and osteogenic differentiation of human mesenchymal stem cells (MSC), and this effect was modulated by LOX/BMP1. Collectively, these data demonstrate that we have developed a convenient protocol to enhance the capacity of in vitro cell cultures to deposit collagen in the ECM, representing this approach a promising technology for application in tissue engineering.

Genipin-crosslinked adipose stem cell derived extracellular matrix-nano graphene oxide composite sponge for skin tissue engineering.

Autologous skin grafts, which can cause donor site morbidity, are currently used to treat deep wounds. To improve the regeneration of poorly healing wounds, cell-derived extracellular matrix (ECM) scaffolds are garnering great research interest due to their associated lower risks of pathogen transfer and immune rejection. However, the mechanical properties of cell-derived ECM scaffolds are inferior when compared to those of tissue-derived ECM scaffolds. To overcome this drawback, different amounts (10, 20, 50, and 100 μg mL-1) of graphene oxide (GO) and genipin (1% w/v) were applied to adipose stem cell (ASC)-derived ECM sponges. There are still only a few studies employing cell-derived extracellular matrices as biomimetic scaffolds for biomedical applications. The aim of our study was to develop biocompatible, biodegradable, low immunogenic, and genipin-crosslinked ASC-derived ECM sponges containing a suitable amount of GO for skin-tissue engineering. Sponges were fabricated using cultures of ASCs, cell sheets, and decellularization of an ASC cell sheet, freeze-thawing, and crosslinking in a sequential manner. Scanning electron microscopic analyses of the sponges demonstrated a highly porous microstructure with a pore size of 71.22 ± 19.52 μm. The in vitro degradation rate was found to be significantly higher in the non-crosslinked ECM sponges and pure ECM sponges than in the genipin-crosslinked ECM sponges. During an in vivo study, we investigated the material feasibilities and degradability of the constructed ECM sponges as a suitable skin tissue-engineering scaffold in a xenogenic animal (rat) model for 4 weeks. After subcutaneous implantation, the ECM sponges containing a medium amount of GO showed appropriate biodegradation with a lower inflammatory reaction. Hence, the fabricated ECM sponges might be a suitable xenogenous skin substitute for full-thickness skin defects and in other future soft-tissue engineering applications, such as healing partial tears of the anterior cruciate ligament.

Comprehensive proteomic characterization of stem cell-derived extracellular matrices

In the stem-cell niche, the extracellular matrix (ECM) serves as a structural support that additionally provides stem cells with signals that contribute to the regulation of stem-cell function, via reciprocal interactions between cells and components of the ECM. Recently, cell-derived ECMs have emerged as invitro cell culture substrates to better recapitulate the native stem-cell microenvironment outside the body. Significant changes in cell number, morphology and function have been observed when mesenchymal stem cells (MSC) were cultured on ECM substrates as compared to standard tissue-culture polystyrene (TCPS). As select ECM components are known to regulate specific stem-cell functions, a robust characterization of cell-derived ECM proteomic composition is critical to better comprehend the role of the ECM in directing cellular processes. Here, we characterized and compared the protein composition of ECM produced invitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts from different donors, employing quantitative proteomic methods. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential survival and gene-expression profiles among the cell types and as compared to TCPS, indicating that the cell-derived ECMs influence each cell type in a different manner. This general approach to understanding the protein composition of different tissue-specific and cell-derived ECM will inform the rational design of defined systems and biomaterials that recapitulate critical ECM signals for stem-cell culture and tissue engineering.

Natural Extracellular Matrix for Cellular and Tissue Biomanufacturing.

Natural extracellular matrices (ECM) derived from native tissues or cultured cells are extensively employed to fabricate biocompatible scaffolds or living tissue constructs for the application in cellular and tissue engineering. The composition and structure of ECM are not only heterogeneous, but also tissue or cell specific. Recapitulating the unique cell or tissue niche, ECM-based products are promising to quickly integrate with host tissues and accelerate restoration of tissue function. A variety of natural ECM-based scaffolds and tissue constructs have been biomanufactured using different approaches. Native tissue derived ECM is typically grounded into powders that can be further processed into hybrid composites in the form of hydrogels, foams, nanofibers, and 3D-printed complex constructs. Cell-derived ECM follows different biomanufacturing methods. Usually, cells are seeded on a scaffold to deposit ECM resulting in ECM-ornamented materials. The employment of resolvable scaffolds and cell sheet engineering technique enables production of complex 3D constructs exclusively composed of ECM with/without cells. In order to enhance mechanical strength, in vivo stability, and biological performance of ECM-based products, cross-linking reagents or bioactive factors are often used for modification. The major focus of this article is to provide an overview of current biomanufacturing approaches that utilize either native tissue or cell-derived natural ECM in the field of cellular and tissue engineering. Furthermore, the existing challenges for translational application of ECM-based products and the potential resolutions are discussed.

Driving iPSC-derived progenitor fate using in vitro synthetized kidney extracellular matrices assembled under macromolecular crowding

Event Abstract Back to Event Driving iPSC-derived progenitor fate using in vitro synthetized kidney extracellular matrices assembled under macromolecular crowding Valentina Magno1, Bella Roßbach2, Krithika Hariharan2, Jens Friedrichs1, Andreas Kurtz2 and Carsten Werner1, 3 1 Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Germany 2 Charité – Universitätsmedizin Berlin, Berlin-Brandenburg Center for Regenerative Therapies, Germany 3 Center for Regenerative Therapies Dresden, Technische Universität Dresden, Germany Introduction: Cell-derived extracellular matrices (ECMs) have shown in the latest years to have broad application in the field of tissue engineering and regenerative medicine. The recent introduction of macromolecular crowding (MMC) for the modulation of in vitro ECM production allowed, furthermore, to better mimic physiological microenvironments[1]. In this work, we aim to explore the effect of MMC on the secretion of ECM by primary mouse mesangial cells and to study the impact of decellularized ECMs produced under standard and MMC conditions on the growth and differentiation of human induced pluripotent stem cell (iPSC)-derived intermediate mesoderm (IM) progenitors[2]. Methods: Covalently attached mesangial cell-derived ECMs were produced on a maleic anhydride co-polymer platform[3]. The ECM secretion was carried out for 8 days either under standard (Std) or MMC culture conditions by supplementing the medium with the synthetic polysaccharide Ficoll. During the ECM deposition phase, cell viability was assessed using a PrestoBlue assay. Cell layers were decellularised using an hypotonic solution and DNA remnants were removed by DNase treatment. The impact of MMC on ECM composition and morphology was investigated by colorimetric assays, immunohistochemistry, atomic force microscopy and scanning electron microscopy. IM progenitors were seeded in parallel on the mesangial cell-derived ECMs as well as on Geltrex and collagen IV as control substrates, with or without the supplementation of the ROCK inhibitor Y-27632. Adhesion and proliferation of IM progenitors were analyzed respectively using a Crystal Violet and a PrestoBlue assay. Furthermore, the expression of relevant kidney markers was examined via real time PCR. Results and Discussion: PrestoBlue assay showed that MMC did not affect the viability of the mesangial cells during ECM deposition. MMC was found to influence the composition, the mechanical properties and the supramolecular assembly of the mesangial cell-derived ECM. IM progenitors showed significantly higher cell viability and adhesion on both types of ECM (Std and MMC) compared to Geltrex and collagen IV. Particularly, in presence of the ROCK inhibitor, a significantly higher adhesion was found on MMC-ECM compared to both Std-ECM and control substrates. Higher proliferation rates were observed on both types of ECM vs. the controls, with the MMC-ECM outperforming Std-ECM. A significant increase in the expression of some mesangial markers and other glomerular markers was found for IM progenitors cultivated on MMC-ECM vs. Std-ECM, with even less mesangial-like characteristics found on the control substrates. Conclusion: Our results indicate that matrix based signals support the differentiation of the IM progenitors toward the mesangial lineage without the addition of soluble cues. In the future, the combination of cell-derived ECM and MMC may be utilized to study kidney progenitor differentiation and to develop bioengineered kidney tissues. A systematic study on different crowder categories and concentrations may also allow to control progenitor differentiation to obtain a reliable cell source for regenerative approaches. This work was supported by EU Marie Curie NephroTools FP7 Initial Training Network (ITN)

Abstract 5089: Desmoplastic stroma affects growth and invasion of progressively mutated human pancreatic cancer cells in vitro

Abstract Desmoplastic (desmo) stroma is a prognostic marker for pancreatic cancer, yet mechanisms behind its tumorigenic effects remain poorly understood. Tumor stroma, known to act permissive to naïve cells, may exacerbate tumor progression in mutated cells, whereas normal stroma acts restrictive to even aggressively mutated cells. The mechanistic details of these responses remain unclear. Previous studies suggested that a 3D microenvironment (i.e. Matrigel) is necessary for the manifestation of the metastatic phenotype in vitro in aggressively K-Ras mutated pancreatic cancer cells. Here we describe a new 3D in vitro model to study how human pancreatic stromal extracellular matrices (ECMs) interact with pancreatic epithelial cells, how these interactions activate specific integrins and downstream signaling pathways such as PI3K and ERK1/2, and how these signaling pathways may synergize with intrinsic mutational cues to enhance progression of pancreatic cancer. Our model is comprised of physiologically relevant desmo and normal 3D cell-derived matrices as well as polyacrylamide gel-based bioengineered ECMs. The influences of cell-derived and bioengineered ECMs were tested using a series of isogenic pancreatic ductal epithelial cells (PDEC) displaying different aggressive behavior. In preliminary studies, we established that physiological desmo ECMs are tumor permissive (i.e. induce growth and/or invasion) for all PDEC cells (especially kRas mutated cells) when compared with matched restrictive/normal ECMs. In addition, K-Ras cells cultured on stromal activated (i.e. desmo) myofibroblast-derived 3D ECMs resulted in enhanced ERK1/2 activity, similar to what had been previously observed with Matrigel based system. In order to discern distinct cellular and signal responses mediated by cell-derived ECM in the various PDEC cells, we independently modulated microenvironmental cues such as stiffness, topography, and biochemical composition in the bioengineered ECMs. Our initial data suggests that the various PDEC cells differentially respond to alterations in microenvironmental cues. We anticipate that our studies will lead to better understanding of ECM-PDEC interactions in more detail, and help in identification of novel biomarker for early detection or prevention of pancreatic cancer. Citation Format: Ruchi Malik, Tiffany Luong, Seda Karamil, Peter Lelkes, Edna Cukierman. Desmoplastic stroma affects growth and invasion of progressively mutated human pancreatic cancer cells in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5089. doi:10.1158/1538-7445.AM2015-5089

Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells

BackgroundChanges in fibronectin (Fn) matrix remodeling contribute to mammary tumor angiogenesis and are related to altered behavior of adipogenic stromal cells; yet, the underlying mechanisms remain unclear due in part to a lack of reductionist model systems that allow the inherent complexity of cell-derived extracellular matrices (ECMs) to be deciphered. In particular, breast cancer-associated adipogenic stromal cells not only enhance the composition, quantity, and rigidity of deposited Fn, but also partially unfold these matrices. However, the specific effect of Fn conformation on tumor angiogenesis is undefined. MethodsDecellularized matrices and a conducting polymer device consisting of poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) were used to examine the effect of Fn conformation on the behavior of 3T3-L1 preadipocytes. Changes in cell adhesion and proangiogenic capability were tested via cell counting and by quantification of vascular endothelial growth factor (VEGF) secretion, respectively. Integrin-blocking antibodies were utilized to examine varied integrin specificity as a potential mechanism. ResultsOur findings suggest that tumor-associated partial unfolding of Fn decreases adhesion while enhancing VEGF secretion by breast cancer-associated adipogenic precursor cells, and that altered integrin specificity may underlie these changes. Conclusions and general significanceThese results not only have important implications for our understanding of tumorigenesis, but also enhance knowledge of cell-ECM interactions that may be harnessed for other applications including advanced tissue engineering approaches. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.