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Biophysical cues synergize with biochemical cues to drive differentiation of pluripotent stem cells through specific phenotypic trajectory. Tools to manipulate the cell biophysical environment and identify the influence of specific environment perturbation in the presence of combinatorial inputs will be critical to control the development trajectory. Here we describe the procedure to perturb biophysical environment of pluripotent stem cells while maintaining them in 3D culture configuration. We also discuss a high-throughput platform for combinatorial perturbation of the cell microenvironment, and detail a statistical procedure to extract dominant environmental influences.

Biophysical cues synergize with biochemical cues to drive differentiation of pluripotent stem cells through specific phenotypic trajectory. Tools to manipulate the cell biophysical environment and identify the influence of specific environment perturbation in the presence of combinatorial inputs will be critical to control the development trajectory. Here we describe the procedure to perturb biophysical environment of pluripotent stem cells while maintaining them in 3D culture configuration. We also discuss a high-throughput platform for combinatorial perturbation of the cell microenvironment, and detail a statistical procedure to extract dominant environmental influences.

Exploiting gene delivery from the surfaces of bio-functionalized implants is a unique strategy to facilitate tissue regeneration and integration. However, it has been challenging, due to the difficulty for incorporation and delivey of sufficient payloads of plasmid DNA (pDNA) to cells from the implant surface allowing efficient gene expression. Herein, we describe a novel and simple coating approach using nanostructured silicate substituted calcium phosphate pDNA complexes termed as NanoSiCaPs, that preferentially adsorb onto titanium (Ti) substrates coated with poly (diallyldimethylammonium chloride) (PDADMAC). The Ti-polyelectrolyte NanoSiCaPs assemblies aptly called PNA, deliver pDNA to MC3T3-E1 cells (pre-osteoblast cell line), induce protein production, without eliciting any cytotoxicity, while simultaneously encouraging cell attachment on the substrate.

Human pluripotent stem cells (hPSCs) have enormous potential to alleviate cell needs for regenerative medicine, however these cells require expansion in cell colonies to maintain cell-cell contact, thus limiting the scalability needed to meet the demands of cell therapy. While the use of a Rho-associated protein kinase (ROCK) inhibitor will allow for culture of single cell hPSCs, typically only 50% of cells are recovered after dissociation. When hPSCs lose cell-cell contact through E-cadherin, dissociation induced apoptosis occurs. In this study, we hypothesized that the extracellular E-cadherin domain of hPSCs will bind to synthetic E-cadherin peptides presented on a hydrogel substrate, mimicking the required cell-cell contact and thereby retaining single-cell viability and clonogenicity. Hence, the objective of this study was to functionalize alginate hydrogels with synthetic peptides mimicking E-cadherin and evaluate peptide performance in promoting cell attachment, viability, maintaining pluripotency, and differentiation potential. We observed that alginate conjugated with synthetic E-cadherin peptides not only supported initial cell attachment with high viability, but also supported hPSC propagation and high fold expansion. hPSCs propagated on the peptide modified substrates maintained the hPSC characteristic pluripotency and differentiation potential, characterized by both spontaneous and directed differentiation. STATEMENT OF SIGNIFICANCE: Human pluripotent stem cells (hPSCs) have enormous potential to alleviate cell needs for regenerative medicine and cell therapy. However, scalable culture of hPSCs is challenged by its need for maintenance of cell-cell contact, dissociation of which triggers the apoptotic pathway. Hence hPSCs are commonly maintained as colonies over Matrigel coated culture plates. Furthermore, use of xenogenic and undefined Matrigel compromises the translational potential of hPSCs. In this work we have developed a completely defined substrate to enable adherent culture of hPSCs as single cells. This substrate prevents apoptosis of the single cells and allows significant fold expansion of hPSCs while maintaining pluripotency and differentiation potential. The developed substrate is expected to be a cost-effective and translatable alternative to Matrigel.

Pluripotent stem cells are promising source of cells for tissue engineering, regenerative medicine and drug discovery applications. The process of stem cell differentiation is regulated by multi-parametric cues from the surrounding microenvironment, one of the critical one being cell interaction with extracellular matrix (ECM). The ECM is a complex tissue-specific structure which is an important physiological regulator of stem cell function and fate. Recapitulating this native ECM microenvironment niche is best facilitated by decellularized tissue/organ derived ECM, which can faithfully reproduce the physiological environment with high fidelity to in vivo condition and promote tissue-specific cellular development and maturation. Recognizing the need for organ specific ECM in a 3D culture environment in driving phenotypic differentiation and maturation of hPSCs, we fabricated an ECM array platform using native-mimicry ECM from decellularized organs (namely pancreas, liver and heart), which allows cell-ECM interactions in both 2D and 3D configuration. The ECM array was integrated with rapid quantitative imaging for a systematic investigation of matrix protein profiles and sensitive measurement of cell-ECM interaction during hPSC differentiation. We tested our platform by elucidating the role of the three different organ-specific ECM in supporting induced pancreatic differentiation of hPSCs. While the focus of this report is on pancreatic differentiation, the developed platform is versatile to be applied to characterize any lineage specific differentiation.

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the in vitro reproduction of specific in vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to in vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel -facilitated platform ideal for islet organoid engineering.

Human embryonic stem cells (hESCs) are characterized by both their pluripotency and ability to self-renew, rendering them attractive for treatment of degenerative diseases. The cues necessary to induce hESC differentiation to a desired lineage can be categorized as either chemical or biophysical in nature. While chemical cues are used as primary inducers of differentiation, biophysical cues have been found to augment the process. In this regard, we have designed a novel chitosan nanoparticle augmented encapsulated alginate (CNPEA) platform which can not only augment, but induce differentiation of hESCs into the definitive endoderm (DE) lineage in the absence of specific soluble chemical inducers. These endoderm cells were comparable in phenotype with chemically driven DE cells and remained amenable to further maturation into pancreatic lineages. This study demonstrates the feasibility of carefully designed and tailored nanomaterials inducing differentiation, and moreover demonstrating the possibility of replacing growth factors by material cues.

Basement membranes (BMs) are thin sheets of extracellular matrix that outline epithelia, muscle fibers, blood vessels and peripheral nerves. The current view of BM structure and functions is based mainly on transmission electron microscopy imaging, in vitro protein binding assays, and phenotype analysis of human patients, mutant mice and invertebrata. Recently, MS-based protein analysis, biomechanical testing and cell adhesion assays with in vivo derived BMs have led to new and unexpected insights. Proteomic analysis combined with ultrastructural studies showed that many BMs undergo compositional and structural changes with advancing age. Atomic force microscopy measurements in combination with phenotype analysis have revealed an altered mechanical stiffness that correlates with specific BM pathologies in mutant mice and human patients. Atomic force microscopy-based height measurements strongly suggest that BMs are more than two-fold thicker than previously estimated, providing greater freedom for modelling the large protein polymers within BMs. In addition, data gathered using BMs extracted from mutant mice showed that laminin has a crucial role in BM stability. Finally, recent evidence demonstrate that BMs are bi-functionally organized, leading to the proposition that BM-sidedness contributes to the alternating epithelial and stromal tissue arrangements that are found in all metazoan species. We propose that BMs are ancient structures with tissue-organizing functions and were essential in the evolution of metazoan species.

Mutations in glycosyltransferases, such as protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1), causes disruptions of basement membranes (BMs) that results in neuronal ectopias and muscular dystrophy. While the mutations diminish dystroglycan-mediated cell-ECM interactions, the cause and mechanism of BM disruptions remain unclear. In this study, we established an in vitro model to measure BM assembly on the surface of neural stem cells. Compared to control cells, the rate of BM assembly on POMGnT1 knockout neural stem cells was significantly reduced. Further, immunofluorescence staining and quantitative proteomic analysis of the inner limiting membrane (ILM), a BM of the retina, revealed that laminin-111 and nidogen-1 were reduced in POMGnT1 knockout mice. Finally, atomic force microscopy showed that the ILM from POMGnT1 knockout mice was thinner with an altered surface topography. The results combined demonstrate that reduced levels of key BM components cause physical changes that weaken the BM in POMGnT1 knockout mice. These changes are caused by a reduced rate of BM assembly during the developmental expansion of the neural tissue.

Patch clamp is a powerful tool for studying the properties of ion-channels and cellular membrane. In recent years, planar patch clamp chips have been fabricated from various materials including glass, quartz, silicon, silicon nitride, polydimethyl-siloxane (PDMS), and silicon dioxide. Planar patch clamps have made automation of patch clamp recordings possible. However, most planar patch clamp chips have limitations when used in combination with other techniques. Furthermore, the fabrication methods used are often expensive and require specialized equipments. An improved design as well as fabrication and characterization of a silicon-based planar patch clamp chip are described in this report. Fabrication involves true batch fabrication processes that can be performed in most common microfabrication facilities using well established MEMS techniques. Our planar patch clamp chips can form giga-ohm seals with the cell plasma membrane with success rate comparable to existing patch clamp techniques. The chip permits whole-cell voltage clamp recordings on variety of cell types including Chinese Hamster Ovary (CHO) cells and pheochromocytoma (PC12) cells, for times longer than most available patch clamp chips. When combined with a custom microfluidics chamber, we demonstrate that it is possible to perfuse the extra-cellular as well as intra-cellular buffers. The chamber design allows integration of planar patch clamp with atomic force microscope (AFM). Using our planar patch clamp chip and microfluidics chamber, we have recorded whole-cell mechanosensitive (MS) currents produced by directly stimulating human keratinocyte (HaCaT) cells using an AFM cantilever. Our results reveal the spatial distribution of MS ion channels and temporal details of the responses from MS channels. The results show that planar patch clamp chips have great potential for multi-parametric high throughput studies of ion channel proteins.

Type 1 diabetes affects more than a million people in the United States and many more across the world. While pharmaceutical interventions and insulin supplementation are the most commonplace treatment of diabetes, these are not essentially cures and can potentially lead to long-term complications. Transplantation of insulin-producing Islets of Langerhans from donor pancreas has been established as a promising alternative to diabetes therapy. While successful islet transplantation has the potential of providing a cure, the primary hurdles to be overcome for it to be clinically viable are the scarcity of donor islets and immune rejection of transplanted islets. Recent advances in stem cell culture and differentiation techniques have established stem cells as a likely source of transplantable islets. Different stem cell sources have been induced toward pancreatic differentiation using specific chemical perturbations along with use of specific substrates. An approach to overcoming the second hurdle of immune rejection of transplantable islets is to encapsulate the islets in specific biomaterials. In this review, we discuss the extensive use of various substrates for pancreatic differentiation of different stem cell sources, along with different biomaterial designs used for islet transplantation.

Basement membranes (BMs) are physiologically insoluble extracellular matrix sheets present in all multicellular organisms. They play an important role in providing mechanical strength to tissues and regulating cell behavior. Proteomic analysis of BM proteins is challenged by their high molecular weights and extensive post-translational modifications. Here, we describe the direct analysis of an in vivo BM system using a mass spectrometry (MS) based proteomics approach. Retinal BMs were isolated from embryonic chick eyes. The BM macromolecules were deglycosylated and separated by low percentage gradient SDS PAGE, in-gel digested and analyzed by LC-MS/MS. This identified over 27 extracellular matrix proteins in the retinal BM. A semi-quantitative measure of protein abundance distinguished, nidogens-1 and -2, laminin subunits α1, α5, β2, and γ1, agrin, collagen XVIII, perlecan, FRAS1 and FREM2 as the most abundant BM protein components. Laminin subunits α3, β1, γ2, γ3 and collagen IV subunits α5 and α6 were minor constituents. To examine binding interactions that contribute to the stability of the retinal BM, we applied the LC-MS/MS based approach to detect potential BM complexes from the vitreous. Affinity-captured nidogen- and heparin-binding proteins from the vitreous contained >10 and >200 proteins respectively. Comparison of these protein lists with the retinal BM proteome reveals that glycosaminoglycan and nidogen binding interactions play a central role in the internal structure and formation of the retinal BM. In addition, we studied the biomechanical qualities of the retinal BM before and after deglycosylation using atomic force microscopy. These results show that the glycosaminoglycan side chains of the proteoglycans play a dominant role in regulating the thickness and elasticity of the BMs by binding water to the extracellular matrix. To our knowledge, this is the first large-scale investigation of an in vivo BM system using MS-based proteomics.

Basement membranes (BMs) are considered to be uniform, approximately 100 nm-thin extracellular matrix sheets that serve as a substrate for epithelial cells, endothelial cells and myotubes. To find out whether BMs maintain their ultrastructure, protein composition and biophysical properties throughout life the natural aging history of the human inner limiting membranes (ILM) was investigated. The ILM is a BM at the vitreal surface of the retina that connects the retina with the vitreous. Transmission electron microscopy (TEM) showed that the ILM steadily increases in thickness from 70 nm at fetal stages to several microns at age 90. By the age of 20, the ILM loses its laminated structure to become an amorphous and very irregular extracellular matrix layer. Atomic force microscopy (AFM) showed that the native, hydrated ILMs are on average 4-fold thicker than the dehydrated ILMs as seen by TEM and that their thickness is prominently determined by its water-binding proteoglycans. The morphological changes are accompanied by age-related changes in the biochemical composition, whereby the relative concentrations of collagen IV and agrin increase, and the concentration of laminin decreases with age. Force-indentation measurements by AFM also showed that ILMs become increasingly stiffer with advancing age. The data suggest that BMs from other human tissues may undergo similar age-related changes.

Heart valve interstitial cells (VICs) appear to have a dynamic and reversible phenotype, an attribute speculated to be necessary for valve tissue remodeling during times of development and repair. Therefore, we hypothesized that the cytoskeletal (CSK) remodeling capability of the aortic and pulmonary VICs (AVICs and PVICs, respectively), which are dominated by smooth muscle alpha-actin, would exhibit unique contractile behaviors when seeded on collagen gels. Using a porcine cell source, we observed that VIC populations did not contract the gels at early time points (2 and 4 hours) as dermal fibroblasts did, but formed a central cluster of cells prior to contraction. After clustering, VICs appeared to radiate out from the center of the gels, whereas fibroblasts did not migrate but contracted the gels locally. VIC gels treated with transforming growth factor beta1 contracted the gels rapidly, revealing similar sensitivity to the cytokine. Moreover, we evaluated the initial mechanical state of the underlying CSK by comparing AVIC and PVIC stiffness with atomic force microscopy. Not only were AVICs significantly stiffer (p < 0.001) than the PVICs, but they also contracted the gels significantly more at 24 and 48 hours (p < 0.001). Taken together, these findings suggest that the AVICs are capable of inducing greater extra cellular matrix contraction, possibly manifesting in a more pronounced ability to remodel valvular tissues. Moreover, significant mechanobiological differences between AVICs and PVICs exist, and may have implications for understanding native valvular tissue remodeling. Elucidating these differences will also define important functional endpoints in the development of tissue engineering approaches for heart valve repair and replacement.

Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.

Encapsulation of donor islets using a hydrogel material is a well-studied strategy for islet transplantation, which protects donor islets from the host immune response. Replacement of donor islets by human embryonic stem cell (hESC) derived islets will also require a means of immune-isolating hESCs by encapsulation. However, a critical consideration of hESC differentiation is the effect of surrounding biophysical environment, in this case capsule biophysical properties, on differentiation. The objective of this study, thus, was to evaluate the effect of capsule properties on growth, viability, and differentiation of encapsulated hESCs throughout pancreatic induction. It was observed that even in the presence of soluble chemical cues for pancreatic induction, substrate properties can significantly modulate pancreatic differentiation, hence necessitating careful tuning of capsule properties. Capsules in the range of 4–7 kPa supported cell growth and viability, whereas capsules of higher stiffness suppressed cell growth. While an increase in capsule stiffness enhanced differentiation at the intermediate definitive endoderm (DE) stage, increased stiffness strongly suppressed pancreatic progenitor (PP) induction. Signaling pathway analysis indicated an increase in pSMAD/pAKT levels with substrate stiffness likely the cause of enhancement of DE differentiation. In contrast, sonic hedgehog inhibition was more efficient under softer gel conditions, which is necessary for successful PP differentiation. Statement of Significance Cell replacement therapy for type 1 diabetes (T1D), affecting millions of people worldwide, requires the immunoisolation of insulin-producing islets by encapsulation with a semi-impermeable material. Due to the shortage of donor islets, human pluripotent stem cell (hPSC) derived islets are an attractive alternative. However, properties of the encapsulating substrate are known to influence hPSC cell fate. In this work, we determine the effect of substrate stiffness on growth and pancreatic fate of encapsulated hPSCs. We precisely identify the range of substrate properties conducive for pancreatic cell fate, and also the mechanism by which substrate properties modify the cell signaling pathways and hence cell fate. Such information will be critical in driving regenerative cell therapy for long term treatment of T1D.

Approximately 285 million people worldwide suffer from diabetes, with insulin supplementation as the most common treatment measure. Regenerative medicine approaches such as a bioengineered pancreas has been proposed as potential therapeutic alternatives. A bioengineered pancreas will benefit from the development of a bioscaffold that supports and enhances cellular function and tissue development. Perfusion-decellularized organs are a likely candidate for use in such scaffolds since they mimic compositional, architectural and biomechanical nature of a native organ. In this study, we investigate perfusion-decellularization of whole pancreas and the feasibility to recellularize the whole pancreas scaffold with pancreatic cell types. Our result demonstrates that perfusion-decellularization of whole pancreas effectively removes cellular and nuclear material while retaining intricate three-dimensional microarchitecture with perfusable vasculature and ductal network and crucial extracellular matrix (ECM) components. To mimic pancreatic cell composition, we recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured up to 5 days. Our result shows successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to strong up-regulation of insulin gene expression. These findings support biological utility of whole pancreas ECM as a biomaterials scaffold for supporting and enhancing pancreatic cell functionality and represent a step toward bioengineered pancreas using regenerative medicine approaches.

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.

The development of polyelectrolyte multilayered coatings on magnesium alloy substrates that can be used for controlled delivery of growth factors and required biomolecules from the surface of these degradable implants could have a significant impact in the field of bone tissue regeneration. The current work reports on the fabrication of multilayered coatings of alginate and poly-L-lysine on alkaline- and fluoride-pretreated AZ31 substrates using a layer-by-layer (LbL) technique under physiological conditions. Furthermore, these coatings were surface functionalized by chemical cross-linking and fibronectin immobilization, and the resultant changes in surface properties have been shown to influence the cellular activity of these multilayered films. The physicochemical characteristics of these coated substrates have been investigated using attenuated total reflectance Fourier transform infrared spectroscopy, atomic force microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Cytocompatibility studies using MC3T3-E1 osteoblasts show that the fluoride-pretreated, cross-linked and fibronectin-immobilized LbL-coated substrates are more bioactive and less cytotoxic than the hydroxide-pretreated, cross-linked and fibronectin-immobilized LbL-coated samples. The in vitro degradation results show that the multilayered coatings of these natural polysaccharide- and synthetic polyamino acid-based polyelectrolytes do not alter the degradation kinetics of the substrates; however, the pretreatment conditions have a significant impact on the overall coating degradation behavior. These preliminary results collectively show the potential use of LbL coatings on magnesium-based degradable scaffolds to improve their surface bioactivity.

Background Embryonic stem cells (ESCs) have been implicated to have tremendous impact in regenerative therapeutics of various diseases, including Type 1 Diabetes. Upon generation of functionally mature ESC derived islet-like cells, they need to be implanted into diabetic patients to restore the loss of islet activity. Encapsulation in alginate microcapsules is a promising route of implantation, which can protect the cells from the recipient’s immune system. While there has been a significant investigation into islet encapsulation over the past decade, the feasibility of encapsulation and differentiation of ESCs has been less explored. Research over the past few years has identified the cellular mechanical microenvironment to play a central role in phenotype commitment of stem cells. Therefore it will be important to design the encapsulation material to be supportive to cellular functionality and maturation. Results This work investigated the effect of stiffness of alginate substrate on initial differentiation and phenotype commitment of murine ESCs. ESCs grown on alginate substrates tuned to similar biomechanical properties of native pancreatic tissue elicited both an enhanced and incrementally responsive differentiation towards endodermal lineage traits. Conclusions The insight into these biophysical phenomena found in this study can be used along with other cues to enhance the differentiation of embryonic stem cells toward a specific lineage fate.

Recent studies have suggested that extracellular matrix rigidity regulates cancer invasiveness, including the formation of cellular invadopodial protrusions; however, the relevant mechanical range is unclear. Here, we used a combined analysis of tissue-derived model basement membrane (BM) and stromal matrices and synthetic materials to understand how substrate rigidity regulates invadopodia. Urinary bladder matrix-BM (UBM-BM) was found to be a rigid material with elastic moduli of 3-8 MPa, as measured by atomic force microscopy and low-strain tensile testing. Stromal elastic moduli were ∼6-fold lower, indicating a more compliant material. Using synthetic substrates that span kPa–GPa moduli, we found a peak of invadopodia-associated extracellular matrix degradation centered around 30 kPa, which also corresponded to a peak in invadopodia/cell. Surprisingly, we observed another peak in invadopodia numbers at 2 GPa as well as gene expression changes that indicate cellular sensing of very high moduli. Based on the measured elastic moduli of model stroma and BM, we expected to find more invadopodia formation on the stroma, and this was verified on the stromal versus BM side of UBM-BM. These data suggest that cells can sense a wide range of rigidities, up into the GPa range. Furthermore, there is an optimal rigidity range for invadopodia activity that may be limited by BM rigidity.

Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.

Stem cells receive numerous cues from their associated substrate that help to govern their behaviour. However, identification of influential substrate characteristics poses difficulties because of their complex nature. In this study, we developed an integrated experimental and systems level modelling approach to investigate and identify specific substrate features influencing differentiation of mouse embryonic stem cells (mESCs) on a model fibrous substrate, fibrin. We synthesized a range of fibrin gels by varying fibrinogen and thrombin concentrations, which led to a range of substrate stiffness and microstructure. mESCs were cultured on each of these gels, and characterization of the differentiated cells revealed a strong influence of substrate modulation on gene expression patterning. To identify specific substrate features influencing differentiation, the substrate microstructure was quantified by image analysis and correlated with stem cell gene expression patterns using a statistical model. Significant correlations were observed between differentiation and microstructure features, specifically fibre alignment. Furthermore, this relationship occurred in a lineage-specific manner towards endoderm. This systems level approach allows for identification of specific substrate features from a complex material which are influential to cellular behaviour. Such analysis may be effective in guiding the design of scaffolds with specific properties for tissue engineering applications.

The current study investigates the structural and compositional changes of ocular basement membranes (BMs) during long-term diabetes. By comparing retinal vascular BMs and the inner limiting membrane (ILM) from diabetic and non-diabetic human eyes by light and transmission electron microscopy (TEM), a massive, diabetes-related increase in the thickness of these BMs was detected. The increase in ILM thickness was confirmed by atomic force microscopy (AFM) on native ILM flat-mount preparations. AFM also detected a diabetes-induced increase in ILM stiffness. The changes in BM morphology and biophysical properties were accompanied by partial changes in the biochemical composition as shown by immunocytochemistry and western blots: agrin, fibronectin and tenascin underwent relative increases in concentration in diabetic BMs as compared to non-diabetic BMs. Fibronectin and tenascin were particularly high in the BMs of outlining microvascular aneurisms. The present data showed that retinal vascular BMs and the ILM undergo morphological, biomechanical and compositional changes during long-term diabetes. The increase in BM thickness not only resulted from an up-regulation of the standard BM proteins, but also from the expression of diabetes-specific extracellular matrix proteins that are not normally found in retinal BMs.