Development of an Alginate Array Platform to Decouple the Effect of Multiparametric Perturbations on Human Pluripotent Stem Cells During Pancreatic Differentiation.

Human embryonic stem cells (hESC)-derived functional cells hold great promise for regenerative cell therapy. Currently approved strategies for clinical translation requires the isolation of the hESCs-derived cells in materials allowing transfer of reagents but preventing integration with the host. However, hESC fate is known to be sensitive to its local microenvironment, both chemical and physical. Given the complexity of hESC response to environmental parameters, it will be important to evaluate the cell response to multiple combinatorial perturbations. Such complex perturbations are best enabled by exploiting high-throughput screening platforms. In this study, the authors report the effect of multivariate perturbations on hESC differentiation, enabled by the development of high throughput 3D alginate array platform. Specifically, the sensitivity of hESC propagation and pancreatic differentiation to substrate properties and cell culture configuration is analyzed. Cellular response to array perturbations is analyzed by quantitative imaging, and cell sensitivity was determined through statistical modeling. The results indicate that configuration is the stronger determinant of hESC proliferation and differentiation, while substrate properties fine-tune the expression around the average levels. This platform allowed for multiparametric perturbations, and in combination with statistical modeling, allows to identify the sensitivity of hESC proliferation and fate to multiparametric modulation.

Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.

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.

Role of substrates in diabetes therapy: stem cell differentiation and islet transplantation.

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.

Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach.

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.

Sensing and modulation of invadopodia across a wide range of rigidities.

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.

Differences in tissue-remodeling potential of aortic and pulmonary heart valve interstitial cells.

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.