Alexandra Paul

Hybrid Elastin-like Polypeptide–Polyethylene Glycol (ELP-PEG) Hydrogels with Improved Transparency and Independent Control of Matrix Mechanics and Cell Ligand Density

Hydrogels have been developed as extracellular matrix (ECM) mimics both for therapeutic applications and basic biological studies. In particular, elastin-like polypeptide (ELP) hydrogels, which can be tuned to mimic several biochemical and physical characteristics of native ECM, have been constructed to encapsulate various types of cells to create in vitro mimics of in vivo tissues. However, ELP hydrogels become opaque at body temperature because of ELP’s lower critical solution temperature behavior. This opacity obstructs light-based observation of the morphology and behavior of encapsulated cells. In order to improve the transparency of ELP hydrogels for better imaging, we have designed a hybrid ELP-polyethylene glycol (PEG) hydrogel system that rapidly cross-links with tris(hydroxymethyl) phosphine (THP) in aqueous solution via Mannich-type condensation. As expected, addition of the hydrophilic PEG component significantly improves the light transmittance. Coherent anti-Stokes Raman scattering (CARS) microscopy reveals that the hybrid ELP-PEG hydrogels have smaller hydrophobic ELP aggregates at 37 °C. Importantly, this hydrogel platform enables independent tuning of adhesion ligand density and matrix stiffness, which is desirable for studies of cell–matrix interactions. Human fibroblasts encapsulated in these hydrogels show high viability (>98%) after 7 days of culture. High-resolution confocal microscopy of encapsulated fibroblasts reveals that the cells adopt a more spread morphology in response to higher RGD ligand concentrations and softer gel mechanics.

Increased Adipogenesis of Human Adipose-Derived Stem Cells on Polycaprolactone Fiber Matrices

With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells - differentiated to adipocytes in vitro - are frequently used as a model system for white adipocytes, as most of their pathways and functions resemble mature adipocytes in vivo. However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in a more physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics – studied by coherent anti-Stokes Raman scattering microscopy - to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells.

Micro- and nano-patterned elastin-like polypeptide hydrogels for stem cell culture

We show that submicron-sized patterns can be imprinted into soft, recombinant-engineered protein hydrogels (here elastin-like proteins, ELP) by transferring wavy patterns from polydimethylsiloxane (PDMS) molds. The high-precision topographical tunability of the relatively stiff PDMS is translated to a bio-responsive, soft material, enabling topographical cell response studies at elastic moduli matching those of tissues. Aligned and unaligned wavy patterns with mold periodicities of 0.24-4.54 μm were imprinted and characterized by coherent anti-Stokes Raman scattering and atomic force microscopy. The pattern was successfully transferred down to 0.37 μm periodicity (width in ELP: 250 ± 50 nm, height: 70 ± 40 nm). The limit was set by inherent protein assemblies (diameter: 124-180 nm) that formed due to lower critical solution temperature behavior of the ELP during molding. The width/height of the ELP ridges depended on the degree of hydration; from complete dehydration to full hydration, ELP ridge width ranged from 79 ± 9% to 150 ± 40% of the mold width. The surface of the ridged ELP featured densely packed protein aggregates that were larger in size than those observed in bulk/flat ELP. Adipose-derived stem cells (ADSCs) oriented along hydrated aligned patterns with periodicities ≥0.60 μm (height ≥170 ± 100 nm), while random orientation was observed for smaller distances/amplitudes, as well as flat and unaligned wavy ELP surfaces. Hence, micro-molding of ELP is a promising approach to create tissue-mimicking, hierarchical architectures composed of tunable micron-sized structures with nano-sized protein aggregates, which opens the way for orthogonal screening of cell responses to topography and cell-adhesion ligands at relevant elastic moduli.

CCN5/WISP2 and metabolic diseases

Obesity and type 2 diabetes increase worldwide at an epidemic rate. It is expected that by the year 2030 around 500 million people will have diabetes; predominantly type 2 diabetes. The CCN family of proteins has become of interest in both metabolic and other common human diseases because of their effects on mesenchymal stem cell (MSCs) proliferation and differentiation as well as being important regulators of fibrosis. We here review current knowledge of the WNT1 inducible signaling pathway protein 2 (CCN5/WISP2). It has been shown to be an important regulator of both these processes through effects on both the canonical WNT and the TGFβ pathways. It is also under normal regulation by the adipogenic commitment factor BMP4, in contrast to conventional canonical WNT ligands, and allows MSCs to undergo normal adipose cell differentiation. CCN5/WISP2 is highly expressed in, and secreted by, MSCs and is an important regulator of MSCs growth. In a transgenic mouse model overexpressing CCN5/WISP2 in the adipose tissue, we have shown that it is secreted and circulating in the blood, the mice develop hypercellular white and brown adipose tissue, have increased lean body mass and enlarged hypercellular hearts. Obese transgenic mice had improved insulin sensitivity. Interestingly, the anti-fibrotic effect of CCN5/WISP2 is protective against heart failure by inhibition of the TGFβ pathway. Understanding how CCN5/WISP2 is regulated and signals is important and may be useful for developing new treatment strategies in obesity and metabolic diseases and it can also be a target in regenerative medicine.

Cell surface proteoglycan-mediated uptake and accumulation of the Alzheimer's disease peptide Aβ(1–42)

Proteoglycans (PGs) have been found in Alzheimers disease amyloid-β (Aβ) plaques and their glycosaminoglycan chains reportedly influence Aβ aggregation, neurotoxicity and intracellular accumulation in cell and animal models, but their exact pathophysiological role(s) remain unclear. We have studied the cellular uptake of fluorescently labelled Aβ(1-42) and Aβ(1-40) peptides in normal CHO cells (K1) and the mutant cell line (pgsA-745) which lacks all protein-attached heparan and chondroitin sulfate chains. After 24 h of incubation, CHO-K1 accumulates more Aβ(1-42) and Aβ(1-40) compared with CHO-pgsA-745, consistent with the suggested role of PGs in Aβ uptake. However, after short incubation times (≤3 h) there was no difference; moreover, the time evolution of Aβ(1-42) accumulation in CHO-K1 followed an unusual sigmoidal-like trend, indicating a possible involvement of PG-mediated peptide aggregation in Aβ endocytosis. Neither Aβ(1-42) nor Aβ(1-40) could stimulate uptake of a 10 kDa dextran (a general endocytosis marker) suggesting that Aβ-induced upregulation of endocytosis does not occur. CHO-K1 cells contained a higher number of Aβ(1-42)-positive vesicles, but the intensity difference per vesicle was only marginal suggesting that the superior accumulation of Aβ(1-42) stems from a higher number of endocytic events. FRET imaging support that intracellular Aβ(1-42) is aggregated in both cell types. We also report that CHO-pgsA-745 cells perform less endocytosis than CHO-K1 and, albeit this does not explain their difference in Aβ internalisation, we discuss a general method for data compensation. Altogether, this study contributes new insights into the mechanisms of PG-mediated Aβ uptake that may be relevant for our understanding of their role in AD pathology.

Microstructured Elastomer-PEG Hydrogels via Kinetic Capture of Aqueous Liquid-Liquid Phase Separation

Abstract Heterogeneous hydrogels with desired matrix complexity are studied for a variety of biomimetic materials. Despite the range of such microstructured materials described, few methods permit independent control over microstructure and microscale mechanics by precisely controlled, single‐step processing methods. Here, a phototriggered crosslinking methodology that traps microstructures in liquid–liquid phase‐separated solutions of a highly elastomeric resilin‐like polypeptide (RLP) and poly(ethylene glycol) (PEG) is reported. RLP‐rich domains of various diameters can be trapped in a PEG continuous phase, with the kinetics of domain maturation dependent on the degree of acrylation. The chemical composition of both hydrogel phases over time is assessed via in situ hyperspectral coherent Raman microscopy, with equilibrium concentrations consistent with the compositions derived from NMR‐measured coexistence curves. Atomic force microscopy reveals that the local mechanical properties of the two phases evolve over time, even as the bulk modulus of the material remains constant, showing that the strategy permits control of mechanical properties on micrometer length scales, of relevance in generating mechanically robust materials for a range of applications. As one example, the successful encapsulation, localization, and survival of primary cells are demonstrated and suggest the potential application of phase‐separated RLP‐PEG hydrogels in regenerative medicine applications.

Mathematical modeling of white adipocyte exocytosis predicts adiponectin secretion and quantifies the rates of vesicle exo- and endocytosis

Adiponectin is a hormone secreted from white adipocytes and takes part in the regulation of several metabolic processes. Although the pathophysiological importance of adiponectin has been thoroughly investigated, the mechanisms controlling its release are only partly understood. We have recently shown that adiponectin is secreted via regulated exocytosis of adiponectin-containing vesicles, that adiponectin exocytosis is stimulated by cAMP-dependent mechanisms, and that Ca2+ and ATP augment the cAMP-triggered secretion. However, much remains to be discovered regarding the molecular and cellular regulation of adiponectin release. Here, we have used mathematical modeling to extract detailed information contained within our previously obtained high-resolution patch-clamp time-resolved capacitance recordings to produce the first model of adiponectin exocytosis/secretion that combines all mechanistic knowledge deduced from electrophysiological experimental series. This model demonstrates that our previous understanding of the role of intracellular ATP in the control of adiponectin exocytosis needs to be revised to include an additional ATP-dependent step. Validation of the model by introduction of data of secreted adiponectin yielded a very close resemblance between the simulations and experimental results. Moreover, we could show that Ca2+-dependent adiponectin endocytosis contributes to the measured capacitance signal, and we were able to predict the contribution of endocytosis to the measured exocytotic rate under different experimental conditions. In conclusion, using mathematical modeling of published and newly generated data, we have obtained estimates of adiponectin exo- and endocytosis rates, and we have predicted adiponectin secretion. We believe that our model should have multiple applications in the study of metabolic processes and hormonal control thereof.