Hydrogel Platforms for Investigating Microenvironmental Signaling Cues Influencing Human Glomerular Endothelial Cell and Podocyte Behavior

Su, Jimmy

doi:https://doi.org/10.21985/n2-zexp-cj29

Work

Hydrogel Platforms for Investigating Microenvironmental Signaling Cues Influencing Human Glomerular Endothelial Cell and Podocyte Behavior

Public

Download PDF

Download All Files (.zip)

End-stage renal disease, or kidney failure, can result from acute kidney injury or sustained kidney damage in the form of chronic kidney disease. As the prevalence of end-stage renal disease continues to rise, the gold-standard treatment—kidney transplantation—is increasingly restricted by the shortage of transplantable donor kidneys. Bioengineered kidney tissues may potentially alleviate these numbers by advancing understanding of kidney development and progression of diseases, improving the efficiency of drug discovery and toxicity testing, and eventually restoring or replacing lost kidney function in therapeutic applications. However, a number of obstacles remain before bioengineered kidney tissues reach clinical applications. One challenge in particular is the design and fabrication of biomaterial scaffolds or matrices that provide the necessary microenvironmental signaling cues to promote a functional cellular response or phenotype. Hydrogels are water-swollen, crosslinked polymer networks that mimic many of the properties of the native extracellular matrix and therefore may serve as ideal scaffolds or matrices for engineering complex tissues and organs. Here, I will present multiple hydrogel platforms for investigating interactions by the glomerular endothelial cells and podocytes that form the glomerular filtration barrier of the kidney nephron. Hydrogels derived from tissue- and organ-specific decellularized extracellular matrix (dECM) may retain bioactive components from the native tissue or organ that could in turn modulate functional cell response. Therefore, the first hydrogel platform I will present is formulated from porcine kidney dECM that has been processed to form physically-crosslinked hydrogels suitable for cell culture and encapsulation investigations. Scanning electron micrographs of hydrogels demonstrated fibrous ultrastructures with interconnected pores, and rheological characterization revealed rapid gelation times with shear moduli dependent on the hydrogel polymer or protein concentration. Conditionally-immortalized human glomerular endothelial cells (GEnCs) cultured on hydrogel substrates or encapsulated within hydrogel matrices exhibited high cell viability and proliferation over a one-week culture period. However, gene expression analysis of GEnCs encapsulated within kidney dECM hydrogels revealed significantly lower expression of several relevant genes of interest compared to those encapsulated within hydrogels composed of only purified type I collagen. These results were further supported by similar trends obtained through gene expression analysis of GEnCs cultured on the hydrogels as substrates. Unfortunately, the complexity of dECM and limitations of naturally-derived materials hinder additional investigations that require the ability to adequately tailor or tune hydrogel properties. As a result, the second hydrogel platform I will present is a tunable hydrogel substrate with conjugated bioactive peptides to modulate cell binding and growth factor signaling. These hydrogels were formed by employing a poly(ethylene glycol) crosslinker to covalently crosslink gelatin polymers and simultaneously conjugate laminin-derived YIGSR peptides or vascular endothelial growth factor (VEGF)-mimetic QK peptides to the gelatin. Rheological characterization revealed rapid formation of hydrogels with similar stiffnesses across tested formulations, and swelling analysis demonstrated dependency on peptide and crosslinker concentrations in hydrogels. Levels of phosphorylated VEGF receptor 2 in cells cultured on hydrogel substrates illustrated that while human umbilical vein endothelial cells (HUVECs) responded to both soluble and conjugated forms of the QK peptide, GEnCs only responded to the conjugated presentation of the peptide. Furthermore, whereas HUVECs exhibited greatest upregulation in gene expression when cultured on YIGSR- and QK-conjugated hydrogel substrates after 5 days, GEnCs exhibited greatest upregulation when cultured on Matrigel control substrates at the same time point. These results indicate that conjugation of bioactive peptides to these hydrogel substrates significantly influenced endothelial cell behavior in cultures but with differential responses between HUVECs and GEnCs. Within the glomerulus, both GEnCs and podocytes are necessary for the formation and function of the glomerular filtration barrier. Consequently, I will present investigations in which conditionally-immortalized human podocyte response to culture on hydrogel substrates or encapsulation within hydrogel matrices was evaluated. Interestingly, the results suggest that podocytes may be particularly sensitive to encapsulation within hydrogels, specifically type I collagen hydrogels and kidney dECM hydrogels. Although podocytes exhibited enhanced podocyte-specific gene expression when cultured on soft Matrigel or poly(ethylene glycol)-crosslinked gelatin hydrogel substrates, cell survival appeared to be compromised at longer time points. In contrast, podocytes cultured on stiffer, 3D-printed gelatin hydrogel scaffolds maintained high cell viability, adhesion, and spreading even after extended culture. These results imply that hydrogel stiffness and 3D structure or architecture are two microenvironmental signaling cues that must be carefully considered when designing hydrogel platforms for maintaining healthy podocytes in a differentiated state. In summary, these hydrogel platforms and the subsequent evaluation of cell behavior will advance understanding of microenvironmental signaling cues integral for the development engineered kidney tissues.

Creator