Creation and application of tools for probing cell functions in wound healing and disease processes
Date
2023
Authors
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Publisher
University of Delaware
Abstract
Fibrosis is a class of diseases driven by the persistent activation of fibroblasts, where this persistence contributes to excess deposition and crosslinking of collagen, stiffening of tissue, and can lead to organ disfunction and death. The most common type of lung fibrosis, idiopathic pulmonary fibrosis (IPF), is a disease with uncertain etiology that is responsible for over 50,000 deaths each year in the US alone, where 80% of patients die within 5 years of diagnosis. While two FDA approved treatments, pirfenidone and nintedanib, modestly slow disease progression, a substantial number have failed in the pre-clinical or clinical pipeline, highlighting a need for additional understanding of disease mechanisms to improve identification of novel therapeutics. Human in vitro models of fibrosis complement in vivo animal models for both hypothesis testing and drug evaluation. However, a challenge remains in understanding the dynamics of human fibroblast responses to complex microenvironment stimuli, motivating the need for more advanced tools to investigate these mechanisms. This work aimed to 1) investigate fibroblast activation in novel synthetic hydrogel culture models mimetic of the extracellular matrix (ECM) and 2) establish approaches for assessing the temporal dynamics of these responses using genetically-encoded fluorescent reporters, with broader implications for probing a range of cell-microenvironment interactions. ☐ While there are many in vitro culture approaches for investigating cellular mechanisms, synthetic hydrogels are particularly advantageous due to their modularity and the library of chemistries that can be used to achieve a wide range of mechanical properties such as substrate stiffness and viscoelasticity, impart biochemical moieties such as integrin binding sequences and degradable crosslinks, and integrate fibrous structures that can mimic aspects of the ECM. As this library of synthetic approaches expands, it becomes critical to choose a model system that is best suited for testing the hypothesis at hand. We first investigated the utility of three hydrogel geometries for studying fibroblast activation in response to microenvironmental cues, where cell shape and polarity were hypothesized to drive differences in cell response. Transitioning to a hydrogel composition with dynamic covalent boronic-acid based chemistry, we demonstrated that self-healing hydrogels could be utilized to create unique co-cultures for investigating interactions between breast cancer and fibroblasts, where this viscoelastic material allowed for migration and proliferation in the absence of degradable moieties. Finally, with the goal of investigating fibroblast response to materials mimicking the collagen structure of fibrotic lung tissue, we encapsulated lung fibroblasts in hydrogels with covalently incorporated collagen mimetic peptides and found that the imparted fibrillar structure influenced fibroblast elongation and spreading, better mimicking their native spindle-like morphology seen in vivo. ☐ As tools expand for recapitulating these complex cell-matrix interactions in vitro, we need increasingly sophisticated methods to measure the dynamics of cell response to these microenvironmental cues. Genetically-encoded fluorescent reporters present an opportunity for implementing into human cell populations non-destructive assessments of dynamic cell response with single cell and temporal resolution that many traditional end-point assays do not provide. Towards studying fibrosis, we established robust methods for the implementation of a lentiviral-based dual-fluorescence system that reports on alpha smooth muscle actin (αSMA), a well-accepted indicator of fibroblast activation. Specifically, we first optimized protocols for lentivirus production with high titer and for high efficiency transduction to create a library of human lung fibroblast reporter cell lines. For this library, three versions of the reporter were utilized, where for each version, the fluorescent protein was modified to exhibit a different temporal stability, providing temporal resolution of protein expression processes over a range of timescales. ☐ Using this library, we demonstrated that reporters provide insights into population shifts in response to both mechanical and biochemical cues that are not detectable by traditional end-point assessments. Further, with these methods, individual cell tracks can also be considered and isolated, highlighting opportunities for complementary measurements such as FACS and single cell RNA sequencing. Establishment of this reporter toolset enables dynamic cell investigations that can be translated into more complex synthetic culture environments for elucidating disease mechanisms and evaluating therapeutics for lung fibrosis and other challenging diseases more broadly.
Description
Keywords
Wound healing, Disease progression, Extracellular matrix, Fibroblast activation, Microenvironmental cues