Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations
Date
2024-06-24
Journal Title
Journal ISSN
Volume Title
Publisher
APL Bioengineering
Abstract
Activation of fibroblasts is pivotal for wound healing; however, persistent activation leads to maladaptive processes and is a hallmark of fibrosis, where disease mechanisms are only partially understood. Human in vitro model systems complement in vivo animal models for both hypothesis testing and drug evaluation to improve the identification of therapeutics relevant to human disease. Despite advances, a challenge remains in understanding the dynamics of human fibroblast responses to complex microenvironment stimuli, motivating the need for more advanced tools to investigate fibrotic mechanisms. This work established approaches for assessing the temporal dynamics of these responses using genetically encoded fluorescent reporters of alpha smooth muscle actin expression, an indicator of fibroblast activation. Specifically, we created a toolset of human lung fibroblast reporter cell lines from different origins (male, female; healthy, idiopathic pulmonary fibrosis) and used three different versions of the reporter with the fluorescent protein modified to exhibit different temporal stabilities, providing temporal resolution of protein expression processes over a range of timescales. Using this toolset, we demonstrated that reporters provide insight into population shifts in response to both mechanical and biochemical cues that are not detectable by traditional end point assessments with differential responses based on cell origin. Furthermore, individual cells can also be tracked over time, with opportunities for comparison to complementary end point measurements. The 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 complex biological processes more broadly.
Description
This article was originally published in APL Bioengineering. The version of record is available at: https://doi.org/10.1063/5.0166152
© 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0166152
Keywords
biological processes and phenomena, flow cytometry, musculoskeletal system, proteins, stress fibers, therapeutics, cell lines, statistical analysis
Citation
Samantha E. Cassel, Breanna M. Huntington, Wilfred Chen, Pedro Lei, Stelios T. Andreadis, April M. Kloxin; Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations. APL Bioeng. 1 June 2024; 8 (2): 026127. https://doi.org/10.1063/5.0166152