A strategy to quantify myofibroblast activation on a continuous spectrum

Abstract

Myofibroblasts are a highly secretory and contractile cell phenotype that are predominant in wound healing and fibrotic disease. Traditionally, myofibroblasts are identified by the de novo expression and assembly of alpha-smooth muscle actin stress fibers, leading to a binary classification: “activated” or “quiescent (non-activated)”. More recently, however, myofibroblast activation has been considered on a continuous spectrum, but there is no established method to quantify the position of a cell on this spectrum. To this end, we developed a strategy based on microscopy imaging and machine learning methods to quantify myofibroblast activation in vitro on a continuous scale. We first measured morphological features of over 1000 individual cardiac fibroblasts and found that these features provide sufficient information to predict activation state. We next used dimensionality reduction techniques and self-supervised machine learning to create a continuous scale of activation based on features extracted from microscopy images. Lastly, we compared our findings for mechanically activated cardiac fibroblasts to a distribution of cell phenotypes generated from transcriptomic data using single-cell RNA sequencing. Altogether, these results demonstrate a continuous spectrum of myofibroblast activation and provide an imaging-based strategy to quantify the position of a cell on that spectrum.

Chemical Modification of Human Decellularized Extracellular Matrix for Incorporation into Phototunable Hybrid-Hydrogel Models of Tissue Fibrosis

“…On soft hybrid-hydrogels, fibroblasts remained rounded, while fibroblasts on stiffened hybrid-hydrogels began to stretch out protrusions that increased the average aspect ratio from 1.59 ± 0.02 to 2.98 ± 0.20 (Figure C). Similarly, stiffening resulted in a greater proportion of activated (noncircular, αSMA+) fibroblasts from 25.8% on soft hybrid-hydrogels to 49.1% on stiffened samples (Figure D).…”

“…In this study, human fibroblasts were seeded on two-dimensional (2D) soft hybrid-hydrogels and then exposed to dynamic stiffening via dual-stage polymerization. Fibroblast activation was measured by assessing cell morphology based on f-actin expression and myofibroblast phenotype by αSMA expression, the combination of which correlates to high expression of a fibroblast activation genetic program . Immunofluorescence staining for f-actin revealed a significant increase in cellular aspect ratiothe ratio between the major and minor axes of an ellipse fit to the cell area.…”

“…Cell nuclei (DAPI channel for immunofluorescent stains or both total and dead dyes for live/dead analysis) were counted by establishing a signal threshold, performing a watershed operation, and analyzing particles. Activated fibroblasts were counted on the αSMA channel by establishing a signal threshold above background and analyzing for particles with a circularity less than 0.5, indicating increased spreading, a characteristic of activation fibroblasts circularity = 4 π ( area perimete r 2 ) To calculate the percentage of activated cells per sample, this count was divided by the total number of cell nuclei.…”

Chemical modification of human decellularized extracellular matrix for incorporation into phototunable hybrid-hydrogel models of tissue fibrosis

“…On soft hybrid-hydrogels, fibroblasts remained rounded, while fibroblasts on stiffened hybrid-hydrogels began to stretch out protrusions that increased this aspect ratio from 1.59 ± 0.02 to 2.98 ± 0.20 (Figure 4C). Similarly, stiffening resulted in a greater proportion of non-circular, αSMA+ myofibroblasts 68 from 25.4% on soft hybrid-hydrogels to 51.8% on stiffened samples (Figure 4D). These results demonstrate the ability of this fully human hybrid-hydrogel culture model to facilitate investigation of dynamic fibroblast mechanosensing, a critical component of fibrotic progression.…”

“…26 In this study, human fibroblasts were seeded on 2D soft hybrid-hydrogels and then exposed to dynamic stiffening via dual-stage polymerization. Fibroblast activation was measured by assessing cell morphology based on f-actin expression and myofibroblast phenotype by αSMA expression, the combination of which correlates to high expression of a fibroblast activation genetic program 68 . Immunofluorescence staining for f-actin revealed a significant increase in cellular aspect ratio, the ratio between the major and minor axis of an ellipse fit to the cell area.…”