Therapeutic Modulation of Cell Morphology and Phenotype of Diseased Human Cells towards a Healthier Cell State Using Lignin

Abstract

Despite lignin’s global abundance and its use in biomedical studies, our understanding of how lignin regulates disease through modulation of cell morphology and associated phenotype of human cells is unknown. We combined an automated high-throughput image cell segmentation technique for quantitatively measuring a panel of cell shape descriptors, droplet digital Polymerase Chain Reaction for absolute quantification of gene expression and multivariate data analyses to determine whether lignin could therapeutically modulate the cell morphology and phenotype of inflamed, degenerating diseased human cells (osteoarthritic (OA) chondrocytes) towards a healthier cell morphology and phenotype. Lignin dose-dependently modified all aspects of cell morphology and ameliorated the diseased shape of OA chondrocytes by inducing a less fibroblastic healthier cell shape, which correlated with the downregulation of collagen 1A2 (COL1A2, a major fibrosis-inducing gene), upregulation of collagen 2A1 (COL2A1, a healthy extracellular matrix-inducing gene) and downregulation of interleukin-6 (IL-6, a chronic inflammatory cytokine). This is the first study to show that lignin can therapeutically target cell morphology and change a diseased cells’ function towards a healthier cell shape and phenotype. This opens up novel opportunities for exploiting lignin in modulation of disease, tissue degeneration, fibrosis, inflammation and regenerative medical implants for therapeutically targeting cell function and outcome.

Prediction of six macrophage phenotypes and their IL-10 content based on single-cell morphology using artificial intelligence

“…To determine if there were quantitative significant differences in shape descriptors (area, length, width, circularity, aspect ratio, roundness, and solidity), single-cell macrophage analysis was performed using a Fiji-based analysis algorithm ( 13 , 29 ). When comparing different groups of macrophages, the violin box plots ( Figure 5 ) revealed that the GM-CSF/TNFα/IFNγ-M1 (largest) and GM-CSF-M1 macrophages were larger in cell area than the other groups.…”

“…those inflamed and degenerated diseased human cells could be reverted to a healthier cell shape via therapeutic modulation and their healthier cell shape correlated with positive changes in major fibrosisand inflammatory-regulating genes (29). Thus, our method provides a simple and cost-effective means of capturing cellular responses by quantitating cell morphology.…”

“…The panel of morphological descriptors that we used here was successfully used by our group to phenotype differentiated human mesenchymal stromal cells (MSCs) ( 30 – 33 ) and healthy vs. inflamed and degenerating diseased human chondrocytes ( 13 ). Moreover, using this panel as a phenotypic marker, combined with multivariate data analysis, we showed that the cell morphology and phenotype, i.e., the “biological fingerprint” of those inflamed and degenerated diseased human cells could be reverted to a healthier cell shape via therapeutic modulation and their healthier cell shape correlated with positive changes in major fibrosis- and inflammatory-regulating genes ( 29 ). Thus, our method provides a simple and cost-effective means of capturing cellular responses by quantitating cell morphology.…”

“…Single macrophage analysis was performed using a Fiji-based ( 38 ) single-cell shape analysis algorithm that we previously used to phenotype differentiated mesenchymal stromal cells (MSCs) ( 6 , 30 – 33 ) and healthy vs. inflamed and degenerating chondrocytes ( 13 , 29 – 33 ). The fluorescent staining with DAPI and phalloidin visualized the cell’s nucleus and body (F-actin).…”

“…By combining cell imaging with a computational image analysis pipeline, here we tailor an automated high-throughput approach (13,(29)(30)(31)(32)(33) for single-cell morphological profiling of various human macrophage populations. Specifically, we focus on imaging cell morphology and intracellular IL-10 to assess the responsiveness and effector potential of these cells under different polarizing conditions.…”