Chirality-induced Lineage Enforcement of Mechanosensitive Mesenchymal Stem Cells Across Germ Layer Boundaries.

Mesenchymal to epithelial transition (MET) is instrumental in embryogenesis, tissue repair, and wound healing while the epithelial to mesenchymal transition (EMT) plays role in carcinogenesis. Alteration in microenvironment can modulate cellular signaling and induce EMT and MET. However, modulation of microenvironment to induce MET has been relatively less explored. In this work, effect of matrix stiffness in mediating MET in umbilical cord-derived mesenchymal stem cells (UCMSC) is investigated. Differential segregation of cell fate determinant proteins is one of the key factors in mediating altered stem cell fates through MET even though the genesis of apicobasal polarity remains ambiguous. Herein, it is also attempted to decipher if microenvironment-induced asymmetric cell division has a role to play in driving the cells toward MET. UCMSC cultured on stiffer PDMS matrices resulted in significantly (p < 0.05) higher expression of mechanotransduction proteins. It was also observed that stiffer matrices mediated significant (p < 0.05) upregulation of the polarity proteins and cell fate determinant protein, and epithelial marker proteins over lesser stiff substrates. On the contrary, expression of inflammatory and mesenchymal markers was reduced significantly (p < 0.05) on the stiffer matrices. Cell cycle analysis showed a significant increase in the G1 phase among the cells seeded on stiffer matrices. Transcriptomic studies validated higher expression of epithelial markers genes and lower expression of EMT markers. The transition from mesenchymal to epithelial phenotype depending on the gradation in matrix stiffness is successfully demonstrated. A computational machine learning model was developed to validate stiffness-MET correlation with 94% accuracy. The cross-boundary trans-lineage differentiation capability of MSC on bioengineered substrates can be used as a potential tool in tissue regeneration, organogenesis, and wound healing applications. In our present study, we deciphered the correlation between YAP/TAZ mechanotransduction pathway, EMT signaling pathway, and asymmetric cell division in mediating MET in MSC in a substrate stiffness-dependent manner. It is inferred that the stiffer PDMS matrices facilitate the transition from mesenchymal to epithelial state of MSC. Further, our study also proposed a scoring system to sort MSC from an intermediate hybrid E/M population while undergoing graded MET on matrices of different stiffnesses using a machine learning technique. This proposed scoring system can provide information regarding the E/M state of MSC on different bioengineered constructs based on their biophysical properties which may help in the proper choice of biomaterials in complex tissue-engineering applications.

Leveraging Substrate Stiffness to Promote Stem Cell Asymmetric Division via Mechanotransduction-Polarity Protein Axis and Its Bayesian Regression Analysis.

Asymmetric division of stem cells is an evolutionarily conserved process in multicellular organisms responsible for maintaining cellular fate diversity. Symmetric-asymmetric division pattern of mesenchymal stem cells (MSCs) is regulated by both biochemical and biophysical cues. However, modulation of mechanotransduction pathway by varying scaffold properties and their adaptation to control stem cell division fate is not widely established. In this study, we explored the interplay between the mechanotransduction pathway and polarity protein complex in stem cell asymmetry under varied biophysical stimuli. We hypothesize that variation of scaffold stiffness will impart mechanical stimulus and control the cytoskeleton assembly through RhoA, which will lead to further downstream activation of polarity-related cell signaling and asymmetric division of MSCs. To establish the hypothesis, umbilical cord-derived MSCs were cultured on polycaprolactone/collagen scaffolds with varied stiffness, and expression levels of several important genes (viz., Yes-associated protein [YAP], transcriptional coactivator with PDZ-binding motif [TAZ], LATS1, LATS2, Par3, Par6, PRKC1 [homolog of aPKC] and RhoA), and biomarkers (viz. YAP, TAZ, F-actin, Numb) were assessed. Support vector machine polarity index was employed to understand the polarization status of the MSCs cultured on varied scaffold stiffness. Furthermore, the Bayesian logistic regression model was employed for classifying the asymmetric division of MSCs cultured on different scaffold stiffnesses that showed 91% accuracy. This study emphasizes the vital role of scaffold properties in modulating the mechanotransduction signaling pathway of MSCs and provides mechanistic basis for adopting facile method to control stem cell division pattern toward improving tissue engineering outcome.

Substrate-dependent control of the chiral orientation of mesenchymal stem cells: image-based quantitative profiling.

Stem-cell (SC) chirality or left-right (LR) asymmetry is an essential attribute, observed during tissue regeneration. The ability to control the LR orientation of cells by biophysical manipulation is a promising approach for recapitulating their inherent function. Despite remarkable progress in tissue engineering, the development of LR chirality in SCs has been largely unexplored. Here, we demonstrate the role of substrate stiffness on the LR asymmetry of cultured mesenchymal stem cells (MSCs). We found that MSCs acquired higher asymmetricity when cultured on stiffer PCL/collagen matrices. To confirm cellular asymmetry, different parameters such as the aspect ratio, orientation angle and intensity of polarized proteins (Par) were investigated. The results showed a significant (p < 0.01) difference in the average orientation angle, the cellular aspect ratio, and the expression of actin and Par proteins in MSCs cultured on matrices with different stiffnesses. Furthermore, a Gaussian support-vector machine was applied to classify cells cultured on both (2% and 10% PCL/Collagen) matrices, with a resulting accuracy of 96.2%. To the best of our knowledge, this study is the first that interrelates and quantifies MSC asymmetricity with matrix properties using a simple 2D model.