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Mechanical Stretch Mobilizes Lgr6+ Epidermal Stem Cells To Drive Skin Growth
Yingchao Xue, PhD1, Ainsley L. Taylor, BS1, Chenyi Lyu, MS1, Amy van Ee, BS/MSE candidate1, Ashley Kiemen, MS1, YoungGeun Choi, BS1, Nima Khavanian, MD1, Dominic Henn, MD2, Chaewon Lee, BS candidate1, Lisa Hwang, BS candidate1, Eric Wier, PhD1, Saifeng Wang, PhD1, Sam Lee, PhD1, Ang Li, MD, PhD1, Charles Kirby, BS1, Pei-Hsun Wu, PhD1, Denis Wirtz, PhD1, Luis Garza, MD, PhD1, Sashank K. Reddy, MD, PhD1.
1The Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2University of Texas Southwestern Medical Center, Dallas, TX, USA.

PURPOSE: Current research in the field of wound regeneration has identified distinct populations of stem cells within specific skin components that regularly replenish the epidermis and hair follicles. The purpose of this study was to identify specific cellular and molecular determinants of skin growth to establish a platform for designing therapeutic strategies for conditions of excess or inadequate skin growth.
METHODS: Young adult mice underwent controlled tissue expansion of back skin. Lgr5/6-EGFP-CreERT2;tdTomato mice were used to lineage trace Lgr5/6+ stem cell progenitors and progeny. Ki67 co-staining quantified the proliferative index of these epidermal stem cells and their descendants. YAP gain- and loss-of-function mouse models were used to interrogate how Hippo pathway signaling contributed to skin growth. 3D skin model reconstructions characterized morphometric changes in growing skin. RNA microarray and single-cell RNA sequencing identified metabolic pathways changes. Pseudotime analysis assessed lineage trajectory of Lgr6+ cells.
RESULTS: Skin growth from mechanical tension is driven by proliferation of the epidermis. Morphometric changes include a 2-3-fold increase in keratinocyte cell layers, an overall increase in average cell size, a decrease in hair follicle density and sebaceous gland volume, and a compensatory increase in the dermal vascular plexus. Epidermal growth is achieved through preferential activation and differentiation of Lgr6+, but not Lgr5+, stem cells of the interfollicular epidermis. As measured by Ki67 staining, keratinocytes showed a maximal 10-fold increased proliferation, of which more than half was from Lgr6+ stem cell progeny. Pearson correlation coefficient confirmed Lgr6+ stem cells are biased toward differentiation rather than renewal. Tension modulates the activity of Hippo effector YAP to drive proliferation of Lgr6+ stem cells for creation of new skin. Gene pathway analysis suggested a robust tension-induced skin growth transcriptome program including up-regulation of growth regulatory (PI3K AKT) and inflammatory (cell chemotaxis, cytokine production, TNF and Il-17 signaling) pathways, as well as a hypoxia state of the skin. Down-regulated genes were associated with cell junctions and regulation of cell proliferation. Psuedotime analysis found that Lgr6+ stem cells of the bulge and interfollicular epidermis rapidly expand, activate, and differentiate to drive skin growth.
CONCLUSION: By defining the differential responses of skin elements to tension-dependent growth, we begin to identify targets to improve skin generation. Methods that may lead to more effective expansion include promoting fibroblast expansion, increasing the number of Lgr6+ stem cells, and direct activation of tension-responsive elements such as YAP. Up-regulated pathways offer additional molecular targets for enhancing skin growth. Future research may investigate the role of mechanical signaling as a driver of growth in other organs such as gut and lung.



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