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Differential Marker Expression between Keratinocyte Stem Cells and Their Progeny Generated from a Single Colony.

ABSTRACT: The stemness in keratinocyte stem cells (KSCs) is determined by their gene expression patterns. KSCs are crucial in maintaining epidermal homeostasis and wound repair and are widely used candidates for therapeutic applications. Although several studies have reported their positive identifiers, unique biomarkers for KSCs remain elusive. Here, we aim to identify potential candidate stem cell markers. Human epidermal keratinocytes (HEKs) from neonatal foreskin tissues were isolated and cultured. Single-cell clonal analysis identified and characterized three types of cells: KSCs (holoclones), transient amplifying cells (TACs; meroclones), and differentiated cells (DSCs; paraclones). The clonogenic potential of KSCs demonstrated the highest proliferation potential of KSCs, followed by TACs and DSCs, respectively. Whole-transcriptome analysis using microarray technology unraveled the molecular signatures of these cells. These results were validated by quantitative real-time polymerase chain reaction and flow cytometry analysis. A total of 301 signature upregulated and 149 downregulated differentially expressed genes (DEGs) were identified in the KSCs, compared to TACs and DSCs. Furthermore, DEG analyses revealed new sets of genes related to cell proliferation, cell adhesion, surface makers, and regulatory factors. In conclusion, this study provides a useful source of information for the identification of potential SC-specific candidate markers.

SUBMITTER: Ali D

PROVIDER: S-EPMC8509450 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation in vitro during the process of nascent epithelial sheet formation as triggered by the calcium switch model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated soft (nominal E = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on stiff (nominal E = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on soft substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two ad hoc analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly cooperative behavior of keratinocytes cultured on soft versus stiff gels during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned along the periphery of an evolving cell colony.

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Differential Marker Expression between Keratinocyte Stem Cells and Their Progeny Generated from a Single Colony.

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