Abstract
The balance between stem cell renewal and differentiation is determined by the interplay between intrinsic cellular controls and extrinsic factors presented by the microenvironment, or ‘niche’. Previous studies on cultured human epidermis have utilised suspension culture and restricted cell spreading to investigate regulation of differentiation in single keratinocytes. However, keratinocytes are typically adherent to neighbouring cells in vivo. In this thesis we developed experimental models to investigate the combined effect of cell-ECM adhesion and cell-cell contact on keratinocyte differentiation.We utilized lipid-modified oligonucleotides to form clusters of keratinocytes which were subsequently placed in suspension to induce terminal differentiation. In this experimental model cell-cell contact had no effect on suspension-induced differentiation of keratinocytes. We next developed a high-throughput platform for robust geometrical confinement of keratinocytes to hexagonal ECM-coated islands permitting direct cell-cell contact between single cells. As in the case of circular islands, differentiation was stimulated on the smallest hexagonal islands. However, the percentage of involucrin-positive cells on small bowtie islands was significantly lower than on single islands, demonstrating that cell-cell contact was affecting differentiation in response to decreased substrate adhesion. None of the small bowtie islands contained two involucrin-positive cells. Rather, in every case one of the cells was involucrin-positive and the other involucrin-negative. This suggests that there is intrinsic asymmetry in the effect of cell-cell contact in reducing differentiation. Thus, our reductionist approaches provide new insight into the effect of the niche on keratinocyte differentiation.
Finally, we explored Notch signalling regulation using a single-cell RNA sequencing dataset. We identified DTX2 and NEDD4L as potential regulators of Notch activity. Our analysis revealed that DTX2 and NEDD4L were highly expressed in transitional cell populations with markers of commitment and Notch targets. We performed validation experiments to confirm the expression of DTX2 and NEDD4L in human keratinocytes in vivo and in vitro. DTX2 knockdown resulted in an increase in involucrin expression and a decrease in colony-forming ability, suggesting that DTX2 activity may negatively regulate differentiation in vitro. NEDD4L expression resembled expression of Notch targets in keratinocyte populations separated by differential adhesion to collagen IV and in keratinocytes induced to differentiate in suspension, confirming our single-cell RNA sequencing analysis. Our findings identify DTX2 and NEDD4L as potential regulators of Notch activity and differentiation in human keratinocytes.
| Date of Award | 1 Nov 2022 |
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| Original language | English |
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| Supervisor | Fiona Watt (Supervisor) & Shukry Habib (Supervisor) |