Research Article: Micro-scaled topographies direct differentiation of human epidermal stem cells

Date Published: January 15, 2019

Publisher: Elsevier

Author(s): Sebastiaan Zijl, Aliaksei S. Vasilevich, Priyalakshmi Viswanathan, Ayelen Luna Helling, Nick R.M. Beijer, Gernot Walko, Ciro Chiappini, Jan de Boer, Fiona M. Watt.


Human epidermal stem cells initiate terminal differentiation when spreading is restricted on ECM-coated micropatterned islands, soft hydrogels or hydrogel-nanoparticle composites with high nanoparticle spacing. The effect of substrate topography, however, is incompletely understood. To explore this, primary human keratinocytes enriched for stem cells were seeded on a topographical library with over 2000 different topographies in the micrometre range. Twenty-four hours later the proportion of cells expressing the differentiation marker transglutaminase-1 was determined by high content imaging. As predicted, topographies that prevented spreading promoted differentiation. However, we also identified topographies that supported differentiation of highly spread cells. Topographies supporting differentiation of spread cells were more irregular than those supporting differentiation of round cells. Low topography coverage promoted differentiation of spread cells, whereas high coverage promoted differentiation of round cells. Based on these observations we fabricated a topography in 6-well plate format that supported differentiation of spread cells, enabling us to examine cell responses at higher resolution. We found that differentiated spread cells did not assemble significant numbers of hemidesmosomes, focal adhesions, adherens junctions, desmosomes or tight junctions. They did, however, organise the actin cytoskeleton in response to the topographies. Rho kinase inhibition and blebbistatin treatment blocked the differentiation of spread cells, whereas SRF inhibition did not. These observations suggest a potential role for actin polymerization and actomyosin contraction in the topography-induced differentiation of spread cells.

The epidermis is the outer covering of the skin. It is formed by layers of cells called keratinocytes. The basal cell layer contains stem cells, which divide to replace cells in the outermost layers that are lost through a process known as differentiation. In this manuscript we have developed surfaces that promote the differentiation of epidermal stem cells in order to understand the signals that control differentiation. The experimental tools we have developed have the potential to help us to devise new treatments that control diseases such as psoriasis and eczema in which epidermal stem cell proliferation and differentiation are disturbed.

Partial Text

Human skin is comprised of two layers: the dermis, which is a well vascularised connective tissue, and the epidermis, which is formed of multiple layers of epithelial cells called keratinocytes. While the dermis gives the skin its flexibility and strength, the main functions of the epidermis are to prevent water loss and to protect the body from external threats such as infections and mechanical stress [1]. Within the epidermis, the basal layer contains stem cells that self-renew and generate daughter cells that undergo terminal differentiation as they move through the suprabasal layers towards the tissue surface [2], [3].

Cell-cell and cell-ECM interactions are key components of the epidermal stem cell niche [2], [4]. We have previously shown that restricted spreading on ECM-coated micropatterned islands, soft hydrogels and gold nanodots triggers the terminal differentiation of keratinocytes [7]. We have also shown that substrates that mimic the undulations of the human epidermal-dermal junction control stem cell patterning [29]. However, the response of keratinocytes to micron-scale topographical features has not been examined previously.




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