Mimicking the topography of the epidermal–dermal interface with elastomer substrates

Abstract

In human skin the interface between the epidermis and dermis is not flat, but undulates. The dimensions of the undulations change as a function of age and disease. Epidermal stem cell clusters lie in specific locations relative to the undulations; however, whether their location affects their properties is unknown. To explore this, we developed a two-step protocol to create patterned substrates that mimic the topographical features of the human epidermal-dermal interface. Substrates with negative patterns were first fabricated by exposing a photocurable formulation to light, controlling the topographical features (such as diameter, height and center-to-center distance) by the photomask pattern dimensions and UV crosslinking time. The negative pattern was then translated to PDMS elastomer to fabricate substrates with 8 unique surface topographies on which primary human keratinocytes were cultured. We found that cells were patterned according to topography, and that separate cues determined the locations of stem cells, differentiated cells and proliferating cells. The biomimetic platform we have developed will be useful for probing the effect of topography on stem cell behaviour.

A genome-wide screen identifies YAP/WBP2 interplay conferring growth advantage on human epidermal stem cells

“…Using PDMS substrates that mimic the topography of the epidermal-dermal interface to recreate the patterned distribution of SCs in adult human epidermis14, we observed nuclear YAP and WBP2 localization to be restricted to integrin β1-bright SC clusters at the tips of the topographies (Supplementary Fig. 12a,b).…”

“…PDMS elastomer substrates mimicking the topography of the epidermal-dermal interface were fabricated as described14. Topography S1 was used for experiments14.…”

“…NHKs were seeded onto rat-tail collagen type I (20 μg ml −1 in PBS, BD Biosciences)-coated PDMS substrates in complete FAD medium at a density of 5 × 10 5 cells per cm 2 . After 45 min, substrates were rinsed gently to remove non-adherent cells and transferred to 12-well cell culture plates (Falcon) containing a 3T3-J2 feeder layer14.…”

“…NHK colonies grown in the presence of a 3T3-J2 feeder layer were fixed in 4% (w/v) paraformaldehyde (Sigma) for 45 min and permeabilized with 0.5% (v/v) Triton X-100 for 45 min at ambient temperature. Confluent NHK sheets grown on PDMS substrates with patterned topographies were processed for immunofluorescence microscopy as previously described14. Samples were blocked for 1 h in 10% (v/v) FBS plus 0.25% (v/v) fish skin gelatin (Sigma-Aldrich) in 1 × PBS (blocking buffer), and incubated with primary antibodies (diluted in blocking buffer) in a humid chamber overnight at 4 °C.…”

“…A subset of highy proliferative epidermal cells has the potential to generate large stratified colonies that subsequently fuse to form multi-layered cell sheets, recapitulating the organization of the epidermis9111213. This culture system has been widely used to study human epidermal SCs and their regulation1112131415, and epidermal sheets generated in vitro are used for autologous transplantation in patients suffering from severe burn wounds or hereditary skin blistering diseases1617. The grafted epidermal sheets can persist as a histologically and physiologically normal epidermis for years161718.…”