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

Individual human epidermal cells differ in their self-renewal ability. To uncover the molecular basis for this heterogeneity, we performed genome-wide pooled RNA interference screens and identified genes conferring a clonal growth advantage on normal and neoplastic (cutaneous squamous cell carcinoma, cSCC) human epidermal cells. The Hippo effector YAP was amongst the top positive growth regulators in both screens. By integrating the Hippo network interactome with our data sets, we identify WW-binding protein 2 (WBP2) as an important co-factor of YAP that enhances YAP/TEAD-mediated gene transcription. YAP and WPB2 are upregulated in actively proliferating cells of mouse and human epidermis and cSCC, and downregulated during terminal differentiation. WBP2 deletion in mouse skin results in reduced proliferation in neonatal and wounded adult epidermis. In reconstituted epidermis YAP/WBP2 activity is controlled by intercellular adhesion rather than canonical Hippo signalling. We propose that defective intercellular adhesion contributes to uncontrolled cSCC growth by preventing inhibition of YAP/WBP2

In situ surface functionalisation of scaffolds using block copolymer self-assembly

Understanding human mesenchymal stem cell (hMSC) adhesion and differentiation in three-dimensional matrices in vitro is important for potential regenerative medicine and stem cell therapies. One of the key requirements for the use of scaffolds is that they correctly display the physicochemical properties mimicking those of the native extracellular matrix (ECM), in particular adhesive heterogeneity. Previous studies in 2D provide evidence for the effects of matrix properties such as stiffness, topography and surface chemistry. Yet, there are very few examples to date where ECM heterogeneity has been recapitulated and its effects on hMSCs investigated. The main aim of this research was to design topologically defined three-dimensional porous scaffolds. This was achieved by exploiting the self-assembly of amphiphilic diblock copolymers confined at an interface. Two methods of scaffold fabrication were used in these studies; high internal phase emulsion (HIPE) templating and electrospinning. In both studies, mixtures of two amphiphilic block copolymers were used to induce phase separation between the dissimilar hydrophilic blocks thereby creating distinct copolymer domains in the nanometer length scales on the scaffold surface. In both scaffold fabrication methods the amphiphilic block copolymers used were a combination of cell inert and cell adhesive chemistries, thereby generating matrices with distinct cell binding sites. The functionality and adhesive heterogeneity of these materials were characterised using varying techniques including x-ray photoelectron spectroscopy, chemical force spectroscopy mapping and contact angle measurements. The effect of adhesive heterogeneity of such matrices on human mesenchymal progenitor adhesion and differentiation based on block copolymer domains were investigated. It was found that hMSCs adhered in a block copolymer dependent manner to scaffolds that most closely mimicked the adhesive heterogeneity in native extracellular matrix

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation

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

Micro-scale topography mimics stem cell patterning in human interfollicular epidermal stem cells.</p

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

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.</p

Cell Instructive Microporous Scaffolds through Interface Engineering

The design of novel biomaterials for regenerative medicine requires incorporation of well-defined physical and chemical properties that mimic the native extracellular matrix (ECM). Here, we report the synthesis and characterization of porous foams prepared by high internal phase emulsion (HIPE) templating using amphiphilic copolymers that act as surfactants during the HIPE process. We combine different copolymers exploiting oil–water interface confined phase separation to engineer the surface topology of foam pores with nanoscopic domains of cell inert and active chemistries mimicking native matrix. We further demonstrate how proteins and hMSCs adhere in a domain specific manner

Cell Instructive Microporous Scaffolds through Interface Engineering

The design of novel biomaterials for regenerative medicine requires incorporation of well-defined physical and chemical properties that mimic the native extracellular matrix (ECM). Here, we report the synthesis and characterization of porous foams prepared by high internal phase emulsion (HIPE) templating using amphiphilic copolymers that act as surfactants during the HIPE process. We combine different copolymers exploiting oil–water interface confined phase separation to engineer the surface topology of foam pores with nanoscopic domains of cell inert and active chemistries mimicking native matrix. We further demonstrate how proteins and hMSCs adhere in a domain specific manner

3D Surface Functionalization of Emulsion-Templated Polymeric Foams

We describe the preparation of porous polymeric scaffolds via polymerization of the oil phase in high internal phase water-in-oil-emulsions using amphiphilic block copolymers polystyrene-<i>b</i>-poly­(ethylene oxide), polystyrene-<i>b</i>-poly­(acrylic acid), poly­(1,4-butadiene)-<i>b</i>-poly­(ethylene oxide), and poly­(1,4-butadiene)-<i>b</i>-poly­(acrylic acid) as surfactants. We show that the block copolymers anchor to the polymerized oil phase via the lipophilic block, which can occur by chemical and/or physical entanglement and consequent presentation of the hydrophilic block on the pore surfaces. The <i>in situ</i> polymerization enables the full surface functionalization of the porous materials with the final surface chemistry dictated by the hydrophilic block. Furthermore, the foam physical architecture may be tailored through controlling emulsion parameters such as the initiator, shear rate, and aqueous phase volume fraction