Final Report Summary - S.CE.N.E. (Deconstructing the stem cell niche in human interfollicular epidermis in vitro.) There are great expectations that somatic (or adult) stem cells can be used to treat disease, and those expectations have moved somatic stem cells into the focus of research. Somatic stem cells retain their unique functions only if they are in intimate contact with an instructive microenvironment, termed the stem cell niche. In these niches, stem cells integrate a complex array of molecular signals that, in concert with cell-intrinsic gene regulatory networks, control their function and balance their numbers in response to physiologic demands. To shed light on the mechanisms that regulate stem cell behaviour, approaches that allow the study of stem-cell functions in response to isolated components of a complex system are therefore crucial. The focus of this research project (Stem Cell Niche Exploration, S.CE.N.E) was to deconstruct the stem cell niche in human inter-follicular epidermis (IFE). The IFE constitutes the outermost layer of the skin that protects us against the physical, chemical, and thermal assaults of our environment, and represents a self-renewing tissue that is maintained by proliferation of stem cells and terminal differentiation of their progeny. By taking adult human IFE stem cells out of their natural microenvironment and placing them ex vivo into artificial microenvironments, we aimed to define the relative importance of cell-intrinsic and microenvironmental stimuli in regulating commitment to terminal differentiation, and to discover how intrinsic, genetic changes in human IFE stem cells impact on the niche. We anticipated that contact between cells and consequently cell-cell communication play an important role in regulating human IFE stem cell fate decisions, given the multi-cellular, multi-layered organisation of the IFE. Key questions to be addressed were whether cell-cell and cell-substrate signals had synergistic or antagonist effects, and whether one type of signal could over-ride another. To expose large numbers of single human IFE stem cells to distinct niche signals in a controllable experimental environment, we designed a (custom-manufactured) glass microchip that contains two different arrays of micro-patterned circular adhesive islands, each able to capture tens of thousands of single human IFE stem cells. This unique design allowed us to monitor IFE stem cell fate decisions ex vivo: on one type of adhesive islands (20 um diameter), IFE stem cells adhere but cannot spread, forcing them to commit to terminal differentiation within 24 hours, whereas on the other type of islands (50 um diameter) IFE stem cells spread out and remain in a proliferative and undifferentiated state. Combined with the possibility to functionalize the adhesive islands with mixtures of different proteins of choice, the microchip provides a versatile platform to monitor the impact of different niche signals on fate decisions of single human IFE stem cells, including cell-cell and growth factor signalling. High content imaging analysis made it possible to monitor (in an automated, unbiased fashion) the fate decisions of every single IFE stem cell in the entire cell population captured on one microchip, thus generating statistically highly significant datasets. In addition, we were also successful in functionalizing fluorescent microbeads with recombinant proteins, which will enable us to study the effects of receptor-ligand interactions on the surface of single IFE stem cells in real time in the near future. For this purpose, we have also started to generate fluorescent reporter cell lines to monitor IFE stem cell fate decisions in live cells. To study how extrinsic signals from the micro-environment modulate the activity of a key factor controlling IFE stem cell fate, we focused on the transcriptional co-activator YAP. We found that in human IFE stem cells, similar to other stem cell types, YAP is, at least in part, regulated by the physical properties of the underlying substrate, as well as by growth factor signalling. We could also show that manipulation of cell-intrinsic signalling by overexpression of YAP can overcome differentiation-commitment signals from the niche. Thus, we have identified situations in which an intrinsic stem cell signal modifies the response to a microenvironmental signal and vice versa. To understand the impact of cell-cell communication on human IFE stem cell fate decisions, we initially focused our research on cell-cell adhesion proteins. However, we found that functionalizing the micro-patterned adhesive islands on our microchip with distinct cell-cell adhesion molecules had no impact on human IFE stem cell fate decisions. We then focused on Notch receptor-ligand interactions and discovered that by functionalizing the micro-patterned adhesive islands on our microchip with distinct Notch ligands (known to be expressed in the human IFE stem cell niche) we can manipulate IFE stem cell fate decisions. We found that some Notch ligands induced strong Notch receptor activation and consequently commitment to terminal differentiation in IFE stem cells growing under proliferating conditions (i.e. on 50 um diameter islands), whereas others induced week Notch receptor activation and were instead able to partially block differentiation (of IFE stem cells growing on 20 um diameter islands). We feel confident that our research has increased our understanding about how somatic stem cells integrate cell-intrinsic gene-regulatory networks with extrinsic signals received from their microenvironment to maintain tissue homeostasis. Moreover, we strongly believe that our research will also have translational applications in the near future. Given the conservation of intrinsic and extrinsic stem cell regulators in different tissues, our in vitro platform can be used to probe niche interactions in other adherent stem cell populations and as a platform for drug discovery. As outlined in the EU Directive 2010/63/EC (Legislation for the Protection of Animals used for Scientific Purposes, in full effect since 1 January 2013), European research should increasingly focus on developing new research models, tools and approaches with reduced reliance on animal use and improved animal welfare. We envisage that our in vitro platform can provide a useful alternative to animal testing, allowing for the fast and direct analysis of potential toxic effects of substances on human IFE stem cells.
