| AVS 55th International Symposium & Exhibition | |
| Biomaterial Interfaces | Tuesday Sessions |
| Session BI+NC-TuA |
| Session: | Protein and Cells Interactions on Micro- and Nanofabricated Substrates |
| Presenter: | M.P. Lutolf, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland |
| Authors: | M.P. Lutolf, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland R. Doyonnas, Stanford University H.M. Blau, Stanford University |
| Correspondent: | Click to Email |
A complex mixture of extracellular cues delivered by support cells is critical for adult stem cell maintenance and regulation of self-renewal in their microenvironment, termed niche. Despite recent progress in the identification of relevant niche proteins and signaling pathways in mice, to date, hematopoietic stem cells (HSCs) cannot be efficiently cultured in vitro without rapidly differentiating. We are developing novel in vitro culture paradigms that allow fate decisions of individual stem cells to be monitored under well-controlled conditions and in real time. We have engineered microarrayed artificial niches based on a combination of biomolecular hydrogel and microfabrication technologies that allow key biochemical characteristics of adult stem cell niches to be mimicked and the physiological complexity deconstructed into a smaller, experimentally amenable number of distinct signaling interactions. Several thousand single stem cells were tracked by fluorescent time-lapse microscopy in these microarrays over a period of several days. Image analysis allowed individual cell fate changes and growth kinetics of entire populations to be statistically analyzed. Subsequent retrospective single cell RT-PCR and transplantation experiments were performed in order to correlate kinetic behavior with phenotype and function. Screening of ca. 20 putative soluble HSC regulators, including Wnt-3a and TPO, as well as surface-tethered cell-cell adhesion proteins such as N-Cadherin, allowed to identify factors that dictate distinct HSC cell cycle kinetics. Based on patterns in kinetic behavior and single cell gene expression profiles induced by stimulation with a few of these candidates, we distinguished hallmarks of self-renewal from differentiation divisions, and validated these disparate behaviors in vivo by subsequent HSC transplantation into lethally irradiated mice. Therefore, the systematic deconstruction of a stem cell niche may serve as a generalizeable paradigm for defining and reconstructing artificial niches to accelerate the transition of stem cell biology to the clinic.