The extracellular milieu plays a critical role in cell fate.  The niche, the microenvironment in which a cell resides in the living organism, is characterized by cell-cell adhesion and extracellular matrix molecules as well as soluble growth factors and morphogens. For example, whether a stem cell remains quiescent, or undergoes a symmetric or asymmetric division is influenced by the niche.  Key examples under study in our laboratory are muscle, neural and hematopoietic stem cells (HSC)that are capable of self-renewal and differentiation.  These cells exist in adult tissues for the purpose of regenerating damaged tissues. Niches are difficult to study in mammals in vivo.  Thus, our goal is to create such microenvironments that impact cell behavior and gene expression in vitro.

We are using bioengineering technologies to construct artificial in vitro microenvironments that recapitulate key biochemical and structural characteristics of muscle, neural and HSC niches.  The goal is to distill the physiological complexity of the niche into a smaller subset of distinct signaling interactions. This is possible because we can monitor the behavior of large numbers of single live cells by time lapse microscopy in microwell arrays of topographically structured polymer hydrogel surfaces.  By systematically analyzing the effects on cells of both bound insoluble and soluble components of the niche, we hope to define the extracellular regulators that alter nuclear function and cell fate determination.  An  elucidation of the microenvironmental molecular cues that control stem cell fate should prove useful in expanding or differentiating stem cells in a controlled manner.

Bioengineered microwell arrays

Culture of single stem cells in microwell arrays facilitates experiments designed to dissect the interplay of soluble and insoluble molecules that comprise the stem cell niche and allow analyses of fate changes of large numbers of single cells in a high-throughput manner. Microwells can be functionalized with different protein and combination of proteins up to 500 microwells/single well of a 96-well plate (50,000 microwells/plate). Statistical analysis of dynamic stem cell fate changes is obtained using automated time-lapse microscopy and classification analytic software.

Microwell chips on which single stem cells have been sorted and tracked by time-lapse microscopy. Eight time points are shown here. Heterogeneity in cell division kinetic is evident, with single cells either remaining quiescent, undergoing one, or multiple divisions to fill the microwell volume