Stem cells are exposed to a range of molecular cues in vivo, such as soluble growth factors and crosslinked ECM components, biophysical cues such as substrate elasticity, and metabolic cues (Figure 1A) (8). The complexity of these signaling networks has motivated the adaptation of microarray platforms to screen tens to hundreds of putative stem cell microenvironments. Such microarrays consist of robotically spotted combinations of cell adhesion molecules and growth factors on cell-repellent substrates, which eliminates the problem of migration of the seeded cells between spots. Due to the restricted cell movement, any observed change in cell behavior could be attributed to the unique microenvironmental combination to which cells were initially exposed (Figure 2). Pioneering work on protein microarrays was performed by Flaim et al., who fabricated a microarray consisting of 32 combinations of collagen I, III, and IV; fibronectin; and laminin (9). By screening for liver-specific differentiation in embryonic stem (ES) colonies seeded onto these ECM arrays, they found a ~140-fold difference between the least and the most efficient protein combinations. Other groups extended the microarray concept from a pure ECM protein screen to cell adhesion molecules (CAM) and various growth factors that were physically adsorbed to a spin-coated layer of poly-dimethylsiloxane (PDMS) or covalently bound to aldehyde-derivatized glass slides (10,11). Using this strategy, Soen et al. could, for example, demonstrate that Notch ligands only led to an effect on primary human neural progenitor cell growth when covalently immobilized onto the substrate; incubation with soluble protein did not induce differentiation. They could also begin to dissect the effects of single versus combinations of growth factors, showing that Wnt and Notch co-stimulation maintained a progenitor cell state, whereas bone morphogenetic protein-4 induced the expression of both gliogenic and neuronal markers, a previously uncharacterized phenotype (10). LaBarge and colleagues systematically screened pairs of proteins for the induction of human mammary gland progenitor cell differentiation (11). Because each constituent was present in at least five different microenvironments, dominant compounds could be identified based on trends in these unique, but related substrates. Quiescence of cells could, for example, be attributed to laminin-1, and differentiation of myoepithelial cells (MEP) to P-cadherin. Notably, microarrays were fabricated to screen for a variety of cell types and target signal libraries, including synthetic polymers and mediators of cell-intrinsic programs (12,13). More recently, the integration of microarrays into a 96-well footprint using a custom-build gasket and microarray holder was described to screen for combinations of ECM molecules and soluble factors (14). These ground-breaking studies illustrate the need to dissect complex niche signaling into its individual components that can then be reassembled in a controlled fashion.
Micropatterning technologies can also be used to dissect spatial or temporal effects in microenvironmental stem cell regulation. In several studies, stem cell colonies of various sizes or shapes were fabricated in high throughput by micro-contact printing (15,16,17). For example, the control over colony size and separation revealed that cell shape influences human mesenchymal stem cell differentiation via a tension-based mechanism (15), or that embryonic stem cell (ESC) colony size and distribution influence ESC differentiation (16), suggesting an underlying colony size dependent paracrine signaling mechanism (17). To investigate these paracrine signaling effects on a single-cell level, an inverted micro-contact printing method termed FlipChip was developed to precisely pattern single ESCs (18). With this method, various spatial and neighboring effects on colony growth efficiency were tested, demonstrating that efficiency from single cells was very low (~40%) and could not be improved significantly by patterning multiple cells on one spot. However, when multiple cells were patterned close to each other but far enough to prevent cell-cell contact, colony forming efficiency reached nearly 100%. Thus micro-contact printing is a useful method for studying niche-related questions in stem cell biology.
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