NEWS AND VIEWS Microarraying the cellular microenvironment

The in vivo stem cell microenvironment or niche is composed of an intricate blend of extracellular matrix proteins, soluble protein factors, immobilized protein factors, proteoglycans, small molecule signals, in some cases mineralized tissue, and numerous adjacent cell types, all of which likely vary in space and time. In concert, these many components present the cells with an ‘array’ of biochemical and biomechanical signals, such that the cell is continually faced with the complex tasks of sensing these inputs, processing the signals through complex signal transduction and gene regulation networks, and executing cell behavioral or fate choices. This native microenvironment therefore poses a daunting challenge to the fields of stem cell biology and engineering: how does one analyze the relative importance of the individual signals of this medley in regulating cell function? In addition to advancing our basic knowledge of the central question of stem cell fate choice, addressing this question will be necessary to achieve the control over cell behaviors needed for regenerative medicine efforts. Reductionist biological approaches have consistently made major strides in elucidating the importance of individual signaling factors; however, cells in general sense and respond to combinations of extracellular signals rather than any single one (Janes et al, 2005). In an article currently published in Molecular Systems Biology, Theo Palmer, Patrick Brown, and colleagues reported on a novel, high-throughput approach to address this problem (Soen et al, 2006). As the number of combinations to be investigated rapidly increases with the number of individual factors, highthroughput and density approaches to sample this signaling space will be of fundamental importance to this challenge. Arrays of cDNA (Ziauddin and Sabatini, 2001), biomaterials (Anderson et al, 2004), and extracellular matrix proteins (Flaim et al, 2005) have proven to be powerful approaches to exploring cell responses to varying signals and microenvironments. Soen et al (2006) have further extended array technologies by building a microarray system to systemically investigate the effects of numerous morphogens, growth factors, cell adhesion molecules, and extracellular matrix components on the fate of primary bi-potent human neural precursor cells that can differentiate into neurons or glial cells (Figure 1). A non-contact, piezoelectric arrayer was utilized to print combinations of factors onto an aldehyde-activated surface and thereby generate arrays of 350–500 mm diameter spots of factors covalently grafted to the surface. Human neural precursors were then isolated from tissue, seeded onto the surface, and cultured under uniform soluble media conditions. Because all cells on the array were exposed to uniform culture medium, this approach focused on the effects of the immobilized ligands on cell function, with the possible exception of local autocrine or paracrine effects that could be modulated by varying the distance between spots. Cellular behaviors (i.e. differentiation and proliferation) were then monitored by immunostaining for bromodeoxyuridine and cell fate markers, followed by high-throughput quantitative imaging analysis at the single-cell level. Utilizing strong experimental analysis, they found numerous interesting phenotypic outcomes. Some factors that bias cell differentiation into glia exerted additive effects when utilized in combination (e.g. the Notch ligand Jagged-1 and ciliary neurotrophic factor). However, others that would be anticipated to exert conflicting effects on cell differentiation (e.g. Wnt-3A versus Notch ligands) unexpectedly resulted in enhanced cell proliferation with low expression of cell fate