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Stem cell colony interspacing effect on differentiation to neural cells.
Efforts to enhance the efficiency of neural differentiation of stem cells are primarily focused on exogenous modulation of physical niche parameters such as surface topography and extracellular matrix proteins, or addition of certain growth factors or small molecules to culture media. We report a novel neurogenic niche to enhance the neural differentiation of embryonic stem cells (ESCs) without any external intervention by micropatterning ESCs into spatially organized colonies of controlled size and interspacing. Using an aqueous two-phase system cell microprinting technology, we generated pairs of uniformly sized isolated ESC colonies at defined interspacing distances over a layer of differentiation-inducing stromal cells. Our comprehensive analysis of temporal expression of neural genes and proteins of cells in colony pairs showed that interspacing two colonies at approximately 0.66 times the colony diameter (0.66D) significantly enhanced neural differentiation of ESCs. Cells in these colonies displayed higher expression of neural genes and proteins and formed thick neurite bundles between the two colonies. A computational model of spatial distribution of soluble factors of cells in interspaced colony pairs showed that the enhanced neural differentiation is due to the presence of stable concentration gradients of soluble signalling factors between the two colonies. Our results indicate that culturing ESCs in colony pairs with defined interspacing is a promising approach to efficiently derive neural cells. Additionally, this approach provides a platform for quantitative studies of molecular mechanisms that regulate neurogenesis of stem cells.
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