Stem cell colony interspacing effect on differentiation to neural cells

Abstract

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.

Aqueous Two-Phase Systems and Microfluidics for Microscale Assays and Analytical Measurements

“…The first use was with noncontact printing of embryonic stem cells onto support cells that induce differentiation (13). This has since been expanded to show that spacing between cell colonies can have a profound effect on differentiation (134). Tissue constructs can be formed using ATPSs, in analogy to cell sheets used in tissue engineering.…”

Novel approach for neuronal stem cell differentiation using aqueous two‐phase systems in 3D cultures

“…Alternatively, several studies have reported the use of PA6, a stromal cell line derived from mouse skull bone marrow, immersed in the PEG phase in which DEX droplets containing stem cells are printed to differentiate them into neural cells, demonstrating that by having direct contact with PA6 cells is sufficient to induce neural differentiation without the addition of external factors 21,30–33 …”

“…31 Alternatively, several studies have reported the use of PA6, a stromal cell line derived from mouse skull bone marrow, immersed in the PEG phase in which DEX droplets containing stem cells are printed to differentiate them into neural cells, demonstrating that by having direct contact with PA6 cells is sufficient to induce neural differentiation without the addition of external factors. 21,[30][31][32][33] Cell density, colony size and interconnectivity are geometrical parameters to consider during the construction of 3D cultures (Fig. 2) to enhance cell survival, migration, neurite outgrowth and lineage commitment.…”

“…Hence, computational models could be designed to identify the regulatory roles of them at different stages of neural differentiation. 33 Studies have shown that a space of 9 mm between each cell colony modulates stem cell fate through paracrine signaling, and therefore had a positive effect on neural differentiation. 21 Taking all the aforementioned factors together during the construction of ATPS-3D cultures and considering the advantage of using traditional laboratory equipment, the efficiency of neural differentiation from stem cells has been compared using Petri dishes and 96-well plates.…”

“…Interconnectivity of colonies is also essential to evaluate the exchange of soluble signaling factors. Hence, computational models could be designed to identify the regulatory roles of them at different stages of neural differentiation 33 . Studies have shown that a space of 9 mm between each cell colony modulates stem cell fate through paracrine signaling, and therefore had a positive effect on neural differentiation 21 …”