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.

Keywords: colony interspacing; concentration gradient; embryonic stem cells; neural differentiation; soluble factors; stromal cells.

Figure 1

Aqueous two-phase system microprinting of mESCs on PA6 stromal cells to generate a pair of mESC colonies with a defined interspacing. (a) Dispensing pins are loaded with mESCs suspended in the aqueous DEX phase. (b) Pins are lowered into the culture plate containing PA6 cells monolayer immersed in the aqueous PEG phase and the pins content autonomously dispenses. (c) The process is repeated once more to form two isolated drops confining mESCs and with a defined interspacing. (d) Microprinted mESCs from both drops attach to the stromal layer and proliferate to form two colonies. (e) A pair of distinct DEX phase drops confining mESCs. (f,g) Colony pairs at two different interspacing distances on day 8 of culture. (h) Measured edge-to-edge distance between the two colonies formed varies linearly with center-to-center distance between the corresponding DEX phase drops. (i, j) Neurite processes extending out between the two colonies in panels b and c, respectively. * p < 0.01. n=18. Error bars represent mean ± S.E.M.

Figure 2

TuJ expression of interspaced mESC colonies. (a–c) Immunostained images of TuJ-positive colony pairs at three different interspacing distances on day 8 of culture. (d) Magnified view of the boxed portion of panel (b) showing the neural processes. (e) TuJ fluorescent intensity normalized to the total perimeter of both colonies shows the highest intensity at the interspacing of 0.66D, followed by the 0.02D and then 1.40D spacing. (f) Total neurites density normalized to the total colony perimeter shows results similar to those from the fluorescent intensity measurements in panel (e). * p < 0.01, n=18. Error bars represent mean ± S.E.M.

Figure 3

Nestin expression of interspaced mESC colonies. (a–c) Immunostained images of Nestin-positive colony pairs at three different interspacing distances on day 8 of culture. (d) Magnified view of a portion of panel (b) showing Nestin stained neural processes. (e) Nestin fluorescent intensity normalized to the total perimeter of both colonies shows the highest intensity when the two colonies are interspaced at 0.66D, followed by at 0.02D and then at 1.40D. * p < 0.01, n=18. Error bars represent mean ± S.E.M.

Figure 4

GFAP and TH expression of interspaced mESC colonies. (a–c) Immunostained images of GFAP-positive colony pairs at three different interspacing distances on day 14 of culture. (d) Magnified view of a portion of panel (b) showing GFAP stained astroglial progenitors. (e) GFAP fluorescent intensity normalized to the total perimeter of both colonies increases with increasing the colony interspacing. (f) An image of TH-positive cells at the periphery of a colony on day 14. (g) TH-positive cell count per colony shows the highest number at 0.66D interspacing, followed by at 0.02D and then at 1.40D. * p < 0.01, n=18. Error bars represent mean ± S.E.M.

Figure 5

Finite element modeling of spatial distribution of mESCs-secreted soluble factors. (a–c) Concentration profiles of diffusive signaling factors of mESC colonies at different interspacing distances. (d–f) Overlay of magnified concentration maps and TuJ expression of cells in the colony pairs. (g) Line scans of concentration profiles for colony pairs along the dashed lines in panels (d–f).

Figure 6

Average normalized mRNA fold change of neural genes from the two-week culture in three different colony interspacing distances. * p < 0.05. Error bars represent mean ± S.E.M.