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. 2013 Feb 25;9(4):585-95.
doi: 10.1002/smll.201202208. Epub 2012 Oct 26.

Microfluidic investigation of BDNF-enhanced neural stem cell chemotaxis in CXCL12 gradients

Hui Xu  1 , Sarah C Heilshorn
Affiliations

Microfluidic investigation of BDNF-enhanced neural stem cell chemotaxis in CXCL12 gradients

Hui Xu et al. Small. .

Abstract

In vivo studies have suggested that gradients of CXCL12 (aka stromal cell-derived factor 1α) may be critical for neural stem cell (NSC) migration during brain development and neural tissue regeneration. However, traditional in vitro chemotaxis tools are limited by unstable concentration gradients and the inability to decouple cell migration directionality and speed. These limitations have restricted the reproducible and quantitative analysis of neuronal migration, which is required for mechanism-based studies. Using a microfluidic gradient generator, nestin and Sox-2 positive human embryonic NSC chemotaxis is quantified within a linear and stable CXCL12 gradient. While untreated NSCs are not able to chemotax within CXCL12 gradients, pre-treatment of the cells with brain-derived neurotrophic factor (BDNF) results in significant chemotactic, directional migration. BDNF pre-treatment has no effect on cell migration speed, which averages about 1 μm min(-1). Quantitative analysis determines that CXCL12 concentrations above 9.0 nM are above the minimum activation threshold, while concentrations below 14.7 nM are below the saturation threshold. Interestingly, although inhibitor studies with AMD 3100 revealed that CXCL12 chemotaxis requires receptor CXCR4 activation, BDNF pre-treatment is found to have no profound effects on the mRNA levels or surface presentation of CXCR4 or the putative CXCR7 scavenger receptor. The microfluidic study of NSC migration within stable chemokine concentration profiles provides quantitative analysis as well as new insight into the migratory mechanism underlying BDNF-induced chemotaxis towards CXCL12.

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Figures

Figure 1
Figure 1
CXCL12 gradient verification and NSC characterization. (A) Structure of the microfluidic gradient generator device. A cell culture chamber is connected to sink (blue) and source (red) channels via micro-capillaries (not observable at this magnification). (B) At equilibrium, a linear and stable CXCL12 gradient was generated across the cell culture chamber, as quantified by the fluorescence intensity of a tracer molecule (Texas Red-conjugated dextran, 10 kDa). Confocal microscopy of NSCs derived from human embryonic stem cells revealed positive immunostaining for two NSC markers nestin (C) and Sox-2 (D) and counter-staining with DAPI to visualize cell nuclei (E).
Figure 2
Figure 2
Experimental assay and data analysis. (A) Human embryonic NSCs were loaded into microfluidic devices mounted on chamber slides, resulting in random cell seeding. The migratory path of each individual cell within a stable CXCL12 gradient was tracked for up to 17 hrs. (B) The migration speed (D/time) and chemotactic index (X/D) were calculated for each cell track. (C) CXCL12 and/or BDNF did not significantly increase NSC proliferation upon 48-hr exposure (p > 0.05).
Figure 3
Figure 3
NSC cell tracks and angular histograms of cell endpoint positions. Individual cell tracks (A,C,E,G,I) are plotted with all cell initial positions at the origin (0,0). The number of cells migrating towards (red tracks) or away from (black tracks) the CXCL12 source is given for each condition. The center of mass for the endpoint positions is marked with a gold star. For angular histogram plots (B,D,F,H,J), the Rayleigh p-value of each endpoint distribution is shown, with p < 0.05 suggesting a statistically significant asymmetric distribution. No significant NSC chemotaxis towards CXCL12 was observed during 4-hr (A,B) or 17-hr (C,D) observations. A 1-hour BDNF pretreatment induced chemotaxis towards CXCL12 during both 4-hr (E,F) and 17-hr (G,H) observations. Treatment with AMD 3100 for 1 hr following a 1-hr BDNF exposure caused a loss of directional cell migration towards CXCL12(I, J).
Figure 4
Figure 4
Quantification of cell chemotactic index and migration speed. (A) Chemotactic indices showed broad population distributions at 4 hr and became narrower at 17 hr. BDNF treatment significantly increased chemotactic indices, while AMD 3100 treatment significantly attenuated NSC chemotaxis. (B) Cell migration speed was similar across all observation times and BDNF treatments, but decreased upon treatment with AMD 3100. * p<0.05, ***p<0.001
Figure 5
Figure 5
Evaluation of NSC chemotaxis within various CXCL12 concentrations. (A) To evaluate the range of absolute CXCL12 concentrations that induced cell polarization, cell tracks were divided into groups based on their initial positions within zones I, II, III, or IV. Absolute CXCL12 concentration increases across the four zones, while gradient steepness is identical. (B) Cells in all four zones exhibited similar chemotactic indices. (C) Cell migration speed was similar in zones I, II, and III, while cells in zone IV migrated more slowly, due to cell interactions with culture chamber wall, which impedes further migration and results in an artificially lowered average cell speed, * p<0.05.
Figure 6
Figure 6
Analysis of receptor and integrin expression. (A) qPCR analysis shows no significant changes in mRNA transcript levels for CXCR4 or CXCR7 upon 1-hr BDNF exposure. (B) Upon 24-hr BDNF treatment, mRNA transcripts for CXCR4, CXCR7, and the α6 integrin sub-unit were marginally upregulated (less than two fold), while the β1 integrin sub-unit had no change * p<0.05. Flow cytometry analysis demonstrated that cell surface receptor expression of CXCR4 (C) and CXCR7 (D) was not significantly altered by BDNF treatment for 1hr.
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