In vitro formation of neuroclusters in microfluidic devices and cell migration as a function of stromal-derived growth factor 1 gradients

Abstract

Central nervous system (CNS) cells cultured in vitro as neuroclusters are useful models of tissue regeneration and disease progression. However, the role of cluster formation and collective migration of these neuroclusters to external stimuli has been largely unstudied in vitro. Here, 3 distinct CNS cell types, medulloblastoma (MB), medulloblastoma-derived glial progenitor cells (MGPC), and retinal progenitor cells (RPC), were examined with respect to cluster formation and migration in response to Stromal-Derived Growth Factor (SDF-1). A microfluidic platform was used to distinguish collective migration of neuroclusters from that of individual cells in response to controlled concentration profiles of SDF-1. Cell lines were also compared with respect to expression of CXCR4, the receptor for SDF-1, and the gap junction protein Connexin 43 (Cx43). All cell types spontaneously formed clusters and expressed both CXCR4 and Cx43. RPC clusters exhibited collective chemotactic migration (i.e. movement as clusters) along SDF-1 concentration gradients. MGPCs clusters did not exhibit adhesion-based migration, and migration of MB clusters was inconsistent. This study demonstrates how controlled microenvironments can be used to examine the formation and collective migration of CNS-derived neuroclusters in varied cell populations.

Keywords: SDF-1; central nervous system; chemotaxis; collective migration; medulloblastoma; retinal progenitors; stromal-derived growth factor.

Figure 1.

µLane microchannel device to measure chemotactic cell migration. (A) Device schematic. (B) Gradient profile based on measurements of fluorescent dextran (MW = 10kDa) at steady-state, t = 18 hours. Position x =0 is assigned to the sink well.

Figure 2.

Cell cluster formation 48 h after plating. Suspensions of MGPCs (Row A) RPCs (Row B), and MB (Row C) at 10⁴ cells/mL (Col 1), 10⁵ cells/mL (Col 2), and 10⁶ cells/mL (Col 3) were plated and photographed at 48 h. Representative single cells and clusters (arrows) are shown for each cell type.

Figure 3.

Changes in cluster number and size with time in culture. Cluster number (Column 1) and size by area (Column 2) are shown for suspensions of MGPCs (Row A), RPCs (Row B) and MB (Row C) at cell densities indicated in each panel. Data show mean and standard deviation, n > 4 wells.

Figure 4.

Chemotactic migration to SDF-1 assessed by transwell assay. Average number of cells migrated through an 8μm porous membrane toward 100 ng/mL SDF-1. Data show total migrated cell count, n > 3 wells.

Figure 5.

Cells in the µLane microfluidic device. Images of MGPC in µLane device 124, (A2) 10⁵, (A3) 10⁶ cells/mL seeding density. Likewise for RPCs (B1–3) and MB (C1–3). Representative clusters demarcated by arrows.

Figure 6.

Spatial plots of single cell and neurocluster trajectory. Representative and mean trajectories for both single cells (Column 1) and clusters (Column 2) along with length of center of mass (L_c) distribution for clusters vs single cells (Column 3) over 24 hr toward an increasing (0–100 ng/mL) SDF-1 gradient in RPCs (Row A) and MB (Row B). Positive y-axis indicates direction of increasing SDF-1 concentration.

Figure 7.

Immunocytochemistry of CXCR4 expression. ICC images of CXCR4 staining at 10⁴ (Column 1), 10⁵ (Column 2), and 10⁶ cell/mL (Column 3) stimulated with 100 ng/mL SDF-1 and comparison of relative fluorescence intensity to unstimulated control (Column 4) for MGPCs (Row A), RPCs (Row B) and MB (Row C). Green staining denotes CXCR4 for MGPCs. Red staining denotes Connexin 43 for RPCs and MB. Blue staining denotes cell nuclei. All scale bars 50 µm. *statistical significance (p<0.05).

Figure 8.

Immunocytochemistry of Connexin 43 expression. ICC images of Cx43 staining for both single cells (Column 1) and clusters (Column 2) stimulated with 100 ng/mL SDF-1 and comparison of relative fluorescence intensity to unstimulated control (Column 3) for MGPCs (Row A), RPCs (Row B) and MB (Row C). Green staining denotes Connexin 43 for MGPCs. Red staining denotes Connexin 43 for RPCs and MB. Blue staining denotes cell nuclei. All scale bars 50 µm. Statistical significance (p<0.05)** or (p<0.1)* between respective single cell or cluster SDF- and SDF+ groups.