The effect of nanofiber-guided cell alignment on the preferential differentiation of neural stem cells

Abstract

Stem cells display sensitivity to substrate presentation of topographical cues via changes in cell morphology. These biomechanical responses may be transmitted to the nucleus through cytoskeletal-linked signaling pathways. Here we investigate the influence of aligned substratum topography on the cell morphology and subsequently, the neuronal differentiation capabilities of adult neural stem cells (ANSCs). ANSCs that were cultured on aligned fibers elongated along the major fiber axis. Upon induction of differentiation with retinoic acid, a higher fraction of cells on aligned fibers exhibited markers of neuronal differentiation as compared with cells on random fiber or unpatterned surfaces. This effect was in part due to substrate selectivity, whereby aligned fiber substrates were less receptive to the attachment and continued survival of oligodendrocytes than random fiber or unpatterned substrates. Substrate-induced elongation alone was also effective in upregulating canonical Wnt signaling in ANSCs, which was further potentiated by retinoic acid treatment. These findings suggest a mechanism by which morphological control of stem cells operates in concert with biochemical cues for cell fate determination.

Figure 1

SEM images of aligned and random electrospun PCL fiber meshes; images were acquired at 10000×, scale bar = 1 μm. Fibers were electrospun from 12 wt%/R18 solution (a, b); 12 wt% solution (c, d); 14 wt% solution (e, f). The average fiber diameter for each condition is noted on the figure. (g) Histogram of fiber diameter distribution at each solution concentration, combining the diameters of aligned and random fibers. An approximate diameter value (260 nm, 480 nm, 930 nm) was used to refer to each of the groups in the text.

Figure 2

Representative fluorescent images of ANSCs on planar surfaces and fiber substrates. Images were acquired at 200× magnification and the scale bar = 50 μm. (a–c) GFP+ ANSCs under continued proliferation conditions on planar TCPS (a), aligned 480-nm PCL fibers (b), and random 480-nm PCL fibers (c). (d–f) ANSCs after 5 days of differentiation following a single dose of 0.5 μM RA and 0.5% FBS treatment. Cell nuclei were labeled with SYTOX Green and early neurons were immunostained with anti-Tuj1 antibody (red). (d) Cells on planar TCPS; (e) aligned 480-nm PCL fibers; (f) random 480-nm PCL fibers. Arrows on images indicate direction of fiber alignment on relevant substrates.

Figure 3

Quantification of early neuronal differentiation on various substrates. Columns represent the mean ±standard error (n=5) for each type of substrate. (a) % of Tuj1+ cells 5 daysafter RA/FBS differentiation at 12,500 cells/cm². (b) % of Tuj1+ and nestin+ cells on planar and 480-nm substrates 5 days after RA/FBS differentiation at 30,000 cells/cm².

Figure 4

Quantification of substrate selectivity to pre-differentiated cells. (a–d) ANSCs were differentiated using 100 ng/ml IGF and 100 ng/ml noggin for 5 days, and then seeded onto the substrates at 25,000/cm². Cells were analyzed and fixed for immunostaining after 24 h. (a) Total cell number on various substrates (dark bars: totalnumber of RIP+ cells); (b) percentage of RIP+ cells that were also apoptotic; (c) representative image cells on aligned fiber substrate; (d) cells on random fiber substrate. (e–h) ANSCs were differentiated using RA/FBS for 5 days, and then seeded at 25,000/cm². Cells were analyzed and fixed for immunostaining after 24 h. (e) Total cell number on various substrates (dark bars: total number of Tuj1+ cells); (f) percentage of Tuj1+ cells that were also apoptotic; (g) cells on aligned fiber substrate; (h) cells on random fiber substrate. Arrows indicate direction of alignment and arrowheads indicate doubly-stained (RIP+/apoptotic or Tuj1+/apoptotic) cells.

Figure 5

Activation of canonical Wnt/β-catenin signaling as measured using the TOPFLASH luciferase reporter assay. Luciferase readings were normalized to protein concentration of the sample. Readings are presented as fold-increase over the luciferase activity of untreated ANSCs seeded onto planar TCPS substrates (dotted line on each graph). (a) ANSCs without any treatment; (b) ANSCs treated with 0.5 μM RA and 0.5% FBS; (c) ANSCs treated with 150 ng/ml Wnt3a.