Nanofiber matrices promote the neuronal differentiation of human embryonic stem cell-derived neural precursors in vitro

Abstract

The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs.

FIG. 1.

Scanning electron microscopy characterization of electrospun fiber matrices. Aligned (A) and random (A′) microfiber scaffolds prepared with 12 wt% polycaprolactone solution. Aligned (B) and random (B′) nanofiber scaffolds fabricated with a 12 wt% solution/0.01% octadecyl rhodamine B chloride. Images were acquired at high magnification (10,000 ×). Scale bars are 1 μm.

FIG. 2.

Scanning electron microscopy images of human NPs seeded on poly-L-ornithine/laminin-coated fiber meshes for 3 days. (A) Cells cultured on an aligned nanofiber substrate. Human NPs are attached on the scaffold and acquire an elongated shape when they grow on aligned fibrous substrates. (B, C) Cells on a random nanofiber substrate. NPs spread across the fiber mat surfaces, forming a continuous cell layer (B) where multiple cell–cell interactions are observed (C). Panels (B) and (C) are low- and high-power photographs, respectively, of cells on randomly oriented nanofibers. Scale bars are 10 μm. NPs, neural precursors.

FIG. 3.

Immunofluorescent images of nestin (+) human NPs cultured onto different substrates. The cytology of human NPs cultured on aligned (A, A′) and random (B, B′) nanofiber matrices was examined following nestin immunocytochemistry (NPs are in red) and epifluorescence (A, B) or confocal microscopy (A′, B′). DAPI counterstaining (blue) was used to observe cell nuclei. Scale bars are 50 μm. DAPI, 4′,6′-diamidino-2-phenylindole. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Effect of fiber alignment and diameter on human NPs viability. (A) The MTT assay was used to assess the viability of cultured human NPs on different fibrous scaffolds in the presence of mitogens. Overall variance was studied with ANOVA and was significant (p < 0.05). Significant differences between groups based on Tukey's post hoc multiple comparison test are indicated by an asterisk. (B) Quantitative analysis of nestin-positive cells cultured on different substrates in the absence of growth factors. As detailed in the Materials and Methods section, experiments were performed three times; 10 visual fields were randomly selected per culture and over 1500 cells were manually counted. Results were grouped per substrate type and studied with ANOVA. Variance was significant (p < 0.05). Significant differences between groups based on post hoc testing are indicated by an asterisk. Bars represent mean ± standard error in both panels. ANOVA, analysis of variance; TCPS, tissue culture polystyrene surface; ALN nano, aligned nanofibrous substrate; RN nano, random nanofibrous substrate; ALN micro, aligned microfibrous substrate; RN micro, random microfibrous substrate.

FIG. 5.

Effect of various types of substrates on the neuronal differentiation of human NPs. Fluorescence microscopy images of cells cultured on aligned (A) and random (B) fibrous substrates and on TCPS (C) for 15 days in the absence of growth factors. The phenotypic differentiation of human NPs was assessed by immunocytochemistry for TUJ1, a class III β-tubulin protein that marks early neurons (green). DAPI (blue) was used for counterstaining of nuclei. The fractions of TUJ1+ cells are shown in (D). Data represent three independent experiments, and 10 randomly selected fields were counted per culture. Significant differences between groups based on post hoc testing (p < 0.05, ANOVA test) are indicated by an asterisk. Bars represent mean ± standard error of the mean. Color images available online at www.liebertonline.com/ten.