Engineering Nanofiber Scaffolds with Biomimetic Cues for Differentiation of Skin-Derived Neural Crest-like Stem Cells to Schwann Cells

Abstract

Our laboratory reported the derivation of neural crest stem cell (NCSC)-like cells from the interfollicular epidermis of the neonatal and adult epidermis. These keratinocyte (KC)-derived Neural Crest (NC)-like cells (KC-NC) could differentiate into functional neurons, Schwann cells (SC), melanocytes, and smooth muscle cells in vitro. Most notably, KC-NC migrated along stereotypical pathways and gave rise to multiple NC derivatives upon transplantation into chicken embryos, corroborating their NC phenotype. Here, we present an innovative design concept for developing anisotropically aligned scaffolds with chemically immobilized biological cues to promote differentiation of the KC-NC towards the SC. Specifically, we designed electrospun nanofibers and examined the effect of bioactive cues in guiding KC-NC differentiation into SC. KC-NC attached to nanofibers and adopted a spindle-like morphology, similar to the native extracellular matrix (ECM) microarchitecture of the peripheral nerves. Immobilization of biological cues, especially Neuregulin1 (NRG1) promoted the differentiation of KC-NC into the SC lineage. This study suggests that poly-ε-caprolactone (PCL) nanofibers decorated with topographical and cell-instructive cues may be a potential platform for enhancing KC-NC differentiation toward SC.

Keywords: NRG1; S100b; Schwann cell; keratinocyte derived neural crest; nanofiber; neural crest; poly-ε-caprolactone; proteolipid protein PLP1; topographical cues.

Figure 1

The schematic of designing a cell-instructive fiber for directing the KC-NC fate. (A) Electrospinning of PCL to obtain aligned fibers. (B) Preparation of PCL-PDA via oxidative polymerization of dopamine in the presence of PCL fibers; simplified structure of PDA is shown here. (C) Conjugation of Hep-thiol to PCL-PDA via Michael addition chemistry at physiological pH (7–8), (D) Immobilization of biological cues via their HBD. (E) Confocal microscopy image of the KC-NC grown on aligned NRG1 functionalized fibers and stained for S100b (green), p75 (red), and nuclei (blue). Double-arrow: Aligned fiber direction.

Figure 2

Surface modification and characterization of the aligned fibers. (A) ¹H NMR analysis of Hep-thiol. (B) Chemical conjugation of Hep-thiol to PCL-PDA via Michael-addition chemistry. (C) PCL and PCL-PDA mounted in polystyrene chamber with movable glass base. (D) FTIR spectrum of PCL, PCL-PDA, and PCL-PDA-Hep.

Figure 3

Fiber formation, size distribution, orientation, and mechanical strength. (A) SEM analysis of electrospun PCL (i,iv), PCL-PDA (ii,v), or PCL-PDA-Hep (iii,vi) fibers at two magnifications (top images were taken at 3000×; bottom images were taken at 5000×; scale bars = 10 µm). (B) Diameter distribution of PCL fibers. (C) The orientation angle of the PCL-PDA-Hep fibers was measured using image J software. (D) Stress-strain curve of PCL-PDA-Hep (n = 3 measurements are shown in red, blue, and black colors respectively).

Figure 4

Effect of different cell substrates to guide KC-NC attachment and spreading on the fibers. (A–H) Confocal microscopy images of the KC-NC cells stained for their cytoskeletal microfilaments with actin phalloidin (green) on fibers with no coating (Control), two native ECM glycoproteins (laminin and fibronectin), two recombinantly designed bidomain fusion peptides (HBD-REDV and HBD-RGD), and three bioactive macromolecules (PDGF-BB, FGF2, and NRG1). Scale bar: 100 µm. (I) Quantification of the number of attached cells from n = 3 randomly selected fields per condition. Data are presented as mean ± SD. One-way analysis of variance (ANOVA) with Tukey post hoc test, n = 3 independent experiments. (J) Analysis of cell spreading quantified as eccentricity for each condition. Data are presented as mean ± SD of randomly selected cells. One-way analysis of variance (ANOVA) with Tukey post hoc test. n = 35 randomly selected cells from different fields, ns: p ≥ 0.05, **: p < 0.005, ***: p < 0.0005, ****: p < 0.0001). (K) Cell eccentricity, φ is calculated from measurements of the major and minor axis length using ImageJ. Double-arrow: direction of aligned fibers.

Figure 5

Evaluation of Schwann differentiation on the fibers conjugated with biological cues. Confocal microscopy images for the expression of astrocyte marker, glial fibrillary acidic protein, GFAP (A–D), and Schwann marker myelin proteolipid protein, PLP1 (E–H), on electrospun fibers with no coating (Control) (A,E), Fibronectin (B,F), HBD-REDV (C,G), and NRG1 (D,H), respectively. Scale bar = 100 µm. (I,J) Graph showing the quantification of the mean fluorescence intensity (MFI) for GFAP or myelin PLP1 per cell. Data are presented as Mean ± SD, One-way analysis of variance (ANOVA) with Tukey post hoc test. n = 20 randomly selected cells from three different fields. ns: p ≥ 0.05), ***: p < 0.0005), ****: p < 0.0001. Double-arrow: direction of aligned fibers.

Figure 6

Effect of PCL immobilized NRG1 on KC-NC differentiation and proliferation toward towards Schwann cells. Immunofluorescence images of the expression of Schwann marker, S100b, GFAP, p75, and myelin PLP1 on (A–C) tissue culture plates (TCP), (D–F) electrospun fibers with no coating (Control), or (G–I) immobilized NRG1. (A–C) scale bars: 50 µm; (D–I) scale bars: 20 µm. (J) Quantification of the mean immunofluorescence intensity (MFI) of the indicated SC markers normalized per cell on TCP, control fibers, and NRG1 modified fibers. n = 50 randomly selected cells from different fields. (K) Quantification of average cell density at two-time points (3 DIV and & DIV, DIV = Day in-vitro) on TCP, control, or NRG1 modified fibers. Data are presented as Mean ± SD, One-way analysis of variance (ANOVA) with Tukey post hoc test. ns: p ≥ 0.05), ****: p <0.0001.