Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro

Abstract

In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d-hDPSCs), which promoted survival and neurite outgrowth in 2-dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d-hDPSCs in a 3-dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d-hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell-based therapies as treatment for peripheral nerve injury.

Keywords: cellular hydrogel; glial cell; myelination; nerve repair; neural regeneration.

Figure 1.

Phenotyping hDPSCs after Schwann cell differentiation. Brightfield imaging (A–C) and immunocytochemistry (D–R) were performed on hDPSCs (A, D, G, J, M, P) and d-hDPSCs (B, E, H, K, N, Q) for the typical Schwann cell markers laminin (D–F), p75 (G–I), GFAP (J–L), CD104 (M–O), and nestin (P–R). Nuclei were counterstained with DAPI (blue). Primary rat Schwann cells (C, F, I, L, O, R) were used as positive controls for differentiation. Scale bars = 200 μm (A–C); 50 μm (D–I, L–R); 100 μm (J, K).

Figure 2.

Transmission electron micrographs of hDPSCs (A–C) and d-hDPSCs (D, E). Ultrastructural characteristics of d-hDPSCs differ markedly from those of hDPSCs, with cell-cell contacts (E, oval) between neighboring cells. Scale bars = 20 μm (A, B, D); 2 μm (C, E).

Figure 3.

Neurotrophic factor secretion. A) ELISAs indicated a significant increase in BDNF, b-NGF, NT-3, and GDNF levels after differentiation (n=9). B, C) Neural survival (B; n=7) and neurite outgrowth (C; n=4) were significantly improved with conditioned medium from hDPSCs and d-hDPSCs, with the latter being more potent. Scale bar = 50 μm. Data represent means ± sem. **P < 0.01, ***P < 0.001.

Figure 4.

d-hDPSC alignment in EngNT guides neurite outgrowth in coculture. A–C) Confocal images represent d-hDPSCs (S100, red) and neurites (b-III-tubulin, green). Nuclei were counterstained with DAPI (blue). Scale bars = 100 μm. D) Frequency distribution of neurite angles compared to mean angle of d-hDPSC alignment in each field (field volume: 9.8×10⁶ μm³). Data represent means ± se (in 10° bins; n=4).

Figure 5.

Ultrastructural analysis of myelination capacity of d-hDPSCs. A) Multiple cell-cell contacts (encircled) and enfoldment of neurites by pseudopodial processes of d-hDPSCs (arrows) were frequently observed. B–C) Myelin sheaths were present in the culture (B), showing typical periodic and intraperiodic lines (C). D) Total number of d-hDPSCs and neurites per square millimeter of hydrogel. Scale bars = 1 μm (A, B); 0.2 μm (C).