Newly Generated 3D Schwann-Like Cell Spheroids From Human Adipose-Derived Stem Cells Using a Modified Protocol

Abstract

Peripheral nerve injury (PNI) is a relatively frequent type of trauma that results in the suffering of many patients worldwide every year. Schwann cells (SCs) are expected to be applied in cell therapy because of their ability to promote peripheral nerve regeneration. However, the lack of clinically renewable sources of SCs hinders the application of SC-based therapies. Adipose-derived stem cells (ADSCs) have generated great interest in recent years because of their multipotency and ease of harvest, and they have already been verified to differentiate into Schwann-like cells (SLCs) in vitro. However, the efficiency of differentiation and the functions of SLCs remain unsatisfactory. We newly generated three-dimensional (3D) SLC spheroids from ADSCs using a modified protocol with human recombinant peptide (RCP) petaloid μ-piece. Morphological analysis, gene expression analysis by qRT-PCR, ELISA measurement of the secretion capabilities of neurotrophic factors, and neurite formation assay were performed to evaluate the functions of these 3D SLCs in vitro. Motor function recovery was measured in a sciatic nerve injury mouse model to analyze the nerve regeneration-promoting effect of 3D SLCs in vivo. The differentiation efficiency and the secretion of neurotrophic factors were enhanced in 3D SLCs compared with conventional SLCs. 3D SLCs could more effectively promote neurite growth and longer neurite extension in a neuron-like SH-SY5Y model. Additionally, 3D SLCs had a better therapeutic effect on nerve regeneration after transplantation into the sciatic nerve injury mouse model. These findings demonstrated that the potential of ADSC-derived SLCs to promote nerve regeneration could be significantly increased using our modified differentiation protocol and by assembling cells into a 3D sphere conformation. Therefore, these cells have great potential and can be used in the clinical treatment of PNI.

Keywords: Schwann-like cells; adipose-derived stem cells; nerve regeneration; three-dimensional model.

Figure 1.

Three-dimensional (3D) SLC spheroids generated from ADSCs. (A) Representative image of cell morphology under light microscopy during the differentiation process. (B) The morphology of 3D SLC spheroids is shown after hematoxylin and eosin staining. (C) Gene expression levels of the Schwann cell markers S100B and NGFR were detected in ADSCs, conventional SLCs, 3D SLCs, and primary Schwann cells using RT-qPCR analysis (n = 4). Data are expressed as means ± SD. **P < 0.01, one-way ANOVA with Tukey’s post-test. Scale bar in A, 400 μm; B (left), 500 μm; B (right), 50 μm. ADSCs: Adipose-derived stem cells; NGFR: nerve growth factor receptor; SLC: Schwann-like cell; S100B: S100 calcium-binding protein B.

Figure 2.

Three-dimensional (3D) SLCs exhibit enhanced therapeutic potential in vitro. The relative secretion of NGF (A) and GDNF (B) in the supernatant was analyzed by an enzyme-linked immunosorbent assay in the ADSC, conventional SLC, and 3D SLC groups (n = 4). Data are expressed as means ± SD. *P < 0.05; **P < 0.01, one-way ANOVA with Tukey’s post-test. (C) Representative images of SH-SY5Y cells after direct co-culture with ADSCs, conventional SLCs, and 3D SLCs. The neurites were visualized by immunofluorescent labeling of βIII tubulin (green). (D) Statistical analyses of the number of neurites per cell (n = 4). Data are expressed as means ± SD. *P < 0.05; **P < 0.01, one-way ANOVA with Tukey’s post-test. (E) Statistical analyses of the relative length of each neurite (n = 4). Data are expressed as means ± SD. *P < 0.05; **P < 0.01, one-way ANOVA with Tukey’s post-test. Scale bar in C, 50 μm. SLCs: Schwann-like cells; NGF: nerve growth factor; GDNF: glial cell-derived neurotrophic factor; ADSC: Adipose-derived stem cell; DAPI, 4,’6-diamidino-2-phenylindole, dihydrochloride.

Figure 3.

Three-dimensional (3D) SLCs promote motor function and structural recovery in vivo. (A) Representative images of the right hind paw prints of the control, ADSC, conventional SLC, and 3D SLC groups before injury and 1, 2, 3, and 4 weeks after injury. (B) Change in the SFI from 1 week to 4 weeks after injury (n = 3). Data are expressed as means ± SD. *P < 0.05 vs. control; **P < 0.01 vs. control; ##p < 0.01 vs. conventional SLCs, two-way ANOVA with Tukey’s post-test. (C) Statistical analyses of the differences between 1 week and 4 weeks post-surgery (n = 3). Data are expressed as means ± SD. **P < 0.01, one-way ANOVA with Tukey’s post-test. (D) Representative images of the gastrocnemius muscle of the injured limb in the control, ADSC, conventional SLC, and 3D SLC groups at 4 weeks post-surgery. (E) The relative weights of gastrocnemius muscles in the control, ADSC, conventional SLC, and 3D SLC groups at 4 weeks post-surgery (n = 3). Data are expressed as means ± SD. *P < 0.05; **P < 0.01, one-way ANOVA with Tukey’s post-test. (F) Representative image of sciatic nerve regeneration after 3D SLC transplantation into the injured nerve region for 4 weeks. (G) βIII tubulin (green) expression was determined by immunofluorescence staining in the regenerated sciatic nerve. The dotted lines indicate the borders of injury sites. Scale bar in G, 200 μm. SLCs: Schwann-like cells; ADSC: Adipose-derived stem cell; SFI: Sciatic Function Index; DAPI, 4,’6-diamidino-2-phenylindole, dihydrochloride.