Harnessing 3D collagen hydrogel-directed conversion of human GMSCs into SCP-like cells to generate functionalized nerve conduits

Achieving a satisfactory functional recovery after severe peripheral nerve injuries (PNI) remains one of the major clinical challenges despite advances in microsurgical techniques. Nerve autografting is currently the gold standard for the treatment of PNI, but there exist several major limitations. Accumulating evidence has shown that various types of nerve guidance conduits (NGCs) combined with post-natal stem cells as the supportive cells may represent a promising alternative to nerve autografts. In this study, gingiva-derived mesenchymal stem cells (GMSCs) under 3D-culture in soft collagen hydrogel showed significantly increased expression of a panel of genes related to development/differentiation of neural crest stem-like cells (NCSC) and/or Schwann cell precursor-like (SCP) cells and associated with NOTCH3 signaling pathway activation as compared to their 2D-cultured counterparts. The upregulation of NCSC-related genes induced by 3D-collagen hydrogel was abrogated by the presence of a specific NOTCH inhibitor. Further study showed that GMSCs encapsulated in 3D-collagen hydrogel were capable of transmigrating into multilayered extracellular matrix (ECM) wall of natural NGCs and integrating well with the aligned matrix structure, thus leading to biofabrication of functionalized NGCs. In vivo, implantation of functionalized NGCs laden with GMSC-derived NCSC/SCP-like cells (designated as GiSCs), significantly improved the functional recovery and axonal regeneration in the segmental facial nerve defect model in rats. Together, our study has identified an approach for rapid biofabrication of functionalized NGCs through harnessing 3D collagen hydrogel-directed conversion of GMSCs into GiSCs.


Supplemental Fig. 2. GMSCs cultured on the top surface of the methacrylated 3D-collagen hydrogel. a
The 2D plastic 4-well chambered cell culture slides were pre-coated with 4mg/mL methacrylated collagen hydrogel. GMSCs then were seeded on the top surface of the solidified hydrogel and cultured in complete α-MEM for 48h. b The cellular morphology of GMSCs cultured under 2D plastic culture, encapsulation in 3Dcollagen hydrogel, or on the top surface of the solidified hydrogel, respectively. c Immunofluorescence staining showed no increase in the expression of p75 NTR in GMSCs cultured on the top surface of the solidified collagen hydrogel. Scale bar=50µm (b, c). Images are representative of three independent experiments (biological replicates). Supplemental Fig. 3. BMSCs cultured in methacrylated 3D-collagen hydrogel. a Flow cytometric analysis of MSC-associated cell surface markers on human bone marrow-derived mesenchymal stem cells (hBMSCs). b Adipogenic and osteogenic differentiation of hBMSCs as determined by Red Oil O and Alizarin Red S staining, respectively. Scale bar=50µm. c hBMSCs were encapsulated in 3D-collagen hydrogel (4mg/mL) at a cell density of 2×10 6 /mL and cultured in complete α-MEM for 48h. Cryosections of the 3D-collagen gels laden with hBMSCs were immunostained with a specific antibody for SOX9 or p75 NTR followed by incubation with Alexa Fluor 488-conjugated secondary antibody. Nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI; white or blue). Scale bar=20µm. Data are representative of three independent experiments (biological replicates).

Supplemental Fig. 5. GMSC-derived NCSC/SCP-like cells encapsulated in 3D-collagen hydrogel transmigrated into the wall matrix of nerve conduits and expressed neurotrophic factors.
GMSCs were encapsulated in 3D-collagen hydrogel at a final concentration of 4mg/mL and a cell density of 2×10 6 /mL and filled into AxoGuard Nerve protector or connector (10mm in length and 2mm in inner diameter). Then, the constructs (nerve conduits containing 3D collagen hydrogel encapsulated with GMSCs) were cultured for 24h in complete α-MEM for 24h. Cryosections of nerve conduits were prepared for immunostaining with specific antibodies for GDNF (a), BDNF (b), or NGF (c), followed by incubation with Alexa Fluor 488-conjugated secondary antibodies. Nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI; white or blue). Scale bars=50µm. Images are representative of three independent experiments (biological replicates).
Supplemental Fig. 7. The fate of GiSCs integrated in the nerve conduits following implantation in vivo. 14 weeks post-injury and implantation, the newly regenerated facial nerves were harvested and cross-sectional cryosections (10µm in thickness) were cut for immunofluorescence (IF) studies on expression of GDNF (green color) and human nuclei (red color). Nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI; blue). Scale bars=20 µm. Images are representative of three independent experiments. hNuclei, human nuclei.
Supplemental Fig. 8. Uncropped Western blotting images for Fig. 2e and Fig. 6d. All blots were derived from the same experiment and processed in parallel, where GAPDH was used as an internal loading control and the size markers were labeled.

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