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We have previously reported that nucleus pulposus (NP) progenitor cell-derived spheroid colonies possess pluripotency. Furthermore, we have confirmed that the NP progenitor cells express neural stem cell markers: Nesthin, Musashi-1, and Sox2. Moreover, they could differentiate into Schwann cell-like phenotypes in vitro. However, its functional potential is unknown. The purpose of this study is to evaluate the effect of these Schwann-like cells derived from NP progenitor cells on nerve regeneration in the mouse sciatic nerve transection model.Materials and Methods   Animals We used P0-Cre/Floxed-EGFP-transgenic mice (8-week-old female, n = 40) in this experiment. We have previously reported that EGFP positive cells in this mouse was observed in NP cells in intervertebral discs. Therefore, we used this mouse for the trace of NP cells during glial differentiation in vitro. Culture of NP Cell-Spheroid Colonies NP tissues from tail intervertebral discs of these mice were collected using a surgical microscope. These NP tissues were digested and cultured in αMEM for 7days. NP cells were then transferred to methylcellulose medium for 10 days to induce spheroid colonies. We collected the NP cell-spheroid colonies by diluting the methylcellulose medium with αMEM and pipetting. Differentiation of NP Progenitor Cells The NP cell-spheroid colonies were incubated in 0.25% trypsin-EDTA for 30 minutes, and triturated until a single-cell suspension was obtained. These cells were performed according to the method of neural induction described by Dezawa et al. Transplantation of differentiated NP progenitor cells: differentiated NP progenitor cells were suspended in matrigel (BD Biosciences) at the concentration of 1 × 107 cells/mL, and were filled into Poly-96L/4D-Lactide tubes (8 mm length) to make an artificial graft. Under general anesthesia, a 3-mm segment was completely removed from the left sciatic nerve of severe combined immunodeficient mice (NOD-SCID, 8-week-old female, n = 10) at the middle thigh level. The artificial graft was anastomosed with proximal and distal nerve tips using 10-0 nylon sutures at both ends. We also transplanted lactide tubes with matrigel only as a comparison group ( n = 10). Immunohistochemical Analysis of Transplanted Grafts At 9 weeks postsurgery, the nerve conduits were harvested and nerve regeneration was examined by immunohistochemistry with the following primary antibodies: a marker of primitive neurons; βIII tubulin, a marker of early Schwann cells; S100, a marker of mature Schwann cells; GFAP and GFP; a marker of the present differentiated NP progenitor cells. Evaluation of Functional Recovery The recovery of motor and sensory function of the mouse's hindlimb was assessed by walking track analysis as described previously at 3, 6 and 9 weeks after transplantation. The recovery of sensory function was assessed by the hot water behavior test as described previously.Results   Immunohistochemical analysis: 9 weeks after transplantation, the GFP-expressing cells were visualized in the joined sciatic nerve. In the central and marginal area of the joined nerve, many of the GFP and GFAP, S100-double expressing differentiated NP progenitor cells formed a myelin sheath surrounding GFP-negative and βIII-tubulin-positive host regrowth axons. The evaluation of functional recovery: The mean values of the print length factor in the NP group were significantly lower than in the control group at 3, 6, and 9 weeks, respectively. Regards to the recovery of sensory function, the average time of foot withdrawal in the NP group ( n = 10) was significantly shorter than that in the control group ( n = 10) at 3, 6, and 9 weeks, respectively. These results indicate that the nerve conduits filled in with differentiated NP progenitor cells accelerate the functional recovery of sciatic nerve gaps in mice.Conclusion   The treatment of peripheral nerve defects is primarily aimed at promoting the axonal regrowth into the nerve stump distal to the lesion. This axonal regrowth always needs a scaffold including Schwann cells in the gap. In clinical practice, the frequently used approaches for the reconstruction of peripheral nerve defects include nerve autografts, allografts, and nerve conduits. However, it is often difficult to obtain a good clinical result with these methods. Thus, as an alternative to these therapies, efforts have been made to research the cell therapy for the regeneration of nerve. Several recent studies have reported that the cell therapy including iPS cells and ES cells conduced to the good functional recovery in damaged peripheral nerve. However, several problems remain, such as significant ethical issues and potential malignancy. We have confirmed that the NP progenitor cells can differentiate into Schwann cell-like phenotypes in vitro. In the current study, we evaluated effect of these Schwann-like cells derived NP progenitor cells on nerve regeneration in the transection model of the mice sciatic nerve to assess its functional potential. NP progenitor cell-derived Schwann-like cells transplanted in severed sciatic nerve formed a myelin sheath surrounding the host regrowth axons. Furthermore, this morphological regrowth of the severed nerve also brought initiated good functional restoration. These results suggest that NP progenitor cell-derived Schwann-like cells have similar property to schwann cells. The advantage of these spheroid colonies-forming NP cells over other stem cells is that they are adult stem cell that can evade ethical issues and potential malignancy. Thus, NP cells may be considered to be one of the candidates for the cell therapy for peripheral nerve defects, although ideal donor cell supply may be the major obstacle. Disclosure of Interest None declared

Sciatic Nerve Regeneration in Mice Induced by Transplantation of in Vitro Differentiated Nucleus Pulposus Progenitor Cells