Abstract
Cell therapy for peripheral nerve injury is a promising strategy as regenerative medicine that restores neurological function. However, challenges remain in producing suitable and sufficient amounts of autologous cells for promoting nerve regeneration. This study aimed to identify the characteristics of neural lineage cells (NLCs) differentiated from dental pulp stem cells (DPSCs) and reveal their effect on functional recovery and nerve regeneration after cell transplantation into an immunodeficient rat using a nerve guide conduit. Here we report a protocol of neural induction in monolayer culture and characterize NLCs in vitro. Furthermore, NLCs were transplanted into an immunodeficient rat model with a 10-mm sciatic nerve defect, and cell survival and differentiation were investigated in vivo. Outcomes of nerve regeneration were also assessed using the remyelinated axon numbers, myelin sheath thickness, electrophysiological activities, and gastrocnemius muscle mass. NLCs comprised neuronal, astrocyte, oligodendrocyte, and neural crest lineage cells. NLCs enhanced the activities of endothelial cells, Schwann cells, and neurons in a paracrine-dependent manner in vitro. At 2 weeks post-transplantation, numerous transplanted NLCs differentiated into platelet-derived growth factor receptor alpha (PDGFRα) + oligodendrocyte progenitor cells (OPCs) and a few PDGFRα + /p75 neurotrophin receptor + Schwann cell-like cells derived from OPCs were observed. At 12 weeks post-transplantation, human Schwann cell-like cells survived, and axon growth, remyelination, electrophysiological activities, and muscle atrophy were improved. This study demonstrates the broad application of our protocol of neural induction of DPSCs and portrays the efficacy of transplantation of NLCs derived from human DPSCs as a promising strategy for peripheral nerve regeneration.
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Acknowledgements
We thank Dr. Toshiaki Tachibana (Division of Fine Morphology, Core Research Facilities, Jikei University School of Medicine) for guidance with electron microscopy.
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The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant Numbers 16K15817, 19K09450, and 20H03761) and Translational Research Program (University of Tsukuba, A18-36) for the Japan Agency for Medical Research and Development.
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ST: Contributed to the study conception and design, data acquisition and analysis, and drafting of the manuscript. FU: Contributed to the study conception and design, data analysis, and drafting of the manuscript. HI and HB: Contributed to the study conception and design, data analysis and interpretation, and drafting and critical revision of the manuscript. JT, AO, MW, HM, and SI: Contributed to data acquisition, analysis, interpretation, and drafting of the manuscript. AM, TY, and YM: Contributed to data interpretation and critical revision of the manuscript. NIK, SF, and KY: Contributed to data analysis and interpretation, and critical revision of the manuscript. All authors approved the final version of the manuscript and agree to be accountable for all aspects of this work.
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This study was performed in accordance with the guidelines of the research ethics committee of the University of Tsukuba (Approval No. H29-173). Animal experiments were performed in a humane manner after receiving approval from the Institutional Animal Care and Use Committee of the University of Tsukuba (Approval No. 20-232).
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13577_2021_634_MOESM2_ESM.docx
Supplementary file2 (DOCX 391 kb) Characterization of NLCs. (a) NLCs were immunostained with antibodies against STEM123 (human GFAP) and DCX. Coexpression of GFAP and DCX is indicated by white arrows. Scale bar = 50 µm (b) An ELISA was used to determine the VEGF levels in CM from DPSCs and NLCs (n = 3/group). *p < 0.01. (c) Three clusters were correlated with the maturation of GFAP+ cells. The gene expression profiles of NLCs were adapted. Cluster 1, early pseudo-time; cluster 2, middle pseudo-time; cluster 3, late pseudo-time
Supplementary file3 (AVI 2584 kb). Neuronal activities of NLCs using calcium indicators at 50 DIV
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Takaoka, S., Uchida, F., Ishikawa, H. et al. Transplanted neural lineage cells derived from dental pulp stem cells promote peripheral nerve regeneration. Human Cell 35, 462–471 (2022). https://doi.org/10.1007/s13577-021-00634-9
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DOI: https://doi.org/10.1007/s13577-021-00634-9