{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T21:19:21Z","timestamp":1790025561335,"version":"4.0.1"},"reference-count":38,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2018,8,17]],"date-time":"2018-08-17T00:00:00Z","timestamp":1534464000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The reduction of choline acetyltransferase, caused by the loss of cholinergic neurons, leads to the absence of acetylcholine (Ach), which is related to motor nerve degeneration. The aims of the present study were to evaluate the in vitro cholinergic nerve differentiation potential of mesenchymal stem cells from cryopreserved human dental pulp (hDPSCs-cryo) and to analyze the scale of in vivo motor nerve regeneration. The hDPSCs-cryo were isolated and cultured from cryopreserved dental pulp tissues, and thereafter differentiated into cholinergic neurons using tricyclodecane-9-yl-xanthogenate (D609). Differentiated cholinergic neurons (DF-chN) were transplanted into rats to address sciatic nerve defects, and the scale of in vivo motor nerve regeneration was analyzed. During in vitro differentiation, the cells showed neuron-like morphological changes including axonal fibers and neuron body development, and revealed high expression of cholinergic neuron-specific markers at both the messenger RNA (mRNA) and protein levels. Importantly, DF-chN showed significant Ach secretion ability. At eight weeks after DF-chN transplantation in rats with sciatic nerve defects, notably increased behavioral activities were detected with an open-field test, with enhanced low-affinity nerve growth factor receptor (p75NGFR) expression detected using immunohistochemistry. These results demonstrate that stem cells from cryopreserved dental pulp can successfully differentiate into cholinergic neurons in vitro and enhance motor nerve regeneration when transplanted in vivo. Additionally, this study suggests that long-term preservation of dental pulp tissue is worthwhile for use as an autologous cell resource in the field of nerve regeneration, including cholinergic nerves.<\/jats:p>","DOI":"10.3390\/ijms19082434","type":"journal-article","created":{"date-parts":[[2018,8,17]],"date-time":"2018-08-17T10:54:25Z","timestamp":1534503265000},"page":"2434","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Cholinergic Nerve Differentiation of Mesenchymal Stem Cells Derived from Long-Term Cryopreserved Human Dental Pulp In Vitro and Analysis of Their Motor Nerve Regeneration Potential In Vivo"],"prefix":"10.3390","volume":"19","author":[{"given":"Soomi","family":"Jang","sequence":"first","affiliation":[{"name":"Department of Oral and Maxillofacial Surgery, College of Medicine, University of Ulsan, Ulsan 44033, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Young-Hoon","family":"Kang","sequence":"additional","affiliation":[{"name":"Department of Dentistry, Gyeongsang National University School of Medicine and Institute of Health Science, Jinju 52727, Korea"},{"name":"Department of Oral and Maxillofacial Surgery, Changwon Gyeongsang National University Hospital, Changwon 51472, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4259-2844","authenticated-orcid":false,"given":"Imran","family":"Ullah","sequence":"additional","affiliation":[{"name":"Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sharath Belame","family":"Shivakumar","sequence":"additional","affiliation":[{"name":"Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gyu-Jin","family":"Rho","sequence":"additional","affiliation":[{"name":"Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yeong-Cheol","family":"Cho","sequence":"additional","affiliation":[{"name":"Department of Oral and Maxillofacial Surgery, College of Medicine, University of Ulsan, Ulsan 44033, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Iel-Yong","family":"Sung","sequence":"additional","affiliation":[{"name":"Department of Oral and Maxillofacial Surgery, College of Medicine, University of Ulsan, Ulsan 44033, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bong-Wook","family":"Park","sequence":"additional","affiliation":[{"name":"Department of Dentistry, Gyeongsang National University School of Medicine and Institute of Health Science, Jinju 52727, Korea"},{"name":"Department of Oral and Maxillofacial Surgery, Changwon Gyeongsang National University Hospital, Changwon 51472, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2018,8,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.neulet.2012.11.014","article-title":"Human umbilical cord mesenchymal stem cells derived from Wharton\u2019s jelly differentiate into cholinergic-like neurons in vitro","volume":"532","author":"Liang","year":"2013","journal-title":"Neurosci. Lett."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"961","DOI":"10.3727\/096368912X657413","article-title":"Cholinergic neuron-like cells derived from bone marrow stromal cells induced by tricyclodecane-9-yl-xanthogenate promote functional recovery and neural protection after spinal cord injury","volume":"22","author":"Sun","year":"2013","journal-title":"Cell Transplant."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1002\/stem.277","article-title":"Intracerebral transplantation of bone marrow-derived mesenchymal stem cells reduces amyloid-beta deposition and rescues memory deficits in Alzheimer\u2019s disease mice by modulation of immune responses","volume":"28","author":"Lee","year":"2010","journal-title":"Stem Cells"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2183","DOI":"10.1002\/jnr.22054","article-title":"Stem cell-based cell therapy in neurological diseases: A review","volume":"87","author":"Kim","year":"2009","journal-title":"J. Neurosci. Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"103503","DOI":"10.1155\/2012\/103503","article-title":"Neural crest stem cells from dental tissues: A new hope for dental and neural regeneration","volume":"2012","author":"Ibarretxe","year":"2012","journal-title":"Stem Cells Int."},{"key":"ref_6","first-page":"80","article-title":"Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms","volume":"122","author":"Sakai","year":"2012","journal-title":"J. Clin. Investig."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1969","DOI":"10.1007\/s00784-013-1030-3","article-title":"Dental stem cells and their promising role in neural regeneration: An update","volume":"17","author":"Martens","year":"2013","journal-title":"Clin. Oral Investig."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"S42","DOI":"10.1038\/nm1064","article-title":"Stem cell therapy for human neurodegenerative disorders-how to make it work","volume":"10","author":"Lindvall","year":"2004","journal-title":"Nat. Med."},{"key":"ref_9","first-page":"1","article-title":"A review of the emerging potential therapy for neurological disorders: Human embryonic stem cell therapy","volume":"6","author":"Shroff","year":"2017","journal-title":"Am. J. Stem Cells"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1016\/j.expneurol.2006.12.004","article-title":"Transplantation of bone marrow stromal cells for peripheral nerve repair","volume":"204","author":"Chen","year":"2007","journal-title":"Exp. Neurol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1002\/term.404","article-title":"Peripheral nerve regeneration using autologous porcine skin-derived mesenchymal stem cells","volume":"6","author":"Park","year":"2012","journal-title":"J. Tissue Eng. Regen. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"915","DOI":"10.1016\/j.bbrc.2007.05.212","article-title":"Peripheral nerve regeneration by the in vitro differentiated-human bone marrow stromal cells with Schwann cell property","volume":"359","author":"Shimizu","year":"2007","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"14","DOI":"10.46582\/jsrm.1001004","article-title":"Effects of umbilical cord tissue mesenchymal stem cells (UCX\u00ae) on rat sciatic nerve regeneration after neurotmesis injuries","volume":"10","author":"Pereira","year":"2014","journal-title":"J. Stem Cells Regen. Med."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Tamaki, T., Hirata, M., Soeda, S., Nakajima, N., Saito, K., Nakazato, K., Okada, Y., Hashimoto, H., Uchiyama, Y., and Mochida, J. (2014). Preferential and comprehensive reconstitution of severely damaged sciatic nerve using murine skeletal muscle-derived multipotent stem cells. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0091257"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.lfs.2016.04.026","article-title":"In vitro comparative analysis of human dental stem cells from a single donor and its neuronal differentiation potential evaluated by electrophysiology","volume":"154","author":"Ullah","year":"2016","journal-title":"Life Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1247","DOI":"10.1089\/scd.2017.0068","article-title":"Transplantation of human dental pulp-derived stem cells or differentiated neuronal cells from human dental pulp-derived stem cells identically enhances regeneration of the injured peripheral nerve","volume":"26","author":"Ullah","year":"2017","journal-title":"Stem Cells Dev."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1002\/bem.21966","article-title":"Schwann-like cells differentiated from human dental pulp stem cells combined with a pulsed electromagnetic field can improve peripheral nerve regeneration","volume":"37","author":"Hei","year":"2016","journal-title":"Bioelectromagnetics"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1945","DOI":"10.4103\/1673-5374.172309","article-title":"Dental pulp stem cells-derived Schwann cells for peripheral nerve injury regeneration","volume":"10","author":"Kalbouneh","year":"2015","journal-title":"Neural Regen. Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1787","DOI":"10.1634\/stemcells.2007-0979","article-title":"Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues","volume":"26","author":"Arthur","year":"2008","journal-title":"Stem Cells"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"792","DOI":"10.1177\/0022034509340867","article-title":"Mesenchymal stem cells derived from dental tissues vs. those from other sources: Their biology and role in regenerative medicine","volume":"88","author":"Huang","year":"2009","journal-title":"J. Dent. Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1002\/term.1945","article-title":"Cryopreservation of human dental follicle tissue for use as a resource of autologous mesenchymal stem cells","volume":"11","author":"Park","year":"2017","journal-title":"J. Tissue Eng. Reg. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2397","DOI":"10.1002\/jcb.25563","article-title":"DMSO- and serum-free cryopreservation of Wharton\u2019s jelly tissue isolated from human umbilical cord","volume":"117","author":"Shivakumar","year":"2016","journal-title":"J. Cell. Biochem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.diff.2015.10.001","article-title":"Immunomodulatory properties and in vivo osteogenesis of human dental stem cells from fresh and cryopreserved dental follicles","volume":"90","author":"Kang","year":"2015","journal-title":"Differentiation"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1418","DOI":"10.7150\/ijms.22152","article-title":"Stem cells from cryopreserved human dental pulp tissues sequentially differentiate into definitive endoderm and hepatocyte-like cells in vitro","volume":"14","author":"Han","year":"2017","journal-title":"Int. J. Med. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1002\/1097-4547(20000815)61:4<364::AID-JNR2>3.0.CO;2-C","article-title":"Adult rat and human bone marrow stromal cells differentiate into neurons","volume":"61","author":"Woodbury","year":"2000","journal-title":"J. Neurosci. Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1002\/jcb.21029","article-title":"Epigenetic modifiers promote efficient generation of neural-like cells from bone marrow-derived mesenchymal cells grown in neural environment","volume":"100","author":"Alexanian","year":"2007","journal-title":"J. Cell. Biochem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1089\/scd.2007.0197","article-title":"Neuronal differentiation potential of human adipose-derived mesenchymal stem cells","volume":"17","author":"Anghileri","year":"2008","journal-title":"Stem Cells Dev."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"263","DOI":"10.3233\/JAD-2010-091528","article-title":"Severe motor neuron degeneration in the spinal cord of the Tg2576 mouse model of Alzheimer disease","volume":"21","author":"Seo","year":"2010","journal-title":"J. Alzheimer\u2019s Dis."},{"key":"ref_29","first-page":"442","article-title":"D609 induces vascular endothelial cells and marrow stromal cells differentiation into neuron-like cells","volume":"25","author":"Wang","year":"2004","journal-title":"Acta Pharmacol. Sin."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1016\/j.biocel.2007.08.003","article-title":"Cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in the neural differentiation of mesenchymal stem cells in vitro","volume":"40","author":"Wang","year":"2008","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2253","DOI":"10.1016\/j.biocel.2012.09.013","article-title":"Phosphatidylcholine-specific phospholipase C\/heat shock protein 70 (Hsp70)\/transcription factor B-cell translocation gene 2 signaling in rat bone marrow stromal cell differentiation to cholinergic neuron-like cells","volume":"44","author":"Shao","year":"2012","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2502","DOI":"10.1016\/S0022-2275(20)37455-1","article-title":"Tissue-specific coordinate regulation of enzymes of cholesterol biosynthesis: Sciatic nerve versus liver","volume":"37","author":"Toews","year":"1996","journal-title":"J. Lipid Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1111\/odi.12086","article-title":"Immunomodulatory properties of dental tissue-derived mesenchymal stem cells","volume":"20","author":"Li","year":"2014","journal-title":"Oral Dis."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"627","DOI":"10.1002\/stem.1909","article-title":"Concise reviews: Characteristics and potential applications of human dental tissue-derived mesenchymal stem cells","volume":"33","author":"Liu","year":"2015","journal-title":"Stem Cells"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"37","DOI":"10.5214\/ans.0972.7531.1118203","article-title":"Behavioral analysis after sciatic nerve compression in albino rats","volume":"18","author":"Anand","year":"2011","journal-title":"Ann. Neurosci."},{"key":"ref_36","first-page":"51785","article-title":"Behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases","volume":"91","author":"Tatem","year":"2014","journal-title":"J. Vis. Exp."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"343","DOI":"10.5125\/jkaoms.2012.38.6.343","article-title":"Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro","volume":"38","author":"Byun","year":"2012","journal-title":"J. Korean Assoc. Oral Maxillofac. Surg."},{"key":"ref_38","first-page":"287","article-title":"Peripheral mechanisms for the initiation of pain following trigeminal nerve injury","volume":"18","author":"Robinson","year":"2004","journal-title":"J. Orofac. Pain"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/8\/2434\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T15:19:22Z","timestamp":1760195962000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/8\/2434"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,8,17]]},"references-count":38,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2018,8]]}},"alternative-id":["ijms19082434"],"URL":"https:\/\/doi.org\/10.3390\/ijms19082434","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,8,17]]}}}