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Differentiation of Human Dental Pulp Stem Cells Into Dopaminergic Neuron-Like Cells in Vitro

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Differentiation of Human Dental Pulp Stem Cells Into Dopaminergic Neuron-Like Cells in Vitro ORIGINAL ARTICLE Cell Therapy & Organ Transplantation http://dx.doi.org/10.3346/jkms.2016.31.2.171 • J Korean Med Sci 2016; 31: 171-177 Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro So Young Chun,1,* Shay Soker,2,* We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate Yu-Jin Jang,3 Tae Gyun Kwon,4 into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson’s disease and Eun Sang Yoo4 treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the 1BioMedical Research Institute, Kyungpook National University Hospital, Daegu, Korea; 2Wake Forest medium was replaced with KO-ES medium without LIF to allow the formation of the Institute for Regenerative Medicine, Wake Forest neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS University School of Medicine, Winston-Salem, NC, (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 3 USA; Department of Neural Development and 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth Disease, Korea Brain Research Institute, Daegu; 4Department of Urology, Kyungpook National factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-L-ornithine/ University, Daegu, Korea fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into * So Young Chun and Shay Soker contributed equally dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by to this work. morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. Received: 24 February 2015 The expressions of mesenchymal stem cell markers were observed at the early stages. The Accepted: 9 November 2015 expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The Address for Correspondence: Eun Sang Yoo, MD percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was Department of Urology, Kyungpook National University Hospital, 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural 130 Dongdeok-ro, Jung-gu, Daegu 41944, Korea Tel: +82.53-420-5851, Fax: +82.53-427-5447 cells under experimental cell differentiation conditions, showing potential as an autologous E-mail: [email protected] cell source for the treatment of Parkinson’s disease. Funding: This work was supported by a Biomedical Research Institute grant from Kyungpook National University Hospital Keywords: Human Dental Pulp Stem Cells; Dopaminergic Differentiation; Tyrosine (2013). Hydroxylase; Parkinson Disease; Autologous Stem Cell Therapy; Dopaminergic Neural Cells INTRODUCTION cells (hDPSCs) have been demonstrated to be a good source for autologous stem cell. The hDPSCs can be obtained noninva- Parkinson’s disease (PD) is a serious chronic neurodegenera- sively from deciduous teeth, and maintain their self-renewal tive disease that is caused by the loss of dopaminergic neurons capability and multipotency (8-10) including neuron-like cells in the substantia nigra. A depletion of dopamine in the striatum (9,11). hDPSCs can also be derived from cryopreserved dental leads to parkinsonism (1). Different drug treatments have been pulp (12). proposed, but most chemicals have shown limited efficacy (2). In the present study, we identified the potential of hDPSCs to In order to control the emergence of PD, cell therapy has been differentiate into dopaminergic neurons under appropriate con- suggested as a promising alternative treatment. Data from ani- ditions in vitro. These results could provide fundamental infor- mal models and clinical trials have suggested that cell replace- mation regarding the potential of hDPSCs as an alternative stem ment therapies may be an effective treatment for this disorder cell source for PD treatment. (3,4). However, cell replacement therapy needs to consider im- mune rejection, teratoma formation or ethical issues (5,6). MATERIALS AND METHODS Autologous mesenchymal stem cells (MSCs) were frequently tried as the reliable cell source avoiding above limitations for hDPSC culture PD treatment (7). MSCs are multipotent stem cells that can be hDPSCs were kindly provided by Prof. Eui Kyun Park, Kyung- differentiated into neurons. In order to obtain autologous MSCs, pook National University, Korea. The hDPSCs were maintained invasive operations are usually required. However, to establish in α-minimum essential medium (α-MEM; Invitrogen Corp., a standard and applicably safe cell therapy, noninvasive opera- Carlsbad, CA, USA) supplemented with 10% fetal bovine serum tions are ideal approaches for patients. Human dental pulp stem (FBS; Invitrogen), 100 units/mL penicillin, and 100 mg/mL strep- © 2016 The Korean Academy of Medical Sciences. pISSN 1011-8934 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. eISSN 1598-6357 Chun SY, et al. • Dopaminergic Cells from Human Dental Pulp Stem Cells tomycin (HyClone Labs, Thermo Scientific, Rockford, IL, USA). hDPSCs’ differentiation into dopaminergic neurons The cells were maintained at 37°C in a humidified 5% 2CO incu- For differentiation into dopaminergic neurons, hDPSCs were bator. Cells that had undergone less than five passages were used treated with the Human/Mouse Dopaminergic Neuron Differ- for this study. entiation Kit (R&D Systems, Abingdon, UK). At stage 1, hDPSCs were preinduced on gelatin-coated plates with recombinant SSEA4 NESTIN TUJ1 GFAP MBP TH Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Scale bar = 100 μm A H&E staining TH TH Scale bar = 100 μm SSEA4 NESTIN TUJ1 GFAP MBP 1st antibody SSEA4 NESTIN TUJ1 GFAP MBP TH Isotype control Dilution company 1:100 1:500 1:500 1:500 1:500 1:200 1:500 Cell signaling Abcam Abcam Abcam Abcam Pel-Freez Abcam 2nd antibody anti mouse Alexa-488, anti mouse Alexa-594, anti-rabbit Alexa-488 Dilution company 1:250 Invitrogen B Fig. 1. Morphological and immunocytochemical analyses of human dental pulp stem cells (hDPSCs) throughout the various differentiation stages. (A) The neuronal characteris- tics of hDPSCs were verified at the protein level by immunocytochemical analysis. (B) Substantia nigra of ICR mouse brain tissue was used as the positive control for immuno- histochemistry. SSEA4, stage-specific embryonic antigen 4; TuJ1,β -tubulin III; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; TH, tyrosine hydroxylase; H&E, he- matoxylin & eosin. 172 http://jkms.org http://dx.doi.org/10.3346/jkms.2016.31.2.171 Chun SY, et al. • Dopaminergic Cells from Human Dental Pulp Stem Cells mouse leukemia inhibitory factor (LIF)-containing knockout- analyzed for their neural characteristics on a fluorescence-acti- ES (KO-ES/LIF) medium, made up of knockout Dulbecco’s mo- vated cell sorter calibur instrument (BD Biosciences, San Jose, dified Eagle’s medium (DMEM), FBS, MEM non-essential AA CA, USA). solution, penicillin-streptomycin-glutamine, and β-mercapto- ethanol, for 3-4 days. At stage 2, 2 × 106 cells on 10-cm culture Characterization of differentiated hDPSCs by real-time plates were treated with KO-ES medium without LIF for 4 days PCR to induce neurosphere formation. At stage 3, to select for Nes- At the transcript level, the neural characteristics were analyzed tin-positive cells, the neurosphere was settled to the plate with by real-time PCR. Total RNA was extracted from cultured cells KO-ES medium. After neurosphere attachment to the plate, the by using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) medium was replaced with human insulin-transferrin-sodium according to the manufacturer’s instructions. One microgram (ITS)/fibronectin medium containing DMEM/F-12, glucose, L- of RNA was reverse-transcribed with a Maxime RT Premix Kit glutamine, NaHCO3, ITS supplement, and bovine fibronectin (iNtRON Biotechnology, Korea), using a C1000 thermal cycler for 6-8 days. At stage 4, for Nestin-positive cell expansion, 5 × 105 (Bio-Rad Laboratories, Hercules, CA, USA). For the real-time cells/well were transferred into poly-L-ornithine/fibronectin- PCR analysis, the SYBR Green PCR Master Mix and ABI Prism coated 24-well plates with N-2-supplemented (insulin, trans- Sequence Detection System 7000 (PE Applied Biosystems, Fos- ferrin, selenite, putrescine, and progesterone) medium that also ter City, CA, USA) were used. The sequences of the primers (Bi- contained basic fibroblast growth factor (bFGF), FGF-8b, sonic oneer, Korea) are shown in Table 1. The PCR cycling conditions hedgehog-N (Shh-N), and ascorbic acid, for 4-6 days. At stage 5, were 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of to differentiate cells into dopaminergic neurons, the cells were 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds, 60°C cultured in N-2/ascorbic acid medium (without growth factors) for 20 seconds, and 95°C for 15 seconds. for 10-15 days. Tyrosine hydroxylase quantification by enzyme-linked Characterization of differentiated hDPSCs by immunosorbent assay immunocytochemistry To confirm quantitatively the dopaminergic differentiated hDP- During the five differentiation stages, the neural characteristics SCs, the tyrosine hydroxylase enzyme-link immunosorbent as- of the cells were analyzed on the protein level. The hDPSCs were say (TH ELISA) was performed. Total protein extracted from cultured on a chamber slide for 1 day. Subsequently, the cells cells, undifferentiated hDPSCs (stage 1), and fully differentiated were fixed with 4% paraformaldehyde in 0.1 M phosphate buf- hDPSCs (stages 5) were prepared using RIPA lysis buffer (Gen- fer (pH = 7.4) and then treated with 4% bovine serum albumin DEPOT Laboratories, Barker, TX, USA).
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