Altered development of dopaminergic neurons differentiated from stem cells from human exfoliated deciduous teeth of a patient with Down syndrome

Human exfoliated deciduous teeth were provided by Pediatric Dentistry and Special Need Dentistry at Kyushu University Hospital in Japan. After informed parental consent was obtained, deciduous teeth were collected from a normal participant and a patient with DS at 6 and 14 years of age, respectively. The isolation procedure was completed as previously described [15]. Briefly, the pulp tissue was subjected to an enzymatic dissociation in 3 mg/mL collagenase I (Washington, NJ, USA) and 4 mg/mL dispase II (Wako, Osaka, Japan) for 1 h, and then maintained at 37 °C in a humidified 5% CO₂ incubator in the Alpha modification of Eagle’s Minimal Essential Medium (α-MEM; Sigma-Aldrich, MO, USA) containing 15% fetal bovine serum (Sigma-Aldrich), 100 μM L-ascorbic acid 2-phosphate (Wako), 2 mM L-glutamine (Life Technologies, NY, USA), 250 μg/mL Fungizone (Life Technologies), 100 U/mL penicillin (Life Technologies), and 100 μg/mL streptomycin (Life Technologies). Cells of not more than 10 passages were used, but Ctrl-SHED and DS-SHED were not always of the same passage.

Whole-cell lysates were extracted with lysis buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 5% β-mercaptoethanol, and 10% glycerol), and the protein concentration was measured using Bradford ULTRA (Novexin, Cambridge, UK). A total of 5 μg of protein was separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking with 5% non-fat milk for 30 min, the membrane was incubated overnight at 4 °C with anti-nestin (1:1000; Millipore, CA, USA) and anti-HSP90 (1:1000; Santa Cruz Biotechnology, CA, USA) antibodies. Membranes were washed and incubated with HRP-conjugated secondary antibody (1:5000; Santa Cruz Biotechnology) for 1 h at room temperature and visualized with ECL prime (GE Healthcare, Buckinghamshire, UK). The chemiluminescent signals were detected and quantified using LAS-1000 pro (Fuji Film, Tokyo, Japan) with Image Gauge software (Fuji Film). HSP90 was used as an internal control. To normalize the nestin expression, the chemiluminescent signal of nestin was divided by the chemiluminescent signal of HSP90.

DN differentiation was induced as previously described with minor modifications (brain derived neurotrophic factor [BDNF] was excluded in the second step) [16]. In the first step, 1.5 × 10⁵ SHED were plated onto a 6-well culture plate or glass coverslips coated with 0.01% poly-L-lysine (Sigma-Aldrich), in the same culture medium as described above. They were incubated overnight at 37 °C in the presence of 5% CO₂, and were then cultured in serum-free Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 20 ng/mL epidermal growth factor (Sigma-Aldrich), 20 ng/mL basic fibroblast growth factor (Peprotech, NJ, USA), and 1% N2 supplement (Life Technologies) for 2 days at 37 °C, in the presence of 5% CO₂. In the second step, DMEM was replaced with neurobasal medium (Life Technologies) supplemented with 2% B27 supplement (Life Technologies), 1 mM dibutyryladenosine 3,5-cyclic monophosphate (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), and 200 μM ascorbic acid (Nacalai Tesque, Kyoto, Japan), and cells were incubated for 5 days, at 37 °C, in the presence of 5% CO₂.

The cells cultured on coverslips were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10 min. The cells were permeabilized with 0.1% TritonX-100 for 5 min, then blocked with 2% bovine serum albumin (BSA; Wako) in PBS for 20 min at room temperature. Next, cells were stained with primary antibodies against STRO-1 (1:100; Millipore), nestin (1:250; Millipore), β-tubulin III (1:250; Sigma-Aldrich), tyrosine hydroxylase (TH; 1:100; Millipore), N-methyl-d-aspartate receptor subunit 1 (NMDAR1; 1:100; Millipore), and DA (1:200; Abcam) for 90 min. Following this, cells were incubated with Alexa Fluor secondary antibodies (1:500; Life Technologies) for 1 h at room temperature in the dark. The cells were counterstained with 0.1 μg/mL DAPI (Dojindo) for 5 min, and then mounted with ProLong diamond (Life Technologies). The fluorescence images were taken with Nikon C2 confocal microscope (Nikon, Tokyo, Japan) in Fig. 1c and 3a, with Zeiss LSM700 confocal scanning microscope (Zeiss) in Fig. 2a and d, with Zeiss Axio Imager M2 microscope (Zeiss) equipped with ApoTome2 (Zeiss) in Fig. 4c and d.

For morphological analysis, the TH-immunostained images were analyzed with MetaMorph software (Molecular Devices, CA, USA). A morphological analysis of DN was performed as previously described [19]. Briefly, neurite length and number of branches were measured from 100 TH-positive cells in 20 non-overlapping TH-immunostained images selected randomly from three experiments using the Neurite Outgrowth and Multi-Wavelength Cell Scoring module of MetaMorph software (Molecular Devices). Next, the cells were classified into 4 stages based on neurite length and cell diameter as previously described [19].

To measure the number of NMDAR1 puncta per unit length of neurite, DN were immunostained with anti-NMDAR1 and anti-TH antibodies. Thirty neurites that were in focus and clearly observed were chosen from 10 Ctrl-DN and 12 DS-DN that were positive for both NMDAR1 and TH. These cells were randomly chosen from three experiments. The number of NMDAR1 puncta per 25 μm of neurite was analyzed.

Extracellular DA was measured using a Dopamine Research ELISA kit (BA E-5300, LDN, Nordhorn, Germany) according to the manufacturer’s instructions. SHED were plated at a concentration of 5 × 10⁵ cells per 6-cm dish and differentiated into DN. 500 μl of culture medium was collected to measure extracellular DA. To measure extracellular DA under glutamate stimulated conditions, the cells were treated with 30 μM L-glutamate for 1 min at 37 °C before harvesting the medium. Next, the cell culture medium was centrifuged at 20,400 g for 5 min at 4 °C to remove cell debris and immediately stored at − 80 °C until assayed. Subsequently, total protein was extracted from cells using lysis buffer (62.5 mM Tris-HCl pH 6.8 supplemented with 2% SDS, 5% β-mercaptoethanol, and 10% glycerol), and the protein concentration was measured using Bradford ULTRA (Novexin). To normalize the DA amount in each sample, the DA amount was divided by the total protein of that sample.

Total RNA was extracted from the cells using an RNAeasy Mini Kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized using a ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). The sequences of primer sets used in this study were as follows: DAT1: 5’-TGCTGCACAGACACCGTGAG-3′ (forward), 5’-AATGGTCCAGGAGCGTGAAGA-3′ (reverse); VMAT2: 5’-TGAAGAGAGAGGCAACGTCA-3′ (forward), 5’-CGTCTTCCCCACAAACTCAT-3 (reverse); HPRT1: 5’-CCTGGCGTCGTGATTAGTG-3′ (forward), 5’-TCCCATCTCCTTCATCACATC-3′ (reverse). Real-time quantitative PCR was performed using GoTaq qPCR Master Mix (Promega, WI, USA) and analyzed with StepOnePlus Real-Time PCR Systems (Life Technologies). The threshold cycle (Ct) value of HPRT1 was subtracted from the Ct value of the target genes (ΔCt). Statistical analysis was performed using the ΔCt values from four experiments. The relative expressions of the target genes are shown as fold changes determined using the 2^-ΔΔCt method.

Values are represented as mean ± standard error of the mean (SEM) from at least three experiments. Two-tailed Student’s t-tests were used to compare the Ctrl and DS groups. Differences were considered significant if p < 0.05. JMP software (SAS Institute, NC, USA) was used for the statistical analysis.

Methods of figure S1 and S2 are described in Additional file 4.

We isolated SHED from the deciduous teeth of a child with DS (DS-SHED) and a normal participant (Ctrl-SHED). DS-SHED were spindle-shaped and exhibited a fibroblastic cell morphology that was similar to Ctrl-SHED (Fig. 1a). The presence of 3 copies of chromosome 21 in the nuclei of DS-SHED was verified by FISH (Fig. 1b). Next, analysis of cell proliferation showed similar proliferation of DS-SHED and Ctrl-SHED (Additional file 1: Figure S1). SHED have multi-lineage potential and express mesenchymal and neuronal stem cell markers. To examine the stem cell characteristics of DS-SHED, immunofluorescence staining was performed using antibodies against stem cell markers. Both DS-SHED and Ctrl-SHED expressed STRO-1, a mesenchymal stem cell marker (Fig. 1c, upper panel). In contrast, expression of nestin, a neuronal stem cell marker, was reduced in DS-SHED compared to Ctrl-SHED (Fig. 1c; lower panel; yellow arrows denote cells with weak nestin expression). Western blotting (Fig. 1d) and flow cytometry analysis (Additional file 2: Figure S2) were also used to examine nestin expression and showed reduced nestin expression in DS-SHED compared to Ctrl-SHED.

We differentiated Ctrl-SHED and DS-SHED into DN, and immunostained these with antibodies to the neuronal marker β-tubulin III and DN marker TH. DN differentiated from DS-SHED (DS-DN) expressed β-tubulin III and TH (Fig. 2a), but neurite length and branching were reduced compared to DN differentiated from Ctrl-SHED (Ctrl-DN). Quantitative analysis also showed that neurite length and branching were reduced in TH-expressing DS-DN compared to Ctrl-DN (Fig. 2b, c). DN development, evaluated in 4 stages according to cell morphology (Fig. 2d; based on a report by Leach et al.) [19], showed that DS-DN development was reduced compared to Ctrl-DN (Fig. 2e).

A functional analysis of DN was performed by examining DA expression and DA secretion. DA expression was examined by immunostaining cells, which showed that DA expression was present in both Ctrl- and DS-DN (Fig. 3a). Next, DA secretion was examined by measuring extracellular DA and it was observed that extracellular DA of DS-DN was significantly reduced compared with that of Ctrl-DN under basal conditions (p = 0.046; Fig. 3b). Although no significant differences between the DS- and Ctrl-DN were observed (P = 0.506), extracellular DA of DS-DN was lower than that of Ctrl-DN under glutamate stimulated conditions (Fig. 3c).

Possible causes of reduced DA section from DS-DN include the abnormal expression of molecules involved in DA homeostasis, such as dopamine transporter 1 (DAT1) that mediates DA reuptake, vesicular monoamine transporter 2 (VMAT2) that mediates packaging of DA into secretory vesicles, and glutamate receptors. Analysis of DAT1 and VMAT2 mRNA expression showed that DAT1 expression was greater and VMAT2 expression was reduced in DS-DN compared to Ctrl-DN (Fig. 4a, b). Furthermore, expression of NMDAR1, a subunit of glutamate receptor, by immunostaining showed that the number of NMDAR1 puncta per unit length of neurite was reduced in DS-DN compared to Ctrl-DN (Fig. 4c-e).