Immunomagnetic sorting of PDLSCs and PDLSCs identification

PDLs were isolated from normal premolars extracted from four patients (aged from 13–14 years, 2 for males and 2 for females) who sought orthodontic treatment. Written informed consent were provided by all patients and their guardians, and ethical approval had been obtained from the Ethics Committee of Stomatological Hospital of Guangzhou Medica University. PDLs were seperated from the middle third of the roots gently and digested in 3 mg/ml Type I Collagenase as well as 4 mg/ml Dispase for 1 h at 37 °C. Single-cell suspensions were seeded into 25 cm2 flask with a-MEM supplemented with 10% fetal calf serum, 100 μmol/l ascorbic acid 2-phosphate, 2 mmol/l glutamine, 100U/ml penicillin, and 100 μg/ml streptomycin. The cultures were incubated in a humidified atmosphere of 5% carbon dioxide and 95% air at 37 °C. To isolate putative periodontal ligament stem cells, subconfluent secondary cultures of periodontal ligament cells were separated by using immunomagnetic cell sorting technique as previously reported [15]. According to the manufacturer’s instructions, single-cell suspensions of PDLCs were incubated with mouse anti-human STRO-1 IgM primary antibody (Life technologies, USA) on a rotator for 1 h at 4 °C, as STRO-1 was the best-known mesenchymal stem cell marker. Wash the cells by using phosphate buffered saline containing 0.1% bovine serum albumin (BSA) and 2 mM EDTA, and resuspended with Dynabeads rat anti-mouse IgM for 45 min on a rotator at 4 °C. All bead-positive cells were isolated with Dynamag 15 magnetic particle concentrator and cultured in Nutristem MSC Basal Medium supplemented with Nutristem MSC XF Supplement Mix MSC (Biological Industries, Israel), while the bead-negative cells were incubated in a-MEM medium. To assess colony-forming efficiency, after 12 days, subconfluent cultures (second passage) of MSC positive and negative cells were fixed with 70% ethanol and then stained with 0.1% Crystal Violet. Aggregates of over 50 cells were counted as a colony under microscopic observation. To analyze the expression of relative markers of PDLSCs, subconfluent secondary cultures of bead-positive and bead-negative cells were seeded at 1 × 103 cells per well on a eight-chambered slide. All the cells were fixed in 4% paraformaldehyde for 20 min at room temperature and blocked with phosphate buffered saline containing 1% BSA as well as 0.3%Triton-100X for 30 min at room temperature. Then the samples were incubated with primary antibodies at 4 °C overnight. The primary antibodies were as follows, mouse anti-human STRO-1 IgM, mouse anti-human CD146, mouse anti-human vimentin, mouse anti-human cytokeratin (Life Technologies, USA). The cells were incubated with donkey anti-mouse secondary antibodies of IgG-FITC (Jackson, USA) for 45 min at 37 °C subsequently. Cell nuclei were stained with 0.25 μg/l Hoechst 33342 (Life Technologies, USA) for 5 min at room temperature. Cell staining was evaluated using the fluorescence microscope (Leica CTR6000, Germany).

APTG-CM preparation

APTG-CM was made as previously reported [2]. The use of Sprague-Dawley rats (Laboratory Animal Center of Sun Yat-sen University) and the experimental protocols were approved by Animal Care Committee of Guangdong Province. Eight-day postnatal Sprague-Dawley rats were killed by cervical dislocation. Twenty molar germs were removed from mandibles under a stereomicroscope, and Hertwigs epithelial root sheath (HERS) associated with apical mesenchyme was carefully dissected from the apical root of molar germs (Fig. 2A). The apical portions of tooth germ were minced into < 1 mm3, and digested in 3 mg/ml Type I Collagenase as well as 4 mg/ml Dispase for 1 h at 37 °C. Single-cell suspensions were seeded into 75cm2 flask at 1 × 105 cells/ml with a-MEM supplemented with 10% fetal calf serum, 100 μmol/l ascorbic acid 2-phosphate, 2 mmol/l glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cultures were incubated in a humidified atmosphere of 5% carbon dioxide and 95% air at 37 °C. The culture medium of primary apical tooth germ cells containing both epithelial and mesenchymal cells was changed every 48 h until full confluence (Fig. 2B). The medium was collected and centrifuged at 2000g for 15 min 3 days after the last medium changed. Then the supernatants, which were mixed with an equal volume of fresh a-MEM medium, were used as APTG-CM and stored at − 20 °C for PDLSCs culture.

Fig. 2

Isolation and culture of APTGs. A mandibular developing first molar germ of a 8-day-old Sprauge-Dawley rat (A). The cultured epithelial and mesenchymal apical tooth germ-derived stem cells. EC and MC refer to epithelial and mesenchymal cells respectively (B)

Alkaline phosphatase (ALP) activity assay

PDLSCs were inoculated into 12-well culture dish (2 × 103 cells/well) and cultured with or without APTG-CM for 4, 8, 12 and 16 days. The ALP activity was detected with an assay kit (Jiancheng Co, Nanjing, China) according to the manufacture’s instructions. Briefly, cells were harvested using protein lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl as well as 1%NP-40. Then, cells were incubated with the applied phenol standard liquid, buffer and the substrate solution for 15 min at 37 °C. After adding the color-substrate solution, all the experiments were performed in triplicate in three different experiments using the same cells and tested at 520 nm wavelength by a enzyme micro-plate reader.

Mineralization assay

The mineralization assay in vitro were operated as previous described [16]. The PDLSCs (1 × 105/well) were cultured to confluence in Nutristem MSC Basal Medium supplemented with Nutristem MSC XF Supplement Mix MSC in six-well cuture dishes. Then, the cells were cultured in two different medium conditions. The induced cells were grown in APTG-CM, while the non-induced cells were cultured in Nutristem MSC Basal Medium. After 14 and 21 days, the cells were fixed with 4% paraformaldehyde for 20 min, and stained with 2% Alizarin Red Solution for 30 min at room temperature. The mineralized nodules were imaged by microscope (Zeiss, Germany). Then added 1.5 ml 0.5 N HCl into each well for 30 min and collected the supernatent. The absorbance of each supernatent was measured by spectrophotometer at 405 nm wavelength (Thermo, USA).

Real time q-PCR analysis for osteoblastic gene

Total RNAs were isolated from 21-day APTG-CM-induced and non-induced PDLSCs derived from four individual donors (Sample A, B, C and D) using mirVanaTM miRNA isolation kit (Life Technologies, USA) according to the manufacture's instructions. The quantity and purity of the total RNA were verified by spectrophotometer. Reverse transcription reaction was performed using reverse transcriptase M-MLV (Takara, Japan), and mRNA expression levels were quantified using Light Cycler 480 SYBR Green I Master (Takara, Japan). The primers of osteoblastic target genes were listed as in Table 1. Relative quantification presented qPCR data of target genes relying on internal control gene GAPDH as reference. PCR conditions of Light Cycler 480 (Roche, Switzerland) were as follows: pre-denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C 15 s, primer annealing at 56 °C for 15 s, and extension at 72 °C for 25 s.

Table 1 Primers used for Real-time RT-PCR are listedIllumina miRNA sequencing

Total RNAs of 21-day APTG-CM-induced and non-induced PDLSCs derived from three individuals (Sample A, B and C) were isolated by using mirVana™ miRNA isolation kit (Life Technologies, USA) and sequenced for analysis, which were sent to Shanghai Biotechnology Corporation. Briefly, 3' and 5' adapters were sequentially ligated to small RNA, and single-stranded RNA molecules with adapters at both ends were amplified for 11 cycles using a common primer and a primer index. Then the cDNA was purified in 6% Novex TBE PAGE Gel, and the gel slice corresponding to the DNA size was excised and eluted in 1 × gel elution buffer. DNA High Sensitivity Chip of Aglient 2100 (Aglient Technologie, USA) was used in the quality control. After that, the purified cDNA was used for cluster generation and sequencing analysis using Agilent Technologies 2100 Bioanalyzer ( Agilent Technologie, USA).

miRNA qRT-PCR analysis

Total RNAs of 21-day APTG-CM-induced and non-induced PDLSCs derived from Sample D were isolated using mirVanaTM miRNA isolation kit (Life Technologies, USA). The designed miR-146a-5p-specific primer and U6B internal control primers were listed in Table 2, Quantitative RT-PCRs for miRNA were performed with SYBRe PrimeScript miRNA RT-PCR Kit (Takara, Japan). The PCR reaction were conducted utilizing LightCycler 480 (Roche, Switzerland) as following conditions: 1 cycle of pre-denaturation at 95 °C for 30 s, followed by 40 cycles of: 5 s at 95 °C and 20 s at 60 °C.

Table 2 Primers used for qRT-PCRmiRNA transfection

In order to validate the regulatory effects of miR-146a-5p on osteoblast differentiation of PDLSCs in vitro, recombinant lentivirus miR-146-5p (Lenti-miR-146-5p) and lentivirus anti-miR-146-5p (Lenti-anti-miR-146-5p), along with lentivirus miR negative control (Lenti-miR-NC) and lentivirus anti-miR negative control (Lenti-anti-miR-NC), designed and provided by Genechem lnc. (Shanghai, China, http://www.genechem.com.cn), were used to regulate miR-146a-5p levels in PDLSCs. After the cells reached about 30 mlnc./" l lowotathey were transfected with lentivirus at the desired multiplicity of infection (MOI = 1000) with enhanced infection solution (ENI.S) according to the manufacturer’s protocol. Stably transfected cells were selected with 2 μg/ml puromycin (Sigma, German). The expression of stable transformants was observed under fluorescence microscopes and identified by qRT-PCR.

Predicted miRNAs targets

A combination of two online softwares, miRWalk (http://mirwalk.umm.uni-heidelberg.de/) and TargetScan (https://www.targetscan.org/cgi-bin/targetscan/hsa-miR-146a-5p+), was used to predict the potential hsa-miR-146a-5p target genes. We selected the identical target genes indicated in both softwares and then did bioinformatics analyses to determine associations between hsa-miR-146a-5p and target genes by PubMed (http://www.ncbi.nlm.nih.gov/pubmed/).