Healthy teeth were obtained with informed consent from donors (Table I), following protocols approved by the Hiroshima University Ethics Authorities (permit no. D88-2; Hiroshima, Japan). DPCs were grown out of dental pulp tissue explants in the presence of DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biowest LLC, Miami, FL, USA or GE Healthcare Life Sciences, HyClone Laboratories, Logan, UT, USA) and a 1% antibiotic-antimycotic solution (Invitrogen Life Technologies, Carlsbad, CA, USA) in 95% air (5% CO₂) at 37°C (1). Migrating cells were briefly incubated with a 0.05% trypsin-EDTA solution (Sigma-Aldrich) and harvested cells were plated at 4×10⁴ cells/cm² in 10-cm tissue culture dishes (BD Biosciences, San Jose, CA, USA) and incubated with DMEM supplemented with 10% FBS, 1% antibiotic-antimycotic solution and 1 ng/ml fibroblast growth factor-2 (FGF-2) (medium A). These DPCs were confirmed to induce matrix calcification following exposure to osteogenesis induction medium (data not shown).

DPSCs were isolated at Hiroshima University with informed consent from donors (Table I), using the enzyme digestion method (6,7). Pulp tissues were digested with 3 mg/ml collagenase type 1 (Life Technologies, Carlsbad, CA, USA) and 4 mg/ml dispase (Life Technologies), in the presence of DMEM, in 95% air (5% CO₂) at 37°C for 1 h. Dispersed cells were filtered through a 70-µm mesh (BD Biosciences) and seeded at a low density of 1×10⁴ cells/10-cm tissue culture dishes in DMEM supplemented with 20% FBS and a 1% antibiotic-antimycotic solution. Colony-forming cells were incubated briefly with a 0.05% trypsin-EDTA solution (Sigma-Aldrich) and harvested cells were plated at 4×10⁵ cells/cm² in 10-cm tissue culture dishes and incubated with medium A. These DPSCs also induced calcification following exposure to osteogenesis-induction medium (data not shown). In certain studies, cells were incubated with α-MEM supplemented with 10% FBS or 7.5% PRP. PRP was prepared using a kit from AdiStem Ltd. (Hong Kong, China).

Human skin FBs were obtained from Kurabo Industries Ltd. (Osaka, Japan). Human gingival FBs were obtained with informed consent from donors and were isolated as described by Kawahara and Shimazu (12). Bone marrow-derived MSCs (BM-MSCs) were isolated with informed consent from donors (13), or were obtained from Cambrex Bio Science Walkersville Inc. (Walkersville, MD, USA) and PromoCell GmbH (Heidelberg, Germany). Osteoarthritis and rheumatoid arthritis synovium-derived MSCs (OA-MSCs and RA-MSCs) and adipose tissue-derived MSCs (A-MSCs), were obtained from Cell Applications, Inc., (San Diego, CA, USA) and Zen-Bio, Inc., (Research Triangle Park, NC, USA) (Table I).

Different cell types (donor information in Table I) were cultured by incubating the cells with medium A at specific passage numbers, as follows: DPCs (DPCs-2, −3 and −4), BM-MSCs (BM-MSCs-1, −2 and −3), A-MSCs (A-MSCs-1, −2 and −3), OA-MSCs (OA-MSCs-1, −2 and −3) and RA-MSCs (RA-MSCs-1, −2 and −3) at passages 5–9; and skin FBs (FBs-1 and −2) and gingival FBs (FBs-3) at passages 7–14. Cells were cultured under similar conditions using the same batch of FBS. These cells were expanded with FGF-2 to maintain their multipotent nature throughout several mitotic divisions (14). FGF-2 was removed from the culture medium 72 h before the isolation of RNA to decrease a direct effect of the growth factor on gene expression. The total RNA was isolated, 24 h after the cultures reached confluency, using TRIzol (Life Technologies) and an RNeasy Mini kit (Qiagen, Chatsworth, CA, USA). In addition, total RNA was isolated from OBs, ADs and CHs, which were derived from BM-MSC (BM-MSCs-1, −2 and −3) cultures exposed to appropriate differentiation-inducing media for 28 days (13). DNA microarray analyses were carried out by a Kurabo GeneChip Custom Analysis Service with the Human Genome U133 Plus 2.0 chips (Affymetrix Inc., Santa Clara, CA, USA). Raw data were standardized by the global median normalization method using GeneSpring (Silicon Genetics, Redwood City, CA, USA). Normalization was limited by flag values and the median was calculated using genes that exceeded the present or marginal flag restrictions. The raw data have been deposited at the Gene Expression Omnibus database (accession nos. GSE9451 and GSE66084) (15).

First-strand cDNA was synthesized from total RNA using ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan) and oligo(dT) primer. RT-qPCR analyses were carried out using an ABI PRISM® 7900HT Sequence Detection System instrument and software (Applied Biosystems Inc., Foster City, CA, USA), based on the comparative Ct method. The cDNA were amplified using the Universal PCR Master mix (Applied Biosystems Inc.) with a forward and reverse primer (Table II). The PCR cycling conditions included an incubation of 50°C for 2 min, denaturation of 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. TaqMan probes were purchased from Roche Diagnostics (Basel, Switzerland). Data were normalized against 18S rRNA levels.

Data were analyzed by a two-way analysis of variance (ANOVA) and are expressed as mean ± standard deviation. The statistical differences between two groups were evaluated using Bonferroni's test when the ANOVA indicated a significant difference among the groups. In all analyses, P<0.05 was considered to indicate a statistically significant difference.

In DNA microarray analyses, MSX1, MSX2, TBX2 and ENTPD1, which are involved in tooth development and calcification (16–19), were expressed at significantly higher levels in DPCs (>4-fold, P<0,05) compared to FBs, BM-MSCs, A-MSCs, OA-MSCs, RA-MSCs, OBs, ADs and CHs (Fig. 1).

Subsequently, the mRNA levels of the DPC-characteristic gene candidates were quantitated by qPCR in DPCs, DPSCs, FBs and BM-MSCs (Fig. 2). DPCs were isolated from permanent molar teeth by the explant method and DPSCs were isolated from primary incisor and permanent molar teeth by the enzyme digestion method (Table I). These cells consistently showed the enhanced expression of MSX1, MSX2, TBX2 and ENTPD1 mRNA when compared to FBs and BM-MSCs, irrespective of the different isolation procedures, donor age, donor gender and tooth types. Furthermore, the selective expression of the four genes in DPCs/DPSCs was observed with cells from different donors by DNA microarray and qPCR analyses (Fig. 2).

DPSCs were incubated in α-MEM with either 10% FBS or 7.5% PRP, as culture media may affect the expression of the DPC-characteristic genes. In these media, DPSCs showed a higher expression of MSX1, MSX2, TBX2 and ENTPD1 compared to A-MSCs, irrespective of whether FBS or PRP was used (Fig. 3). Therefore, the enhanced expression of these genes in DPSCs could also be observed with α-MEM (Fig. 3) and DMEM (Fig. 2). In the presence of PRP, the average expression levels of these genes were also higher in DPSCs compared to A-MSCs, although the observed differences were not statistically significant.

In a pilot study, whether the passage number affects the DPC-characteristic gene expression was evaluated in DPSCs. The MSX1, MSX2, TBX2 and ENTPD1 genes were expressed at high levels in DPSCs, irrespective of the passage number; at least until the 11th passage (data not shown).

Certain DPC-characteristic genes may be expressed in dental pulp tissue in vivo. MSX1, MSX2, TBX2 and ENTPD1 were expressed at comparable levels in cultured DPCs-1 and in the dental pulp specimens from 4 donors (Fig. 4). The expression levels of these genes were comparable between Pulp-1 and DPCs-1, as these were isolated from the same tooth. However, although Pulp-1 and Gingiva-1 were obtained from the same donor (Table I), the expression levels of these genes were higher in Pulp-1 compared to Gingiva-1.