Characteristic expression of MSX1, MSX2, TBX2, and ENTPD1 in dental pulp cells

Dental pulp cells (DPCs) are a promising source of transplantable cells in regenerative medicine. However, DPCs have not been fully characterized at the molecular level. The purpose of this study was to distinguish DPCs from various source-derived mesenchymal stem cells, fibroblasts, and other cells by the expression of several DPC-characteristic genes. DPCs were isolated from human pulp tissues by the explant method, or the enzyme digestion method, and maintained with media containing 10% serum or 7.5% platelet-rich plasma. RNA was isolated from the cells and from dental pulp tissue specimens. The mRNA levels were determined by DNA microarray and quantitative real-time PCR analyses. The msh homeobox1 (MSX1), msh homeobox 2 (MSX2), T-box 2 (TBX2), and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) mRNA levels in DPCs were higher than the levels found in the following cells: mesenchymal stem cells, derived from bone marrow, synovium, and adipose tissue; and in cells such as fibroblasts, osteoblasts, adipocytes, and chondrocytes. The enhanced expression in DPCs was consistently observed irrespective of donor age, tooth type, and culture medium. Moreover, these genes were expressed at high levels in dental pulp tissue in vivo. We conclude that this gene set may be useful in the identification and characterization of DPCs in basic studies and pulp cell-based regeneration therapy. Dental pulp cells were isolated by the explant outgrowth method (Calcified tissue international (2000) 66:129-138.) from healthy teeth according to protocol approved by ethical authorities at Hiroshima University. Human skin fibroblasts were obtained from Kurabo (Osaka, Japan). Human gingival fibroblasts were isolated as described previously (Journal of periodontal research (2003) 38: 242-246.). Bone marrow-derived MSCs (BM-MSCs) were isolated13 from Hiroshima University with informed consent, or were obtained from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD) and PromoCell Co., Ltd. (Heidelberg, Germany). Osteoarthritis and rheumatoid arthritis synovium-derived MSCs, and adipose tissue-derived MSCs, were obtained from Cell Applications Inc. (San Diego, CA) and Zen-Bio Inc. (Research Triangle Park, NC). Osteoblasts, adipocytes, and chondrocytes, which were derived from BM-MSC, were cultured with appropriate differentiation-inducing media for 28 days (Genes to cells (2009)14:407-424). 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. We removed FGF-2 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, Japan) and an RNeasy Mini Kit (Qiagen, Chatsworth, CA). DNA microarray analysis was performed by KURABO GeneChip Custom Analysis Service with Human Genome U133 Plus 2.0 chips(Affymetrix. Inc., Santa Clara, CA). The CHP data (Microarray Suite version 5.0, Scaling Factor = 1, Normalization Value = 1, Detection Call a1 = 0.05, a2 = 0.065, Tau = 0.015, Affymetrix Inc.) were standardized by the global median normalization method using GeneSpring (Silicon Genetics, Redwood City, CA). The normalization was limited by flag values, and the median was calculated using the genes that exceeded the Present or Marginal flag restriction.