Method: To investigate the stemness of human dental pulp cells, we performed flow cytometry and immunotyping during the expended culture. By decoy immunization, we constructed monoclonal antibodies (MAbs) against undifferentiated pulp progenitors originated from early passages. Differentiated cells were used as a decoy immunogen. 12 Balb/c mice were immunized in right hind footpads with differentiated pulp cells and in the left with undifferentiated cells. After immunization, the left popliteal lymph node cells were collected and used for construction of hybridoma.
Result: The mesenchymal stem cell markers were expressed in early passages of the culture. Stem cell populations were decreased during the expended culture. Under the differentiation condition, mineralization was facilitated within 2 weeks. Dentinogenic markers appeared to be peaked at around passage 5 and decreased with increasing passage number. Based on the passage-specific existence of hDPSC, we performed a screening of surface molecules specifically expressed on progenitors. By decoy immunization, we collected 45 MAbs bound to undifferentiated pulp cells. Of these, 14 MAbs bound to the undifferentiated cells, not only weakly, but not at all to the differentiated cells. 40-60% of newly identified MAbs-positive cells were also positive for known mesenchymal markers. Several MAbs were bound to cancer cells originated from mesenchymal tissues, indicated that these cells might be good sources for identification of specific antigen against these MAbs.
Conclusion: Decoy immunization is an efficient method for MAbs screening against hDPSC progenitors, and these will be helpful for enrichment of hDPSC for dentin regeneration. This work was supported by WCU program (R31-10069) funded by the NRF in Korea.