The clusters were visualized using an inverted

optic micr

The clusters were visualized using an inverted

optic microscope. Dental pulp stem cells were isolated and expanded in vitro from EGFP-transgenic mice. The cells are plastic-adherent and showed rapid expansion and proliferation capacity in vitro after isolation. Approximately 80% of the cells proliferated after 48 h of culture ( Fig. 2). A polymorphic morphology was observed in the cell populations obtained. Initially the cells had rounded or fibroblastoid shapes ( Fig. 1a). Cells with fusiform ( Fig. 1b) and stellate shapes ( Fig. 1c) began to appear amongst fibroblastoid cells after 20–28 days of culture. Curiously, in one batch of cells, some elongated cells acquired the contraction capacity (see supplemental material). Proliferative mDPSC showed a normal karyotype in the passages evaluated ( Panobinostat Fig. 1d). In only one isolate, tetraploidy was found in 40% of the cells in the sixth passage (data not shown). The formation of cell clusters in vitro was also observed (data not shown). More than 90% of the cells expressed GFP ( Fig. 2 and Fig. 3c). After long term cryopreservation, mDPSC are capable of quickly restarting proliferation in culture, in a manner similar to that of recently isolated cells. To investigate the phenotypic characteristics of the mDPSC, cell cultures were analysed

using antibodies against several cell surface and intracellular antigens. In the third passage, flow cytometric analysis revealed the expression of cell surface molecules that characterize mesenchymal stem cells, Oligomycin A in vitro such as CD90,

CD73, STRO-1 and Ly6a/Sca-1 (Fig. 2). In contrast, the percentage of hematopoietic cell markers was low Cyclin-dependent kinase 3 (CD117) or undetectable (CD34, CD11b, or CD45) in this passage. The expression of hematopoietic stem cell markers was detected only in the first passage (data not shown). Approximately 80% of the cells were positive for the endothelial cell marker CD31 (Fig. 2). Similar results were observed with cells cultured until the 18th passage (data not shown). Cells were positive for alkaline phosphatase (Fig. 3a) such as observed in the positive control, a culture of embryonic stem cells (Fig. 3b). Curiously, the expression of others embryonic stem cells markers, such as SSEA-1, was strongly positive in mDPSC cultures (Fig. 3d), whereas SSEA-4 and TRA-1-60 markers were not detected by immunofluorescence analysis (Fig. 3e and f). The transcript ZFP42/Rex-1, but not Nanog, was detected in undifferentiated stem cells by RT-PCR analysis ( Fig. 4). Flow cytometry analysis confirmed that approximately 25% of the cells were positive for Pou5f1/Oct-4 ( Fig. 2). Confluent monolayers of the mDPSC were submitted to conditions known to promote osteogenesis, chondrogenesis and adipogenesis. Control mDPSC were cultured only with growth medium (Fig. 5b, d and f).

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