Differentiation and Immunoregulatory Activity of Dental Pulp-Derived Mesenchymal Cells.

INTRODUCTION. Mesenchymal Stem Cells (MSC) have the ability to renew and differentiate into various lineages of mesenchymal tissue such as bone, cartilage, fat, muscle, etc. Moreover they are not immunogenic and display immunoregulatory activity in preclinical animal models.

The aim of this study was to investigate whether dental pulp MSC (DPMSC) were able to differentiate toward osteogenesis, chondrogenesis and adipogenesis and express the characteristic immunomodulatory activity of MSC derived from known sources such as bone marrow.

METHODS and RESULTS. We isolated MSC from dental pulp and bone marrow samples obtained from fully informed healthy donors. Flow cytometric analysis showed that either DPMSC or bone marrow MSC (BMMSC) expressed the membrane antigens SH2, SH3, SH4, CD29 and CD166, while CD 14, CD 34 and CD 45 were negative.

Cell differentiation was evaluated after PDMSC and BMMSC were cultured in appropriate conditions. To evaluate adipogenesis after 2–3 weeks of culture the cells, containing neutral lipids in fat vacuoles, were fixed in 10% formalin and stained with fresh oil red-O solution.

To demonstrate osteogenic differentiation, the cultures were fixed and subjected to alkaline phosphatase and von Kossa staining. For chondrogenic differentiation pellets were formalin fixed, embedded in paraffin, examined morphologically and immunostained for Type II collagen.

We observed that either PDMSC and BMMSC were able to express clear osteogenic and chondrogenic differentiation as demonstrated by von Kossa staining and Type II collagen immunostaining respectively, but a lower number of adipocytes was obtained, according to morphology and red-oil staining, in DPMSC cultures.

For proliferation assay cells were incubated overnight, then [methyl-3H] Thymidine was added (Time 0) and radioactivity followed for up to 15 days. DPMSC and BMMSc presented a very different behaviour in that DPMSC radioactivity had a steep increase from day 3 to 8, then decreasing at day 15, although still above the baseline value. On the contrary BMMSC radioactivity did not change significantly over the time of observation.

Modulation of T Lymphocyte proliferation was studied by coculturing PHA stimulated T cells in the presence of MSC. Compared to cultures of T cells alone, the uptake of [methyl-3H] Thymidine was inhibited by 75%±3%(BMMSC)or 91%±4% (DPMSC).

CONCLUSIONS:

1. Dental pulp is a source of cells expressing the typical phenotype of MSC.

2. Compared to BMMSC, DPMSC present a higher rate of proliferating cells (S-phase), and lower differentiation capabilities toward adipogenesis. These results suggest that BMMSC and DPMSCs are present at diverse differentiation stages, possibly not evidenced by phenotypic characteristics, and their plasticity in different experimental conditions should be further investigated.

3. DPMSC are able to suppress stimulated T lymphocyte proliferation, as described for BMMSC. Therefore they are good candidates when modulation of T cell activity is required, as shown recently by Le Blanc (2003) in the treatment of GVHD in allogeneic transplantation setting.