EFFECT OF PURMORPHAMINE ON GENE EXPRESSION OF HUMAN OSTEOBLASTS DERIVED FROM BONE MARROW MESENCHYMAL CELLS

FF05

Fabíola Singaretti de Oliveira oliveira@forp.usp.br, Grasiele Edilaine Crippa, Márcio Mateus Beloti, Adalberto Luiz Rosa

Cell Culture Laboratory - FORP - USP

Introduction: Purmorphamine promotes osteogenesis in mesenchymal progenitor cells as showed by the upregulation of the bone transcription factor RUNX2 (Runt-related transcription factor 2) and the bone marker ALP (alkaline phosphatase). Objectives: This study investigated the immunophenotype profile of human bone marrow mesenchymal cells (hBMMCs) exposed to purmorphamine by fluorescence-activate cell sorting (FACS). Furthermore, it was evaluated the effect of purmorphamine on osteogenesis of osteoblasts differentiated from hBMMCs. Methods: Experiments were carried out using cells from two healthy bone marrow donors obtained under approval of Committee of Ethics in Research. For immunophenotype profiling primary and first-passaged cells were cultured in non-osteogenic medium and FACS of representative cell surface makers for mesenchymal, hematopoietic and endothelial cells was conducted. First-passaged cells were exposed or not to purmorphamine (2 µM) for 7 days after what the same cell surface markers were evaluated by FACS. For gene expression assay, subconfluent cells in primary culture were enzymatically harvested and first-passaged cells were subcultured in 75 cm² tissue culture flasks (1.4 x 10⁵ cells/flask) for 7 days in osteogenic medium to allow osteoblastic differentiation. Cells were exposed to purmorphamine (2 µM) or vehicle (dimethyl sulphoxide) for 7, and for the last 4, 3, 2 and 1 days of subculture. Gene expression of ALP, RUNX2, ostepontin (OP) and osteocalcin (OC) were evaluated by quantitative real-time PCR. All experiments were done in triplicate and submitted to Mann-Whitney test. Results: FACS of primary culture revealed at least two distinct cell populations, the predominant hBMMCs (CD90⁺, CD73⁺, CD105⁺, CD166⁺, CD29⁺, CD13⁺, HLA class I⁺, CD146⁺, CD44⁺, CD14^-, CD106^-, CD31^-, CD45^-) and the fewer hematopoietic cells (CD34⁺). After 7 days, FACS demonstrated that purmorphamine treatment reduced hBMMCs surface markers expression (excluding CD44, HLA class I and HLA-DR) while untreated culture did not change their immunophenotype profile being comparable to primary culture. Exposition of osteoblastic differentiated cells to purmorphamine resulted in upregulation of all evaluated genes. RUNX2 was upregulated at all periods (7 days: 1.54-fold; 4 days: 1.27-fold; 3 days: 1.38-fold; 2 days: 1.40-fold;1 day: 1.13-fold). OC and ALP were upregulated at 7 (1.10-foldfor OC; 1.12-fold for ALP) and 4 days (1.54-fold for OC; 1.13-fold for ALP). OP was upregulated at 4 (1.07-fold), 3 (1.23-fold) and 2 days (2.83-fold).  Conclusion: FACS analysis suggests that purmorphamine treatment reduces mesenchymal cell population probably by inducing osteoblastic differentiation as revealed by increased gene expression of osteoblastic markers.