OBJECTIVETo optimize the culture conditions for clonal isolation of rat bone marrow-derived multipotential adult progenitor cells (rMAPC) and identify their surface markers and differentiation potentials.

METHODSBy using a low concentration of fetal bovine serum culture medium, rMAPCs were primarily isolated from bone marrow by attachment culture and clonal-like cells were selected by single cell limiting dilution. The surface antigens of the cloned rMAPC were analyzed by flow cytometry and immunocytochemistry. Multi-differentiation capacities were evaluated by lipoblasts and osteoblasts and neuroblasts differentiation induction. The expressions of Oct-4 and three embryonic germ layer markers were detected by RT-PCR.

RESULTSSingle cell-derived rMAPC could be expanded to passage 20 in vitro which still maintained active proliferation ability. The expanded rMAPCs expressed CD71, alpha-SMA and vimentin, but not CD34, CD44 and CD45. About 83% of the rMAPCs was in the resting phase(G0 + G1) of cell cycle and 17% in S + G2 + M phase. They could be induced to differentiate into adipogenic cells, osteogenic cells and neural like cells. RT-PCR demonstrated that there were expressions of oct-4 gene and three embryonic germ layer markers on the rMAPCs.

CONCLUSIONSCloned rMAPC can maintain the phenotypes of stem cell during in vitro culturing. It might be an potential adult stem cell source for therapeutic stem cell transplanting and tissue engineering.