OBJECTIVEMesenchymal stem cells (MSCs) were adult stem cells which contribute to the regeneration of mesenchymal tissues such as bone cartilage, muscle, ligament, tendon, adipose and stroma. Due to the multipotential ability and self-renewal capacity, the mesenchymal stem cells can be applied in many fields, such as the seed cells in tissues engineering, cell therapy and gene therapy. To enhance the clinical use of MSCs, the investigators studied the isolation and expansion of MSCs from adult bone marrow, fetal bone marrow and human umbilical cord blood, and investigated their biological identities.

METHODSTwo kinds of incubation systems containing L-DMEM or MSC special culture medium were used to purify and expand MSCs. The growth, purification and proliferative abilities of 3 kinds of MSCs were observed and their immunophenotypes were determined by flow-cytometry.

RESULTS(1) The shapes of 3 kinds of cells were same. There was no difference in number and size. The colonies formed early in adult bone marrow MSCs. (2) There was no difference in the expansion speed of the 3 kinds of MSCs, but after the colonies confluenced there had no touching constrain in MSCs from umbilical cord blood and fetal bone marrow. When the colonies confluenced, the cells also had proliferation ability. But in adult bone marrow, the touching constrain was significant. (3) MSCs had strong self-renewal capacity. After primary culture approximately 5 - 6 x 10(5) MSCs were obtained from 8 x 10(6) MNC of bone marrow and 25 x 10(6) MNC of umbilical cord blood. After passage 3, passage 5 and passage 10, the investigators could get 10(7), 10(8) and 10(10) MSCs, respectively. (4) Along with the increase in the passage and prolonging of culture time, the ability of expansion decreased, but they maintained good puripotentiality. After passage 2, passage 3 and passage 5, the purity of MSCs was 90%, 95% and 99%, respectively. (5) Three kinds of MSCs were all positive for CD(29), CD(44), CD(59), CD(90), CD(105), CD(166) and all negative for the markers of hematopoietic cells such as CD(11a), CD(14), CD(33), CD(34), CD(28), CD(45). All the important GVHD correlation markers were negative, such as HLA-DR, B7-1 (CD(80)), B7-2 (CD(86)), CD(40) and CD(40L). There were no differences in the phenotype among the 3 kinds of MSCs cells. (6) The 2 kinds of culture mediums used did not markedly affect isolation and expansion of MSCs, and the biological properties of MSCs.

CONCLUSIONS(1) Human MSCs could be isolated from many kinds of human tissues, and they had no difference in their origin; (2) Human MSCs maintained good puripotentiality and self-renewal capacity. Therefore, they could meet with the need of clinical tissue engineering. (3) The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier but had broad clinical use.