[Study on culture and in vitro osteogenesis of blood-derived human mesenchymal stem cells]

Abstract

Objective: To establish a method of isolating and culturing adult human blood-derived stem cells(MSCs) and to investigate their osteogenic potential in vitro.

Methods: Thirty peripheral blood collected from 30 adult volunteers (15 ml per person). Adult human MSCs derived from peripheral blood from the lymphocyte separation fluid fraction of mononuclear cells, cultured in alpha-Modified Eagle's Medium glucose containing 20% fetal bovine serum, and proliferated through a process of subculturing. The phenotype was analyzed with flow cytometry. For in vitro osteogenic differentiation, MSCs from the second passage presence of osteogenic supplements (100 nmol/L dexamethasone,10 mmol/L beta-glycerophosphate, 50 micromol/L and 10 nmol/L 1,25-2-hydroxide vitamin D3). In the fifth passage cells, the activity of alkaline phosphatase, expression level of collagen type I, osteocalcin and osteonectin were determined. And the calcium tubercle would be examined after the continual one-month culture of the fifth passage.

Results: MSCs existed in blood of adult human. And the clone forming efficiency of blood-derived MSCs was 0.27 +/- 0.22/10(6) mononuclear The MSCs expressed CD44,CD54,CD105,and CD166, but did not CD14, CD34, CD45,and CD31. Under osteogenic supplements, the MSCs were found to be higher activity of alkaline phosphatase and higher expression of collagen type I , osteocalcin and osteonectin. And the calcium tubercle formation was examined through fluorescence labeling method.

Conclusion: The isolation and culture conditions established for adult human select a distinct population of peripheral blood-derived adherent cells. Adult human blood-derived osteogenic potential in vitro, and may be used as seed cells for bone tissue engineering.