Differentiation of amniotic fluid-derived fetal mesenchymal stem cells into osteoblasts / 中国组织工程研究

ABSTRACT

BACKGROUND: Amniotic fluid cells have been widely used in antenatal diagnosis for gene mutation-related diseases. However,there are few reports concerning isolation, culture, surface character identification, differentiation and application perspective of fetal mesenchymal stem cells (MSCs) derived from amniotic fluid.OBJECTIVE: To investigate the differentiation of fetal mesenchymal stem cells (MSCs) derived from second-trimester amniotic fluid into osteoblasts in vitro.DESIGN, TIME AND SETTING: The cytological/n v/tro study was conducted at the Experimental Center of Xinhua Hospital from August 2005 to May 2006.MATERIALS: Ten amniotic fluid samples were obtained from pregnant women (18-22 weeks after conception) or aborted women,The informed consents were obtained from pregnant women.METHODS: Fetal MSCs were separated mechanically from amniocyte culture system and expanded in medium in vitro. At passage 3, fetal MSCs were induced in 100 nmol/L dexamethasone, 10 mmol/L β-glycerophosphoric acid and 50 mg/L vitamin C for 14 days.MAIN OUTCOME MEASURES: Collagen Ⅰ and alkaline phosphatase expression was detected by immunohistochemistry.Collagen Ⅰ protein expression was determined by Western Blot analysis. Calcium tuberoses were measured by Von Kossa staining. The cytoskeletal protein was detected by laser confocal microscopy.RESULTS: The isolated fetal MSCs were uniformly positive for CD44 and HLA-ABC, negative for CD34, CD45 and HLA-DR. After being induced with osteogenic medium for 14 days, 91% cells were positive for alkaline phosphatase, and 87% cells for collagen Ⅰ.Cells expressed collagen Ⅰ protein. Number of calcium tuberoses was increased and became big over time. Cytoplasm microfilament presented green fluorescence and the microfilament surrounding cells formed dense bundle.CONCLUSION: Fetal MSCs derived from amniotic fluid could be induced into osteeblasta and displayed a typical osteoblastic morphology and biological characteristic.