Isolation of human fetal lung mesenchymal stem cells

Objective To investigate the effective methods to isolate human fetal lung mesenchymal stem cells(MSCs). Methods The fetal lung tissues were collected after abortion under aseptic conditions. The cells were isolated by explant culture and enzyme digestion respectively,followed by cell morphology observation under phase contrast microscopy; cell phenotype was detected by flow cytometry,and multipotent differentiation potential via adipogenic and osteogenic induction. Results The morphology of cells isolated by two methods was different in the initial stage of primary culture.However,they became homogeneous fibroblast-like cells in later stage of primary culture and after passages. Flow cytometry analysis showed that both cell populations isolated by two different methods expressed MSCs markers CD13,CD29,CD44,CD90,CD105,CD166and HLA-ABC,but not hematopoietic markers( CD45,CD34,CD14,CD38and CD133),endothelial markers CD31,and other surface markers(CD41a,CD42b,CD49d,CD61,CD106and HLA-DR). Some of the MSCs changed into large,flattened cells after 3-5 days of being exposed to adipogenic differentiation medium. One week after adipogenic induction,accumulated lipid vacuoles were observed in the cytoplasm. After another week of induction,more and larger lipid vacuoles which were stained red with Oil O were observed in the cytoplasm. After osteogenic induction,calcium deposits were gradually observed in the cytoplasm. Two weeks after osteogenic induction,calcium deposits stained red by Alizarin Red S were found in most of the cells. Conclusion Both tissue culture and collagenase digestion methods are effective methods to isolate human fetal lung mesenchymal stem cells.