Identification and Isolation of M esenchymal Stem Cells from Human Fetal Lung

Objective:To isolate and study the phenotype and biological characteristics of lung derived mesenchymal stem cell.Methods:Lung of 4～5 months old aborted fetus was dissected away from connective tissue,and was cut into small pieces,and digested by collagernase(type Ⅱ).The adherent cells were harvested and subcultured,after the third subculture,the cells were used for experiments.Cell cycle was analyzed by measuring DNA content by FACS can flow cytometer.Phenotypical of MSCs was analyzed by immunohistochemical SA technique and differentiated cells were identified by specific staining.The cultured cells dyed with PKH26 were injected into NOD/SCID mice sublethally irradiated(250 cGy).The transplantation of the cells was studied.Results:Fetal lung derived cells gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast like morphology.During the log phase of growth,MSC proliferated with a population doubling time of 21h.MSC can be ex vivo expanded by successive cycles of trypsinization,seeding,and cultured.Under these conditions,MSC had capability of passaging up to 30 times without displaying significant changes in morphology,with 2 fold increase in cell number after each passage.This indicates the high ex vivo expansion potential of MSC.Cell cycle analysis by flow cytometry revealed that more than 94% of cells were in the G0/G1 phases,while a small population of cells were actively engaged in proliferation(S+G2+M=5 6%).It is also showed that more than 90% of cells were positive stained by FITC labeled CD44,CD29,CD105,CD166,Flk?1,and only under 1% of cells positive for CD34,HLA DR.,CD45,CD11a.In the differentiation research,the culture expanded pancreas MSCs can be directed into the osteogenic lineage as determined by osteoblastic morphology,expression of alkaline phosphatase,modulation of osteocalcin mRNA production and the formation of a mineralized extracellular matrix.And it presented as well that MSCs can give rise to the adipogenic as evidenced by accumulation of lipid rich vacuoles within cells and the expression of lipoprotein lipase mRNA.The dyed cells in the lung of NOD/SCID mice 2 months through fluoromicroscopy could be observed.Conclusion:Mesenchymal stem cells exist in human lung and can be isolated by their adherent ability and should be essential to sustain a steady supply of primitive cells in tissue remodeling.And mesenchymal stem cells could lodge in the lung of NOD/SCID mice during a long time.