Effects of epithelial growth factor on colony-forming efficiency of non-adherent bone marrow mesenchymal stem cells*,*,⋆

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Bone marrow mesenchymal stem cells (BMSCs) are a kind of fibroblast-like cells with high adhesion. Some studies showed that BMSCs in non-adherent status have the potential of self-duplicating and multi-directional differentiation.


To explore effects of epithelial growth factor (EGF) on fibroblastic colony forming units (CFU-F) from the non-adherent BMSCs.


The cytological, in vitro study was performed at the Research Center of Bone and Stem cells, Nanjing Medical University from July to December 2007.


Six male C57/BL6 mice aged 4 weeks were supplied by Experimental Animal Center, Nanjing Medical University. Epidermal growth factor (Sigma, USA) was used in this study.


Mouse BMSCs were isolated in vitro by the whole bone marrow method. The third passage of cells at 2×109/L were incubated in adipogenic, osteogenic and chondrogenic mediums, separately. 30 mL BMSC suspension was obtained and averagely assigned into two tubes. Cells in the EGF group were treated with 10 μg/L EGF. Cells in the blank control group were treated with common complete medium. Using repeat transfer non-adherent bone marrow cell method, non-adherent bone marrow cells were moved in the a new Petri dish every day. Adherent cells in the original dish were derived from the total bone marrow. Cells were named PO1 following the first transfer, and PO2 following the second transfer, and so on. Culture was stopped at day 12.


Determination results of BMSCs; methylene blue staining showed that cell clone formation in each dish; percentage of clone forming region to dish bottom area; clone number.


Non-adherent BMSCs could adhere at day 4 following moving into a new dish, and gradually amplify into clones. Following induction, these BMSCs could differentiate into adipocytes, osteoblasts and chondrocytes. Following EGF treatment, both total bone marrow-derived adherent cells and transferred non-adherent BMSCs could produce fibroblast colony forming unit, and the number was significantly more compared with the blank control group (P < 0.05). The number of adherent cells from total bone marrow-derived adherent cells in the EGF group and PO1-PO5 non-adherent BMSCs was 1.54 times, 4.25 times, 2.51 times, 1.56 times, 1.25 times, 2.01 times of that in the blank control group, and the clone number was 1.2 times, 1.8 times, 1.4 times, 1.4 times, 1.1 times of that in the blank control group.


EGF can effectively promote proliferation of non-adherent BMSCs under suspension condition, which significantly elevates enriching efficiency of BMSCs.

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