Studies on Expansion of Erythroid Progenitors and CD34⁺ Cells among MNCs from Umbilical Cord Blood.

Objective: The experiment is to evaluate the effect of FL, SCF, TPO on CD34+ expansion.

Materials and methods: Cord blood samples were collected in heparinized tubes by the Obstetrics and Gynecology Department of Guangzhou first Municipal Peoples hospital. Mononuclear cells(MNCs) were isolated by Ficoll gradient separation. MNCs were culture in 50ml flask containing 6ml serum-free liquid culture system for 14 days at a density of 1*10⁶/ml according to different cytokines combinations. A: control group, no cytokines were added in the culture system. B: cells cultured in the group of SCF+FL+TPO+EPO+IGF-1. C: cells cultured in the group of SCF+FL+TPO. The final concentration of cytokines was 10ng/ml for TPO, 25ng/ml for FL, 25ng/ml for SCF, respectively. Replacing half of media containing the same concentration cytokines on day 6, renewal of Group B and C at day 6, 10 and 14 for final concentration 5U/L EPO, 50ng/ml IGF-1. Part of the cells on day 6, 10 and 14 were counted and detected erythroid progenitors and CFU-GM on semi-solid culture system and the proportion of CD34+, CD34+ CD71+, CD71+ GPA+ cells was detected by FACS. All calculation was performed using SPSS program.

Results:1. Proliferation of the total cells: After 10 days, the total cord blood cells were increased 6.89 folds in group B and 3.06 folds in group C respectively. The latter two groups had highly significant differences with the control group A(p<0.01). There is difference between group B and group C 0n day 10. More cells were gained in group B than in group C. (p<0.05) 2. Proliferation of CD34+ cells: The CD34+ cells were increased 4.83 folds in group B containing cytokine FL+TPO+SCF+EPO+IGF-1 and 2.47 folds in group C containing cytokine FL+TPO+SCF on day 10. There is difference between group B and group C on day 10. More cells were gained in group B than in group C.(p<0.05). 3. Proliferation of colony-forming cells: The CFCs were increased 4.3 folds in group B and 2.5 folds in group C on day 10. There is difference between group B and group C on day 10. More cells were gained in group B than in group C(p<0.05). 4. Proliferation of erythroid progenitors: The BFU-E and CFU-E were increased 5.4 folds in group B and 3.1 folds in group C on day 10. There is difference between group B and group C at examined time point(p<0.05). 5. Proliferation of CD34+CD71+cells: The CD34+CD71+ were increased 8.72 folds in group B and 3.37 folds in group C on day 10. There is difference between group B and group C at examined time point(p<0.05). 6. Proliferation of CD71+ GPA+ cells: The CD71+ GPA+ cells were increased 53.4 folds in group B and 30.29 folds in group C at day 10. There is difference between group B and group C at any time (p<0.05).

Conclusion: Firstly, in the group of FL+SCF+TPO, CD34+cells and CFC could greatly be expanded from cord blood MNCs in the serum-free culture system. Secondly, in the group of FL+SCF+TPO+EPO+IGF-1, erythroid progenitors could be greatly expanded in the serum-free culture system. Supplying EPO on day 0 is better than supplying on day 6. Thirdly, because the largest number of colony-forming cells such as BFU-E and CFU-E were gained in the TPO+SCF+FL+EPO+IGF-1 group onday 10, the harvest time after cultivation should be set on day 10.