| Background: Hematopoietic stem cell(HSC) is the key link of hematopoiesis in all vertebrates for its capability of complete hematopoietic reconstruction after transplantation. Therefore, allogenetic hematopoietic stem cell transplantation(allo-HSCT) is the only cure for many malignant blood diseases(such as leukemia, thalassemia, hematologic neoplasms). Clinically, adult hematopoietic stem cells are mainly extracted from the bone marrow and umbilical cord blood, the clinical application of adult HSC has been greatly hindered due to the limitation of its source and the immunological rejection. Embryonic stem cells(ESCs) possess the potential of infinite proliferation in vitro and differentiating into cells of all three germ layers as well as HSC under certain conditions, soloving the problems of limited source and immune regection and offering a infinite cell resource for human regenerative medicine and cell replacement therapy.ES cells induced hematopoietic differentiation is one of the most in-depth research fields. OP9 cell line is the most frequently used stromal cell line in inducing hematopoietic differentiation in vitro and but conventional inducing cycle takes 2 to 3 weeks, it takes long time. In our work, in order to shorten the time of differentiation and to improve the hematopoietic differentiation system in vitro, the conventional inducing system was utilized and the optimized density of OP9 cells was explored. Through the technique of hematopoietic differentiation system in vitro, we established a platform for the hematopoietic differentiation of h ES cells, grounding a foundation of stem cell therapy and providing a basis for the study of the regulatory mechanism of hematopoiesis.Objective: To explore the optimal condition of OP9 co-culture system to induce hematopoietic differentiation from h ES cells in vitro and to establish a platform for induced hematopoietic differentiation and facilitate mechanism study.Methods: In traditional method of inducing hematopoietic differentiation by co-culture with the mouse bone marrow mesenchymal cells OP9, it often takes totally 2 to 3 weeks for the preparation of OP9 cells and the process of hematopoietic differentiation. In our work, we set two groups. In experimental group, OP9 cells were inoculated to 10 cm culture dishes at six different denstity of 1.5×105, 2.5×105, 3.5×105, 5.0×105, 6.5×105 and 8.0×105 cells/ml. After 24 hours culture, the OP9 cells were co-cultured with HN14(human embryonic stem cell line generated in our lab) for the induced hematopoietic differentiation. In control group, OP9 cells were co-cultured with HN14 for hematopoietic differentiation after inoculated at the denstity of 1.5×105 cells/ml to a 10 cm culture dish and cultured for 4 days. The efficiency of induced hematopoietic differentiation was compared through morphological changes, FACS assay, quantitative real-time PCR and colony-forming assay.Results: Both the experimental group and control group, h ES cells appeared to different degrees of differentiation were observed in each dish. The percentage of CD34 positive cells were tested through FACS assay on day 8?10 and 12. In experimental group, the percentage of CD34 positive cells reached the peak on day 10 while that of the control group was on day 12. In quantitative real-time PCR, the mesoderm-related marker gene BRACHYURY were analyzed. Even though in both group the expression of BRACHYURY appeared to increase at the first time and then decreased, the expression of BRACHYURY reached the peak level on day 6 in the control group while that was on day 4 in the experimental group, which was in keeping with the expression of CD34 positive cells. CFU assay showed that the OP9 cells were capable in differentiating into various hemopoietic progenitor cells and there were no significant differences among each group. Our results demonstrated that compared to control group, as the density of 5.0-6.5×105 cells/ml to plate OP9 cells and just culture 24 hours, the differentiation efficiency on day 10 was the same as that in control group. Importantly, the induced time was shortened greatly for 3 to 5 days, and using the new method could improve the efficiency of differentiation.Conclusion: A platform for the induced hematopoietic differentiation in vitro was established. At the same time, we standardized the OP9 cells inoculation density. The optimized method shortened the time of hematopoietic differentiation and improved the efficiency of differentiation. This system established the foundation of hematopoietic differentiation in vitro and mechanism study. |
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