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Differentitation of the hESCs Clones with hFIX Site-specific Integration into Hematopoietic Cells in Vitro

Author: LeiMing
Tutor: LiangDeSheng
School: Central South University
Course: Genetics
Keywords: human embryonic stem cells hematopoietic cells differentiation site-specific integration
CLC: R457
Type: Master's thesis
Year: 2011
Downloads: 32
Quote: 0
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Abstract


Blood cells transfusion and hematopoietic stem cells transplantation are important methods for cell therapy, and have been widely used in the treatment of genetic diseases, incurable hematological disorder and immunologic deficiency. Hematopoietic cells are major target cells used in ex vivo gene therapy. We have successfully targeted the human coagulation factor IX gene (hFIX) into the rDNA locus of human embryonic stem cells (hESCs) H9 using human rDNA targeting vector (pHrneo-FIX) carrying hFIX. Differentiation of hESCs into hematopoietic cells may provide the experimental basis for the future use of induced pluripotent stem cells (iPSCs) to achieve the autologous gene therapy for hemophilia B.Methods:In this study, we induced the hematopoietic differentiation of hESCs H9 and the hESCs clones with hFIX site-specific integration in vitro respectively. The hESCs were cultured in low cell-binding dishes to form human embryoid bodies(hEBs), and then induced into hematopoietic cells. Three methods were employed for induced differentiation, including spin hEBs, suspended-adherent culture and methylcellulose-liquid medium. The genotypic expression was characterized by semiquantit-ative RT-PCR in day 0 to 26. In addition, the expression of CD34 and CD 38 was examined by flow cytometry at day 11. After 26 days, the morphological characterizations were examined by Wright-Giemsa staining.Results:We didn’t get any hEBs by the method of spin hEBs, but a large number of the hEBs were successfully obtained by using the methods of suspended-adherent culture and methylcellulose-liquid medium. The induced cells showed the feature of hematopoietic cells, expressing hematopoietic specific makers such as CD34, TAL-1/SCL, GATA-2, RUNX-1, BRACHYURY, MIXL-1 and FLK-1/KDR as well as hFIX. The expression of CD34 and CD 38 was detected by flow cytometry, with positive rates of 48.75% and 30.98% respectively. Wright-Giemsa staining showed erythrocyte, granular leukoblast, metagranulocyte, rod-shaped granulocyte, segmented granulocyte, lymphocytes, megakaryoblast and other types of hematopoietic cells.Conclusion:Methods for targeted differentiation of the hESCs clones with hFIX site-specific integration into hematopoietic cells has been established. Providing the experimental basis for the future use of iPSCs to achieve autologous gene therapy for hemophilia B.

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