The Role of Transcription Factor SCL/TAL-1 in the Hematopoiesis of Human Cord Blood Hematopoietic Stem Cell

Abstract 4795

The role of transcription factor SCL/TAL-1 in the hematopoiesis was studied in the CD34+ hematopoietic stem cells purified from human cord blood. The sorted CD34+ cells were transfected by the plasmids pTRIPdU3-RNAiTALh-EF1a-GFP (SCL/TAL1 shRNA to reduce the expression of SCL/TAL-1), pTRIP-EF1a-TAL1 (SCL/TAL-1 cDNA to enhance the expression of SCL/TAL-1) and control plasmid pTRIP-dU3-RNAiluc-EF1-GFP expressing EGFP gene via lentiviral vector system, named the groups of SCL/TAL-1^low, SCL/ TAL-1^high and LUC control, and then cultured in methylcellulose medium. The clone forming unit (CFU) and the expression of hematopoietic related genes and surface markers of transfected CD34+ cells were examined by light microscope, RT-PCR and flow cytometry, and the role of SCL/TAL-1 in regulation of hematopoiesis was explored. After cultured in methylcellulose semi-solid medium with cell factors for 14 day, each 500 CD34+ cell purified from cord blood expanded to about 120 CFU/BFU. FCM detected the surface markers of hematopoietic differentiation including CD71/CD35a, CD33/CD13 and CD41a/CD42b in CD34+ cells cultured at d7. The results showed CD71 was 9.7±5.7%, CD235a 6.7±3.8%, CD33 49.4±12.6%, CD13 46.9±9.6%, CD41a 5.5±2.3% and CD42b 2.0±1.2%. During the hematopoietic differentiation of CD34+ cells, the overall tendency of SCL/TAL-1 mRNA increased significantly, especially after the 7 days. The levels of PU.1, LMO1 and LMO2 mRNA were almost similar with SCL/TAL-1's. The expression of GATA mRNA increased slightly during the differentiation process, and GATA2 showed a gradually increased expression in the first 10 days and then a decrease on the 14th day. However, there were no obvious alterations in RUNX1 mRNA level during the whole differentiation process. After interference of SCL/TAL-1, the CFU of SCL/TAL-1^low significantly decreased in number and showed dysplasia with small sizes,especially in erythroid clones, there was no GEMM-BFU to be found. Hematopoiesis of SCL/ TAL-1^high still developed successfully and was slightly better than LUC control. RT-PCR revealed that the level of PU.1 and GATA2 mRNA significantly decreased following a knockdown of SCL/TAL-1, the mRNA of LMO1 and LMO2 decreased slightly, while GATA1 varied reversely with SCL/TAL-1, and there was no obvious alteration in RUNX1 mRNA. The results of FCM showed that CD235a and CD71 decreased significantly in the group of SCL/TAL-1^low and increased in the group of SCL/TAL-1^high compared with LUC control. CD33 and CD13 expression in SCL/TAL-1^low decreased significantly too, and CD33 and CD13 expressions in SCL/TAL-1^high rose markedly. Expressions of CD41a and CD42b also decreased in SCL/TAL-1^low and increased in SCL/TAL-1^high, but there was no statistical significance. The results conclude SCL/TAL-1 plays a key role in the regulation of hematopoiesis by affecting the differentiation of erythroid and myeloid cells positively. Grant support: National Natural Science Foundation of China (No.30770912), Foundation of the Science & Technology Department of Sichuan Province (No.2008SZ0017).