| Hematopoietic stem cells (HSCs) is a very important kind of tissue stem cell. At present, multipotent HSCs have been reported to be presented in fetal yolk sac, fetal liver, fetal blood, fetal aorta-gonad-mesonephros (AGM) and placenta. Here we examined the committed hematopoietic induction of nuclear transfer embryonic stem cells (nthESC) in vitro, and also investigated the expression of hematopoietic marker of hematopoietic cell and hematopoietic cytokines in yolk sac, liver and placenta in vivo, which will provide theoretical foundation and technical method for clinic disease therapy by stem cells. 1. The isolation, purification, expression, GFP transduction, and differentiation of human embryonic bone marrow MSCs into neurocytes and osteoblasts were examined in vitro. Immunohistochemistry results showed that bone marrow MSC did not express antigens CD34, CD45 and CD38, but express CD29, CD13 and CD59. LSM fluorescence exhibited 60% MSCs with GFP gene. Immunohistochemistry staining showed that cultured MSC cells expressed vimentin and Mallory, and that induced different passage MSCs expressed nestin and calcium nodes were seen by Von Kossa staining. It suggested that human embryonic bone marrow MSCs were capable of expanding rapidly and differentiating into neurocyte and osteoblasts in vitro, which could be a course of feeder steadily. 2. The aim of this study was to investigate the potential effect of MSC on the hematopoietic development of nthESC and the feasibility of a novel system in which nthESC will be cocultured with MSC. After 20d coculture , a lot of CD34+ and CD133+cells were produced. KDR+ cells first appeared on day 5 of culture, and gradually peaked at day 11 with CD133 and CD34.CD34+ cells appeared 2 days later within CD133+ population and gradually increased by day 11 of culture, then decrease tardily to day 20. BMP4, FGF-1 and VEGF could promote hematopoietic induction of nthESC. The timeframe of hematopoiesis onset of nthESC can be defined first KDR+ cell appearing, then CD133+ cell and CD34+ cell lastly. It suggested that human embryonic MSC can induce nthESC into hematopoietic stem cells. 3. EB could be induced with suspension culture by stages appending BMP4 and FGF-1, then HSC of EB be expanded using VEGF. The results showed that low adherence plates could promote EB production efficiently, and numbers of EB be expanded using BMP4 and FGF-1, VEGF also expanded CD34+ cell in EB. It revealed that suppension culture of nthESC by stages could be induced hematopoietic stem cell with cytokines efficiently. 4.To study the expression and distributing of hematopoietic marker, cytokine and translation factors in Yolk Sac of human embryo at different development stage, immunhischemical SP staining and RT-PCR were used. CD34, CD133 and c-kit were not found in yolk sac on day 16 except KDR. The results showed that KDR, CD133, CD34 and c-kit largely appeared on the 30th day, then increased at week 6 , and decreased quickly after week 7. The expression of VEGF, BMP4, FGF-1 and SCF had the same tread. Ihh,SCl, GATA-1, GATA-2 and PU.1 were detected by RT-PCR. The results showed that PU.1 was not expressed on the 16th day, other factors were expressed all the time. It suggested that yolk sac not only have primitive hematopoiesis, but also definitive hematopoiesis. 5. To probe the ability of hematopoiesis in liver of human embryo, immunhischemical SP staining and RT-PCR were used to investigate the expression of hematopoietic marker, cytokine and translation factors in month 5, month 7 and fetal liver. The results showed that 5-month liver hematopoiesis was the strongest, 7-month was the lowest. Ihh,SCl, GATA-1, GATA-2 and PU.1 were detected by RT-PCR. The results showed that Ihh was not expressed in all the groups, other factors expressed in all the three groups. The results demonstrated 5-month fetal liver had a best hematopoietic ability, fetal liver had a better ability than 7-month's. 6. In this study ,we demonstrate the function of human placenta in hematopoiesis by immunocytological study on 7-month and full-term months'placental slices. Positive reactions were observed in 7-month placenta using antibody against SCF, VEGF, BMP-4, CD34, CD133 and KDR. However, full-term months'placenta just reacted slightly with these antibody. The staining for neural marker such as Nestin and MAP2 showed the same result. From isolated adherent cells of placenta we found that they can express AKP, Vimentin and hematopoietic related factors. Nestin and MAP2 neural markers also were expressed in these cells. They showed neural differentiation potential under appropriate conditions. The data suggest that placenta can express hematopoietic related factors to support hematopoiesis and placental-derived cells have neural differentiation potential in term of morphology and cell-surface antigen expression.
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