Effect Of Hypoxia On The Proliferation And Differentiation Of Embryonic Stem Cells In Vitro

Molecular oxygen (O2) is vital to nearly all forms of life on earth via its role in energy homeostasis, embryogenesis and differentiation, and it is also an important physiological regulator for cell function. Hypoxia is an essential environment for life development. At the earlier period of pregnancy, before the formation of blood vessels, embryogenesis precesses in the hypoxia environment. Genital system, embryo and embryonic tissues are all in the hypoxia environment. And as a physiological stimulate factor, hypoxia affects embryogenesis, embryo-development and the maintenance of normal function. Hypoxia activates hypoxia-inducible factor 1 (HIF-1), thus it controls a series of expression of genes correlated with hypoxia. For example, hypoxia induces the increased expression of genes for glucose decomposition, transportation, and regeneration of erythrocyte and blood vessels, which can maintain oxygen microenvironment homeostasis. At present, the researches are focused on the hypoxia injury and the hypoxia adaptation, while little effort at the effect of hypoxia on the proliferation and differentiation of stem cells. Recent years, only few reports are with regard to theeffect of hypoxia on the proliferation and differentiation of embryonic stem cells (ES cells) . The study and research of the proliferation and differentiation of ES cells in vitro are all in normoxia condition (20% O2), and it cannot reflect the real condition in vivo. As a physiological stimulate factor, at present, the effect of hypoxia on the proliferation and differentiation of ES cells in vitro is still unclear, and researchers do not pay enough attention to it. So this paper is about preliminary study for the effect of hypoxia on the proliferation and differentiation of ES cells in vitro. 1. Effect of hypoxia on the proliferation of ES cellsWe observe the proliferation of ES cells using by hematometery and BrdU-labled flow cytometery (FCM), and we also detect the expression of hypoxia inducible factor-1a (HIF-1a ) by RT-PCR and Western-Blot. Results as follows: (1) The number of ES cells cultured in the hypoxia environment (3% O2 and 10% O2) for 24 hours are lesser than that in normoxia (20% O2); (2) The number of ES cells significantly increased after exposed to intermittent hypoxia (3% O2) stimulus for 10 minutes per day for 4 days compared with normoxia control; (3) We also observe the relationship between the expression of HIF-1 a and the proliferation of ES cells by RT-PCR. The results show that the expression of HIF-1 a has no significant change after ES cells were exposed to hypoxia environment (3% O2 and 10% O2) for 24 hours or intermittent hypoxia (3% O2 and 10% O2) 10 minutes per day for 4 days. And ES cells express HIF-1 a in normoxia (20% O2); (4) After persistent hypoxia and insisttent hypoxia ?the expression of HIF-1 a protein has no significent change between the hypoxia group and the normoxia group.2. Methods of differentiating ES cells into neurocytes by RAES cells as routine culture are plated into petri dishes at appropriate cell density. And in the differentiation culture media of 20% FBS, ES cells can differentiate into EB naturally. During the differentiation progress from ES cells into EBs, RA is an important inducer, which can induce EB to differentiate into neurocytes. In this study, we use the method of 4d+4dRA, that is, ES cells plating in the petri dishes differentiate naturally into EB for 4 days, and continue to induce by RA for 4 days. After treated with RA, EBs are collected and plated in cell dishes covering 1% gelatin by single cell. The culture media is DMEM medium of 20% FBS and 5X10-7M RA. At the second day, the media is changed with DMEM/F12 medium of 10% FBS and 5X10-7M RA. At the third day, the media is changed with DMEM/F12 medium of no FBS and 2% N2.Results: (1) The method of 4d+4dRA can induce nestin+ - neural progenitor cells, and the differentiation ratio reachs above 90%; (2) The method 4d+4dRA also can induce NeuN+ - neurocytes, and the differentiation ratio reachs above 60%.3. E...