The Embryonic Stem Cells (ESCs) are characterized by unlimited self-renewal ability and potential to differentiate into all cell types of the body. Those cells are derived from embryos which reside in 3-5 % oxygen environment. This hypoxic condition is physiologically normal not only for ES cells but also for many other types of stem cells, for example Neural Stem Cells. These observations suggest that hypoxic condition plays a very important role in the maintenance of cell stemness. It was also demonstrated that low oxygen tensions are preferential for maintenance of a highly proliferative, pluripotent population of hES cells. Stemness is regulated by Hypoxia Inducible Factors (HIF), which depend on oxygen tensions. HIF2A (HIF-2 alpha) is an upstream regulator of Oct4, which is the main transcription factor used by Yamanaka and his group to generate the first iPSCs (induced Pluripotent Stem Cells). It has been shown that knock-down of HIF-2 alpha or HIF-3 alpha but not HIF-1 alpha, leads to a decrease in the expression of Oct4, Nanog and Sox2, which are important stem cells markers. In this study we are trying to find out the best oxygen conditions for HUCB-NSC (human umbilical cord blood neural stem cells), from which iPS cells will be generated. We investigated the difference between the level of expression of chosen genes in HUCB-NSCs cultured under atmospheric air (21% oxygen) and 5% oxygen (low oxygen tensions). The cells were cultured for two weeks in two incubators with two different oxygen concentrations. HUCB-NSCs were grown in medium containing: DMEM/F12, 1%ITS, 2%FBS, 1%AAS. For comparison of expression levels of Oct4, Sox2 and Nanog from two different oxygen environments Real-Time RTPCR was used. In summary, the cells from low oxygen conditions had higher expression of genes: Oct4, Sox2, and Nanog compared to that of cells cultivated under atmospheric air, which is in agreement with previous observations. These outcomes indicate, that the cells from 5% oxygen conditions are the better source of cells for iPS generation than those which grow in 21% of oxygen. This is due to the higher endogenous expression genes of pluripotency what suggests possible easier generation of iPS cells and more efficient responses to reprogramming program. Thus in our further investigation on reprogramming of HUCB-NSC we will apply low oxygen conditions and epigenetic modifications in order to obtain iPS cells from HUCB-NSC cell line. Sponsored by grants from Polish Ministry of Scientific Research and Higher Education: Nr 0141/B/P01/2008/35 and Nr N N302 597838.
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