Effect Of Hypoxia On The Proliferation And Differentiation Of Stem Cells

Molecular oxygen (O2) is vital to nearly all forms of life on earth and, perhaps via its role in energy homeostasis, embryogenesis and differentiation, it is also an important physiological regulator for the cell function. Currently the researches are focused on the hypoxia injury and the hypoxia adaptation, with little effort at the effect of hypoxia on the proliferation and differentiation of stem cells. In particular, there has been little attention given to the embryonic genesis and local microenvironment hypoxia during the physiology and pathology through the adult. Standard cell culture system generally employs environmental oxygen (O2) levels of 20%, whereas actual tissue O2 levels in both developing and adult brain are of a lower order of magnitude, which might result in a distortion of the condition in vivo. Furthermore, the proliferation and differentiation of different types of stem cell are markedly different, and these cell behaviors under hypoxia are unknown. This study aims at a tentative examination of the effect of hypoxia on the proliferation and differentiation of stem cells.1. Effect of hypoxia on the proliferation and differentiation of neural stem cells in vivoThe male Wistar adult rats (body weight 160-180g) were treated with 3000 and 5000 meters high altitude 4 hours/day for consecutively 2 weeks. 5-Bromo-2-deoxyuridine (BrdU) immunochemistry demonstrated that the number of BrdU-labeled cells in the SVZ and DG increased 62% and 3 5% respectively as compared with control groups, and the BrdU-labeled cells in DG also significantly increased after 3000 metersintermittent hypoxia. The number of BrdU-labeled cells in the SVZ recovered to normal level 4 weeks later after intermittent hypoxia. However, the stem cells in SVZ and DG did not differentiate into NeuN or GFAP at the 2nd and 4th week following a 2-week hypoxia, while the BrdU-labeled cells in the DG were unmistakably higher as compared with 4 weeks before. These results imply that stem cells in different areas responded differently to hypoxia, which means that they may fall into different subgroups. The action of hypoxia in itself may lead to the proliferation of the endogenous stem cells, but does not result in their differentiation.From these data we conclude that intermittent hypoxia facilitates the neurogenesis of the adult rat brain, and the neural precursors in the SVZ and DG have different responses to hypoxia. These findings imply that forebrain neurogenesis may contribute to adaptive changes in intermittent hypoxia.2. Effect of hypoxia on the proliferation and differentiation of neural stem cells in ratsResults from the in vivo experiment on the stem cells in rats showed that lowered (3%, 10%) O2 detectably promoted the formation of the primary cultured neural sphere, which was found to be 1.5 and 2.4 times that in normal conditions following a 3-day culture. The BrdU-positive cells in the lowered O2 also significantly increased compared with the control. The expressions of HIF-1 in neural precursor cells cultured under the lowered O2 were examined by RT-PCR to be noticeably higher than those in normal conditions.We further examined the effect of lowered O2 on the differentiation of neural stem cells. Compared with the normal condition, the cultured stem cells from lowered O2 (3% ,10%) displayed an increase in theabsolute number of neurons and a decrease in the absolute number of GFAP. In addition, 3% lowered O2 promoted the formation of Na+-K+ electric flow in the neural membrane. The data indicate that hypoxia may modify the proliferation and differentiation.3. Effect of hypoxia on the proliferation of myoblast in rats Myoblast has been used in the treatment of cardiac ischemia, and ithas been extensively studied as an engineering-cell for gene therapy. However, the effect of hypoxia on the proliferation of myoblast has not been established. We isolated myoblast from the newborn rat, and examined with Desmin-immunohistochemistry. The number of Desmin-positive cells in lowered (3% , 10%) O2 for 3 days significantly increased compared with the 20% O2. The number of myoblast in lowered (3%, 10%) O2 was 1.5 and 2.5 times than in 20% O2.4. Effect of hypoxia and CoCl2 on the proliferation of hMSCsAt present, human marrow-derived mesenchymal stem cells (hMSCs) are clinically ideal tissue-derived stem cells, on which extensive research work has been undertaken. Here we examined the effect of hypoxia on the proliferation of hMSCs. The number of hMSCs treated with 100 μ M and 200 μ M CoCl2 or in lowered (3%, 10%) O2 for 3 days were detected to be higher than cultures with 20% O2. The differences between groups are of statistical significance (P<0.05). These data show that hypoxia can promote the proliferation of hMSCs in vitro.5. Effect of hypoxia and CoCl2 on the proliferation of ES cells The embryo stem cells ( ES cells) were cultured under the 3%, 10%and 20% oxygen tension or treated with 50μM, 100μM, 200μM CoCl2 for 24 hours, and the proliferation of ES cells were investigated by hematocytometery and BrdU in corporation with flowmetery. The resultsshow that hypoxia did not affect the proliferation of ES cells, and that the cell number of CoCl2 -treated groups was not significantly different from control. Expression of the HIF-1 investigated by RT-PCR did not change under the hypoxia. This suggested that hypoxia did not influence the proliferation of ES cells in vitro.In summary, the effect of hypoxia on the proliferation and differentiation of stem cells is extensive and has cell-type specificity. According to our data, hypoxia has no effect on the proliferation of embryonic stem cell, but it promotes the proliferation of the tissue-derived stem cell. The proliferation of related tissue-derived stem cells may be involved in starting the injured tissue's recovery under some pathological condition. The proliferation and differentiation of stem cells is regulated by a series of factors in vivo or vitro, of which the level of oxygen is an important one. As little is known about the regulating mechanism of stem cells and the consequences of different levels of oxygen as an essential life indicator under physiological and pathological conditions, studying the mechanism for the effect of hypoxia on the proliferation and differentiation of stem cells will be of major theoretical interest. Apart from that the study may also offer a new approach to regulating the proliferation and differentiation of stem cells in vivo by using a physical factor.