Mechanism Research Of Proliferation And Differentiation Enhanced By Hypoxia In Human Bone Marrow-Derived Mesenchymal Stem Cells

Molecular oxygen (O2) is vital to nearly all forms of life on earth via its roles 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. For example, at the earlier period of pregnancy, before the formation of blood vessels, embryogenesis processes 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. 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 the effect of hypoxia on the proliferation and differentiation of stem cells. The study and research of 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. Stem cells, involving bone marrow-derived mesenchymal stem cells (MSCs) 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 MSCs in vitro is still unclear, and researchers do not pay enough attention to it. Moreover, human bone marrow-derived mesenchymal stem cells (hMSCs) are clinically ideal tissue-derived stem cells at present. So this paper is about preliminary study for the effect of hypoxia on the proliferation and differentiation ofhMSCs in vitro.1. Effect of hypoxia on the proliferation of hMSCs in vitroTo explore the role of hypoxia in cell proliferation, we observed the effects of intermittent hypoxia (3% O2or 10% O2) and continuous hypoxia (3% O2or 10% O2, 100 umol/L C0CI2 or 200 umol/L C0CI2) on the proliferation of human bone marrow-derived mesenchymal stem cells (hMSCs) in vitro. Cell numbers were determined by hemacytometer count. The proliferous indexes were detected by flow cytometer (FCM). The results showed that the numbers and proliferous indexes of hMSCs were higher (P<0.05) when treated with 100 umol/L CoCl2 or 200 umol/L C0CI2 or maintained in continuous hypoxia than for those of control group, and that the differences between groups of the intermittent hypoxia experiments are not of statistical significance (P>0.05). These findings demonstrate that hypoxia can promote the proliferation of hMSCs in vitro. It seems reasonable to say that cultivation of hMSCs in low oxygen could play an important role in possible clinical application of these cells.To determine differentially expressed genes at two culture conditions, human genome-wide long oligonucleotide microarray was performed. Labeled oligonucleotide targets were prepared from hMSCs RNA of the control group and the hypoxia group. These were hybridized with the microarray, and 21 329 spots (corresponding to 21 329 genes) that differed by more than 2-fold intensity in at least one pairwise comparison were selected. A total of 282 genes showed temporal gene expression changes during hypoxia, including genes encoding transcriptional regulators and signaling molecules. Semi-quantitative RT-PCR analyses confirmed differential expression at the transcriptional level of several genes identified by oligonucleotide microarray screening. Differential gene expression analysis revealed no change messages for HIF-1 and its almost all target genes. These data demonstrate increased proliferation in lowered O2, making this method an important advance in the ex vivo generation of hMSCs for engineering of artificial tissues. This study also provides novel information on the molecular mechanisms required for proliferation of hMSCs in lowered O2. We also observe the relationship between the expression of HEF-la and the proliferation ofhMSCs by RT-PCR. The results show that the expression of HIF-la has no significantchange between the hypoxia group and the normoxia group.2. Effect of hypoxia on the differentiation of hMSCs induced in vitro2.1 Method of differentiating hMSCs into neuron-like cells by p-mercaptocthanolhMSCs as routine culture are plated into fibronectin-coated dishes at appropriate cell density. Twenty-four hours prior to neuronal induction, media were replaced with preinduction media consisting of DMEM-HG/20%FBS/lmM P-mercaptoethanol (BME). To initiate neuronal differentiation, the preinduction media were removed, and the cells were washed with PBS and transferred to neuronal induction media composed of DMEM-HG/5mM BME. To characterize neuronal differentiation further cells were fixed after 5h and stained for the neuronal marker TUJ-1 and TH, and the percentage of neuronal cells was determined. The hMSCs in this manner exhibited neuronal morphologies and stained positive for TUJ-1 (67.7%±4.8%) and TH (8.6%±0.3%) expression.2.2 Effect of hypoxia on the differentiating hMSCs into neuron-like cellsTo observe the effect of hypoxia on hMSCs differentiated into TH-positive cells, we detected the proportion of TH-positive cells by TH-labeled FCM. hMSCs are inoculated in fibronectin-coated dishes at appropriate cell density. Then we divide them into different groups: control group, BME group and BME/hypoxia group. The BME/hypoxia group means that hMSCs are induced by BME and exposed to hypoxia environment simultaneously. The result of TH-labeled FCM shows that hypoxia facilitates the differentiation of hMSCs, and the result of immunocytochemistry also confirms this phenomenon.2.3 Effect of hypoxia on committed differentiation into dopaminergic neurons of hMSCsTo observe the effect of hypoxia on hMSCs differentiated into dopaminergic neurons, we detected the contents of DA and HVA by high-performance liquid chromatography-electro-chemical detection (HPLC-ECD). The intracellular DA content in the BME group and BME/hypoxia group was higher than the extracellular...