| BackgroundMesenchymal stem cells?MSC?possess the self-renewal capacity,multilineage differentiation potential,immunomodulatory function and trophic action.Although a number of fundamental issues in the MSC biology remain to be elucidated,researchers have a great interest in their potential applications for cell transplantation therapy and tissue-engineering seed cells.Their frequency,however,is extremely low within any tissues,and only a very limited amount of MSC can be collected from a single donor.The in vitro culture is recognized as a hopeful means to obtain sufficient expanded cells for the needs of clinical trials and basic researches of MSC.The investigations of optimal culture conditions for MSC expansion are underway.A critical consideration is the optimal oxygen tension in the culture medium.At the tissue level,MSCs reside in the hypoxic microenvironment where the oxygen concentration is lower than that in the traditional incubators.A lot of studies have offered some evidences for the negative impacts of normoxic culture on MSC,such as early senescence,poor growth kinetics,increased apoptosisand DNA damage.On the other hand,low levels of oxygen had positive effects on the in vitro survival and proliferation of MSC.However,there are still no conclusive results of the effects of hypoxic culture on the vitality,proliferation and differentiation of MSC,and the underlying molecular mechanisms need to be clarified.The transcriptome annotation is an important method of the molecular-level research,and RNA-seq technology is now widely used for transcriptomic analysis.To the best of our knowledge,the transcriptomic assay of rat bone marrow-derived mesenchymal stem cells?rBMSC?cultured under hypoxic condition has not been reported so far.PurposeThis study was focused on analyzing the transcriptomic response of rBMSC exposed hypoxia in the culture by RNA-seq,following the experimental evaluation of the benificial effects of low oxygen on the survival and growth of these stem cells in vitro.It was expected that this study can provide the useful data for the improvement of MSC expansion in vitro and the elucidation of cellular and molecular mechanisms underlying the response and adaptation of MSC to hypoxia.Methods1.The rBMSCs were isolated from rats by whole bone marrow culture method.After subculture,rBMSCs were identified by microscopic morphology,tri-lineage differentiation ability and expression levels of surface molecular markers?CD29,CD90,CD45and CD11b/c?.2.The 3rd-passage rBMSCs were divided into three groups which were exposed to different oxygen concentrations:1%,5%and 18%O2.These cells were cultured for 5 days or 7 days?for growth curve?.Cell counting,cck-8 and CFU-F assay were used to detect the proliferative potential of cells.Cell cycle and apoptosis were detected by flow cytometry.The expression of PHH3 was detected by fluorescence immunoassay.Expression levels of HIF-1?and caspase-3 were detected by western-blotting.3.The passage 3 rBMSC were divided into two groups,namely,hypoxia?1%O2?and control?18%O2?groups.The total RNA samples were collected following a 5-day culture.After mRNA extraction,cDNA library construction and HiSeq sequencing,the transcriptomic databases of rBMSC under two oxygen concentrations were established.The differential gene expression profiling was carried out based on the library screening,comparison of the reference genome,quantification of gene expression and analysis of differential gene expression.Gene function annotation,KEGG pathway and PPI network analyses were performed for the differentially expressed genes?DEGs?.4.On the basis of aformentioned results,10 DEGs were selected for RT-qPCR to verify the RNA-seq data.The passage 3 rBMSCs were cultured at 1%and 18%oxygen concentrations for 5 days according to the above method.Total RNA was collected,and the relative mRNA expression levels of individual genes were detected.Results1.Hypoxic culture promoted the proliferation of rBMSC in an oxygen concentration-dependent manner.The proliferation rate of the passaged rBMSC at 1%O2 and 5%O2 was significantly higher than that of the normoxia group?18%O2?,while the cell proliferation potential of the 1%O2 group was the highest,which was proved by the growth curve,colony formation ability and cck-8 absorbance values.It was revealed for the first time that there were more phosphohistone H3?PHH3?-positive cells in the hypoxic cultures of rBMSCs than in the normoxic culture.The results of flow cytometry showed a significant increase of the proliferation index in the 1%O2 group,as compared with the other two groups.The apoptotic indexes of two hypoxic groups were much lower than that of normoxia group.It was confirmed that hypoxia significantly up-regulated HIF-1?protein expression.2.The clean bases of each sample were 7 Gb or so,and the bases were evenly distributed.The overall sequencing data quality was ideal.The mapping of reads obtained from each sample to the reference genome demonstrated that the percentage of total mapped read was higher than 81%,and the proportion of reads mapped to exons was higher than 93%.3.Between the hypoxia and control?normoxia?groups,there were significant differences?q-value<0.05 and log2?fold change?>2?in the expression levels of 818 genes,and 541 genes were up-regulated while277 genes were down-regulated.Among these genes,the marked changes in expression of Mmp7,Mmp9,Mt1,Mt2A and Ptgs2 have been detected for the first time in MSCs exposed to hypoxia in vitro.4.The gene function analyses,including GO and KEGG,showed that hypoxia significantly regulated genes related to metabolism,proliferation and cytokine secretion.The hypoxic adaptation of rBMSC may be highly correlated with metabolism,HIF-1 pathway,chemokines,and the interaction between cytokines and their receptors.5.The protein-protein interaction network analysis of DEGs suggested that the proteins encoded by Vegfa,Prkcb,Pik3r3,Ptgs2,Nme3,Gnaz,Adcy8,Nrp1,Gucy1b3,Itgb7 and Aldh1a2would play an important role in the protein-protein interaction network.They were mainly related to signaling pathways of cell apoptosis,HIF-1,PI3K-Akt and cGMP-PKG.6.RT-qPCR verified that the expression levels of Vegfa,Itgb7,Ptgs2,Mt1,Mt2A,Mmp7,Mmp9,Il1rl1,Nrp1 and Ogn were consistent with the RNA-seq results.Conclusion1.The hypoxia can promote the proliferation of rBMSCs in vitro in a dose-dependent manner under experimental conditions of this study.Compared with 5%or 18%oxygen concentration,1%oxygen concentration is more suitable for in vitro amplification of rBMSC.The proliferation-promoting mechanisms of the hypoxic culture are involved in the control of cell mitosis and apoptosis.2.RNA-seq analysis reveals that 1%oxygen concentration has significant effects on the transcriptomic profile of rBMSCs in vitro,and the functions of the significantly regulated genes are related to the survival,growth,differentiation of these stem cells,especially related to HIF-1 pathway.3.It has been reported for the first time that Mmp7,Mmp9,Mt1,Mt2A and Ptgs2 in MSCs were significantly regulated by hypoxia,and they may play a role in anti-apoptosis,proliferation promotion and angiogenesis promotion under hypoxia by participating in HIF-1pathway or VEGF pathway. |
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