Investigation Of The Subcellular Localization And Function Of HESRG, A Novel Gene Specifically Expressed In Human Embryonic Stem Cells, By Transgenic Method

Human embryonic stem cells(ES cells)are totipotent cells derived from inner cell mass(ICM)of human blastocyst-stage embryos or primordial germ cells(PGCs),being capable of proliferating unlimitedly and giving rise to cells found in all three germ layers of the embryo. Human ES cells can be induced into mature cells of certain tissues such as nerve cells,cardiomyocytes,islet cells and blood cells,being important cell sources for cell replacement therapy in many diseases including neurodegenerative diseases,cardiac infarctions,diabetes mellitus and hematologic diseases,etc.Human ES cell lines were first established from ICM and PGCs by Thomson et al and Shamblott et al in 1998,respectively.Since then,the human ES cell research has immediately become one of the most attractive hot-spots in biomedical field.Human ES cells can differentiate easily and spontaneously when cultured inappropriately in vitro.Generally speaking,human ES cells culture systems can be divided into feeder-layer culture system and feeder free culture system.In feeder-layer culture system,human ES cells are cultured on mouse embryonic fibroblasts(MEFs)which are known as feeder cells;however it may cause contamination from exogenous mouse proteins and be unsuitable for large scale culture required for clinical applications.In feeder free culture system,human ES cells have to be cultured in MEFs-conditional medium supplemented with basic fibroblast growth factor(bFGF),which is troublesome and also unsuitable for large scale culture.In order to find an optimized and convenient way for culturing human ES cells,it is of great importance to elucidate the molecular mechanisms underlying the self-renewal of human ES cells.Recently,some groups have reported that somatic cells can be reprogrammed back into pluripotent stem cells by ectopic expression of some human ES cells related transcription factors.These studies not only open up a new way for clinical trials of stem cell therapy but also give prominence to the study on mechanisms regarding the self-renewal of human ES cells.Previous evidence indicate that the self-renewal of human ES cells is regulated by many factors including transcript factors(Oct-3/4,Nanog,Sox2,et al),signaling pathways (TGFβ,Wnt,FGF and PI3K,et al),cell cycle,microRNA,chromatin and DNA methylation.It has been confirmed that these transcription factors are tightly regulated by each other and construct a complicated regulatory circuit in human ES cells.Recent researches demonstrat that some other known and/or unknown genes may play important roles in maintaining the self-renewal of human ES cells besides the above-mentioned factors.Recently we identified a highly differentially expressed EST between human ES cells and 7-day embryoid bodies(7-d EBs)by Affymetrix Human Genome Microarray U133A and B.The EST-representing novel gene was then successfully cloned by EST assembling,5'-RACE and Northern blot,and it was denominated human ES cells related gene(HESRG).Bioinformatic analysis predicted that HESRG gene may contain two candidate open reading frames(ORFs), named HESRG1 and HESRG2.HESRG1 comprising 669bp with an initiation codon at 2-4 nucleotides(nt)and a stop codon at 668-670nt encoded a 24 kilo-Dalton protein containing 222 amino acid(aa). HESRG2 comprising 243bp with an initiation codon at 2245-2247nt and a stop codon at 2485-2487nt encoded an 8.7 kilo-Dalton protein containing 80aa.However,bioinformatic predictions need to be validated by experiments.To investigate the proper ORF,subcellular localization and biological functions of HESRG by gene over-expression strategy is the goal of this study.【Detection of the protein expression and subcellular localization of HESRG1,and the changes of biological characteristics of HESRG1-overexpressed human ES cells】HESRG1 is one of the candidate ORFs of HESRG gene.HESRG1 sequence was amplified from undifferentiated human ES cells by RT-PCR.Subsequently,the recombinant plasmids designated pEF/HESRG1 and pcflag-HESRG1 were constructed by inserting the HESRG1 sequence into the pEF/His(C)vector and pCMV-Tag4A vector, respectively.Then the pEF/HESRG1 plasmid was transfected into H9 cells by Fugene 6,a non-toxic transfection reagent to human ES cells. Several G418-resistant cell clones were obtained after G418 selection. We analyzed the expression of HESRG1 at mRNA level by RT-PCR, Real-time quantitative PCR and detected the HESRG1-Xpress fusion protein expression by Western blot using anti-Xpress.Western blot results showed that HESRG1-Xpress fusion protein could be detected in H9 cells transfected with pEF/HESRG1(named H9-HESRG1 cells).To further determine the subcellular localization of HESRG protein,the pcflag-HESRG1 plasmid was transiently transfected into H9 cells and the expression of HESRG-flag fusion protein was detected by indirect immunofluorescence using anti-flag at 72 hours post-transfection.As a result,HESRG-flag fusion protein was found to be distributed in nuclei. Thus,we infer that HESRG1 should be the ORF of HESRG gene and HESRG protein is located in the nuclei of human ES cells,in accordance with previous bioinformatic prediction.Subsequently,the changes in biological characteristics of H9-HESRG1 cells were detected.With over-expression of HESRG1,the compactly growing undifferentiated human ES cells changed into differentiated morphology with a scattered,long and thin phenotype. Pluripotency marker genes including Oct-3/4,Nanog,ectoderm marker genes including Sox1,FGF5 and Nestin,mesoderm marker genes including BRACHYURY(T)and Hand1,endoderm marker genes including GATA4,GATA6,and trophoblast marker genes including CGαand CGβwere detected by Real-time quantitative PCR.The results indicated that Hand1,a mesoderm marker gene,was dramatically up-regulated in H9-HESRG1 cells,while no obvious changes in the expression of marker genes of endoderm,ectoderm and trophoblast were observed.Furthermore,we detected the expression of marker genes including Nestin,Hand1,GATA4 at a protein level by indirect immunofluorescence.Compared to the cells transfected with blank vector(named H9-EF1),the fluorescence signal of Hand1 other than Nestin and GATA4 appeared quite strong in H9-HESRG1 cells.The proliferation ability of Hg-HESRG1 cells was measured by colony formation assay and MTT analysis.Compared with Hg-EF1 cells, H9-HESRG1 cells showed a much lower cloning efficiency in feeder free culture system and displayed much lower proliferation ability,indicating over-expression of HESRG1 could knock down colony formation ability and reduce proliferation rate of human ES cells.【Detection of HESRG2 protein expression and the changes of biological characteristics of HESRG2-overexpressed human ES cells】HESRG2 is another candidate ORF of HESRG gene.HESRG2 sequence was amplified from undifferentiated human ES cells by RT-PCR.Subsequently,pEF/HESRG2 plasmid was constructed by inserting the HESRG2 into the pEF/His(B)vector and transfected into H9 cells by Fugene 6.After G418 selection,several G418-resistant cell clones were obtained.Then RT-PCR,Real-time quantitative PCR and Western blot were used to detect the expression of HESRG2 at mRNA level and protein level.The Western blot results demonstrated that HESRG2-Xpress fusion protein could not be detected in H9 cells transfected with pEF/HESRG2(named H9-HESRG2 cells),suggesting that HESRG2 may not be an alternative ORF of HESRG gene.Subsequently,the biological characteristics of H9-HESRG2 cells were assessed.Similar to human ES cells untransfected or transfected with blank vector(named H9-EF2 cells),H9-HESRG2 cells underwent no visible morphologic changes.MTT assay demonstrated no evident changes in proliferation ability were observed between H9-HESRG2 cells and H9-EF2 cells.Flow cytometry(FCM)analysis also showed that the percentage of H9-HESRG2 cells in G1 phase,S phase,G2/M phase was unchanged, compare to H9-EF2 cells.To illustrate the differentiation ability of H9-HESRG2 cells,EBs formation assay was performed.No distinct morphological differences were found between EBs generated by H9-EF2 cells and H9-HESRG2 cells.Moreover,total RNA of EBs at three time point(7-day,14-day, 21-day)was extracted.RT-PCR analysis revealed that no significant differences in mRNA expression levels of the marker genes representative of three germ layers were found between these two groups of EBs.Based on these data,we speculate that HESRG2 may be located in the untranslated region of HESRG gene and over-expression of HESRG2 alone is dispensable for maintenance of human ES cells self-renewal.In summary,according to our experimental results,HESRG1 is the ORF of HESRG gene and HESRG protein is located in the nuclei of human ES cells.The expression level of HESRG should be restricted within an appropriate range to matain the undifferentiated state of human ES cells.Over-expression of HESRG1 can lead to decrease in proliferation ability of human ES cells and induce human ES cells to differentiate into mesoderm.Nevertheless,HESRG2 lies in the untranslated region of HESRG gene,and its over-expression alone is not essential for maintaining the self-renewal of human ES cells.