Cloning And Characterization Of Novel Genes Involved In The Proliferation And Differentiation Of Multipotent C3H/10T1/2 Cells By Gene Trap Screening

Based on their origins, stem cells fall into two categories, embryonic stem cells (ESCs, ES) and adult stem cells (ASCs). Currently, the clinical application of ESCs is limited by the ethical dispute, the formation of teratomas and the possibility of provoking immune reaction after transplantation of ESCs into a new host. In contrast, mesenchymal stem cells (MSCs), one kind of ASCs, which reside in but not limited to dermis, adipose tissue, muscle, bone marrow and peripheral blood, can be easily isolated and amplified in vitro. In addition, MSCs has the capacity to differentiate or transdifferentiate into endodermal, mesodermal, and ectodermal lineages, preferentially home to the sites of injured tissue and modulate host immunoresponses, and devoid of the ethical concern. Moreover, MSCs secrete bioactive factors that inhibit ischemia-caused apoptosis, prevent the formation of scar tissue, stimulate angiogenesis, and establish a favourable regenerative microenvironment. These factors are mitogenic to tissue-specific stem cells and can enhance the host-mediated differentiation of host-intrinsic stem cells. Therefore, MSCs are ideal cell soureces for treating hereditary or degenerative disease, graft-versus-host disease and inflammatory disease (such as Crohn's disease), autoimmune diseases (like diabetes), scarless regeneration of skin following massive burns or injury, stroke and spinal cord contusion or excision injuries, acute and chronic cardiac events, and acute renal or liver failure. MSCs also has great potential and promising future in the applications in the improving the hematopoietic recovery after radiation injury, tissue replacement with tissue-engineered bone/cartilage, repair of skeletal muscle and vascular injury, leading these cells the hot research focus in tissue engineering and regenerative medicine. There are two critical issues that need to be resolved before the safe and effective clinical application. One is how to obtain enough stem cell more effectively and safely, the other is how to control the differentiation of MSCs into committed effector cells, of which the answers depend on our understanding of the molecular mechanisms that regulate MSCs proliferation and differentiation. The processes of cell proliferation and differentiation are under the spatial and temporal control of cellular restricted expression pattern of genes. Establishing a new cellular phenotype or stimulating the normal cell proliferation requires switching precisely on and off the genes related to cell proliferation and differentiation. Abnormal cell proliferation or even cell transformation may occur once the gene regulation is disorder or deregulated. To understand the molecular mechanisms of MSCs proliferation and differentiation more in detail, it needs not only the accumulation of more experimental results but also the breakthrough of the technology. In recent years an approach based on forward genetic scaning called gene trapping is widely used to identify the location, sequence, expression and function of the trapped genes. Gene trapping is initially designed for random insertional mutagenesis in mouse embryonic stem cells by introducing a DNA vector with a promoterless reporter gene into the host genome. The reporter gene in the gene trap vector can not only trace the expression pattern of the trapped gene but also provide the starting site for the cloning sequences of the trapped genes. Furthermore, the success of insertion mutation and the responses of the trapped genes to various exogenous factors can be readily examined by the reporter gene. There are some distinct advantages when gene trapping is used to study the molecular mechanisms of MSCs differentiation: there are large amount of random insertional mutants after the gene trap constructs are introduced into MSCs chromosomes in one experiment; the expression pattern of endogenous gene can be monitored by the reporter gene at any time, and its sequences can readily be identified by RACE (rapid amplification of cDNA ends); gene trap mutagenesis-based forward genetic approach can be used to explore novel genes, identify the new function of known genes and validate the previous research of MSCs differentiation. So, the results from such experiment will provide more understanding of MSCs differentiation and help to make the clinical intervention more effective.Severe injury leads to dramatically reduced repopulation and dysfunction of tissue-repairing cells, which can impair healing, and cell replacement therapy is one of the feasible strategies in the management of wound healing. Due to their multipotency and widely distribution in various tissues, MSCs represent the most promising candicate cell source for cell replacement therapy. Through the study on the molecular mechanisms of MSC's committed differentiation, we will learn how to direct the cells to generate stem cells (iPS cells) and appropriate specialized cells to improve cell therapies as well as wound healing. The mesenchymal stem cell line C3H10T1/2 (also termed 10T1/2 cells) was isolated from C3H mouse embryo by Reznikoff, and can differentiate into smooth muscle cells, endothelial cells, osteoblasts, chondrocytes, adipocytes, myogenic cells and neurocytes. The multipotential 10T1/2 cells provide a good model for the molecular genetic analysis of mesodermal determination and cell differentiation, especially under the induction of growth factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-β(TGF-β). TGF-β1 is one of cytokines which play very important roles during embryonic development and wound healing by modulating many kinds of cytodifferentiation and cell phenotype change. Therefore, in the present study, 10T1/2 cells are adopted as a model of MSC and TGF-β1 as an inducer of cell differentiation in order to explore the molecular mechanisms of MSC proliferation and differentiation, and provide theoretical basis for MSC application in promoting wound healing, The trapped genes with active transcription will be the key interest.The main results and conclusions are summarized as follows:1. The immunophenotype and multilineage differentiation potential of murine embryonic mesenchymal stem cell line C3H/10T1/2 cells.After analyzed by flow cytometry, the immunophenotype of 10T1/2 cells could be summarized as CD44+, CD73+, CD90+ and CD105+, but CD31-, CD34- and CD45-, which was similar to the reported cell surface markers of mesenchymal stem cells. The model of smooth muscle cell (SMC) differentiation was successfully established based on following findings: 10T1/2 cells tended to be an peak valley-like appearance, an smooth muscle cell-featured growth pattern, and SMC-specific genes such asαSMA, SM22a, SM-MHC, SRF, were upregulated after treated with transforming growth factor-beta1 (TGF-β1) for 24 hours by RT-PCR analysis,αSMA upregulation by immunofluorescence staining analysis, andαSMA, SM22a upregulation by Western blot analysis. After induced by VEGF and bFGF for 9 days, 10T1/2 cells tended to be an obvious cobblestone-like appearance, an endothelial cell-featured growth pattern. Endothelial cell specific markers such as Flt1, Flk1, Ang2, Tie2, VE-cadherin, CD31, Ang1, Vezf1, Notch1, Jagged1 and EphB4/EphrinB2 were upregulated but not Flt4 (a marker of lymphatic endothelial cells) detected by RT-PCR, which suggested the induced endothelial differentiation may not lymphatic endothelial committed. Moreover, the endothial differentiation was successfully established and rendered the cells with endothelial cell function of mature endothelial phenotype as shown by positive immunofluorescence staining for CD31, FactorⅧand VE-cadherin, abundant pinocytotic vesicles and Weibel-Palade bodies under transmission electron microscope, the uptake of Ac-LDL and formation of tubular structures in three-dimensional culture in collagen I gel. Additionally, the constitutively expressedα-SMA in 10T1/2 cells decreased, while significantly CD31 expression occurred after the induction, which did not support the existence of smooth muscle differentiation. When induced by 5-azacytidine, myotube-like cells appeared at day 17. Meanwhile, adipose globelets were also observed in a small proportion of cells at day 17, and Oil Red O staining confirmed adipose accumulation in cytoplasm day 25. After induced to osteoblast differentiation by combining ascorbic acid,β-glycerophosphate, and dexamethasone, bone-like nodules were identified by alizarin red staining at day 21. After induced to adipogenic differentiation by 3-isobutyl-1- methylxanthine (IBMX), dexamethasone, and insulin, Oil Red O staining confirmed adipose accumulation in cytoplasm at day 15. After treated with bFGF,β-mercaptoethanol (β-ME) in combination with DMSO, cytoplasm retracted towards the nucleus and the cell processes prolonged at day 3, and immunofluorescence staining showed that differentiated cells expressed neuron-specific enolase (NSE) at day 15, which indicated the phenotype conversion into neurocytes. Plastic-adherent under culture conditions in vitro, cell surface markers analyzed by flow cytometry, and the potential of multilineage differentiation defined that 10T1/2 cells can be used as an alternative source of mesenchymal stem cells.2. The establishment and identification of gene trap clones from C3H/10T1/2 cells transfected with ROSAFARY vector.The retroviral polyA gene-trap vector with resporter gene LacZ, ROSAFARY, was transfected into packaging cell line Phoenix by lipofectamine. Then the retrovirus particles containing gene-trap construct were collected to infect 10T1/2 cells, and the cell clones with integrated gene trapping vector was selected by hygromycin. The sensitivity of 10T1/2 cells to different concentrations of hygromycin (50, 100, 150, 200, 300, 400, 600 and 800 mg/l) was tested, and the optimized concentration for hygromycin selection on 10T1/2 cells was 150 mg/l because all 10T1/2 cells were killed after cultured with this concentration of hygromycin for 7 days. After the drug selection of 150 mg/l hygromycin, individual drug-resistant colonies were picked and expanded. As a result, totally 103 clones with hygromycin resistance were obtained. Among them, 64 clones were confirmed by genomic DNA PCR that targets LacZ sequence in ROSAFARY vector, including 6 clones with positive LacZ staining, which indicated the active promoter activity of trapped genes, while the other clones with negtive LacZ staining indicated the weak or inactive promoter activity of trapped genes.3. cDNA Cloning and bioinformatic analysis of trapped genes downregulated after transforming growth factor beta1 treatment.Three gene trapped clones with differential expression patterns were obtained by LacZ staining before and after TGF-β1 induced phenotypic modulation of 10T1/2 gene trapped clones to smooth muscle cells. It was found that the trapped genes were Mrps6, a known gene, and two novel genes (mgt-6 and mgt-16) when the RACE-obtained sequences were searched in GenBank using BLAST algorithm. Mgt-6 (murine gene trap clone 6) is mapped to mouse chromosome 14, encodes two protein and has 4 transcripts, which have been confirmed by RT-PCR and submitted to GenBank by its online software BankIt (GenBank accession no. FJ744746, FJ860514, FJ860513 and FJ748867). Transcript 1, 2, 3 have 3 exons but encoded the shorter protein (MGT-6S), whose molecular weight is 1414.68 and isoelectric point is 5.75 calculated by pI/Mw software. Transcript 4 has 2 exons that encoded the longer protein (MGT-6L), whose molecular weight is 4073.57 and isoelectric point is 6.52. Mgt-16 (murine gene trap clone 16) is mapped to mouse chromosome 19, has 2 exons and encodes a protein, whose length is 93 amino acids, molecular weight is 9772.02 and isoelectric point is 6.04. Mgt-16 has been submitted to GenBank (GenBank accession no. GU266552). In order to confirm gene trap vector insertion, proper adaption to the upstream exon of the insertion point, and obtain the precise splicing acceptor(SA) site in ROSAFARY, RT-PCR was performed to detect the fusion transcripts. It was found that ROSAFARY construct were inserted into the intron 1 of both mgt-6 and mgt-16, and the precise splicing acceptor (SA) site is at 2754 nt in ROSAFARY.4. Cloning and molecular characterization of a novel gene, mgt-16, from multipotent 10T1/2 cells.The EGFP fused mgt-16 retroviral vector, pL-EGFP-N1-16, was constructed based on retroviral vector, pL-EGFP-N1, and verified by sequencing. Then they were transfected into packaging cell line Phoenix by lipofectamine. The retrovirus particles were collected to infect 10T1/2 cells and 400μg/ml G418 continuous selection was conducted. The overexpressed EGFP fused mgt-16 in 10T1/2 cells showed the green fluorescence in pan-cytoplasmic distribution which indicated the subcellular localization of EGFP-fused MGT-16 protein expressed in the cytoplasm. This result was consistent with LacZ staining pattern of pan-cytoplasmic distribution in gene trap clone 16.To exclude any possible interference of EGFP on the function of MGT-16, 6×His was used as a small tag to trace MGT-16. The retroviral vector containing the internal ribosome entry site (IRES), pL-IRES-EGFP-N1, was constructed based on two vectors, pL-EGFP-N1 and pIRES2-EGFP-N1, and verified by sequencing. Then 6×His fused mgt-16 retroviral vector, pL-IRES-EGFP-16-His, was constructed based on plasmid pL-IRES-EGFP-N1. Then the two constructed vectors were transfected into packaging cell line Phoenix by lipofectamine. The retrovirus particles were collected to infect 10T1/2 cells and stably overexpressed 6×His fused mgt-16 containing IRES-EGFP construct clones were obtained by 400μg/ml G418 continuous selection. Western blot analysis showed that the overexpression of mgt-16 in 10T1/2 cells attenuated the expression of the SMC-specific markers,αSMA and SM22a which was induced by TGF-β1.Using gene trap clone 16 as a model of mgt-16 gene disrupted, overexpressed 6×His-fused mgt-16 or EGFP-fused mgt-16 10T1/2 cell clones as a model of mgt-16 gene overexpressed, the influence of mgt-16 on the cell proliferation was determined using WST-8 dye (Cell Counting Kit-8) and cell migration by scratch wound on monolayer cells. According to the optical density value measured using WST-8 dye which is proportional to the number of viable cells in the medium, gene trap clone 16 grew more slowly than normal 10T1/2 cells did (P <0.05), which meant the disruption of mgt-16 gene would inhibit cell proliferation. however, the 10T1/2 cells with overexpressed 6×His-fused mgt-16 or EGFP-fused mgt-16 grew more rapidly compared with the corresponding control group (P <0.05), indicating the overexpressed mgt-16 gene would accelerate cell proliferation. The scratch wound assay showed that gene trap clone 16 migrated more slowly than normal 10T1/2 cells did (P <0.05), which implied the disruption of mgt-16 gene could slow down cell migration, while the cell clones with overexpressed 6×His-fused mgt-16 or EGFP-fused mgt-16 migrated more rapidly compared with the corresponding control group (P <0.05), suggesting overexpressed mgt-16 gene would accelerate cell migration. According to the results in disrupted and overexpressed model in cell proliferation and migration tests, it is assumed that mgt-16 gene can positively regulate cell proliferation and migration in 10T1/2 cells.5. The molecular mechanisms of mgt-16 downregulated by TGF-β1In order to elucidate the molecular mechenisms of how mgt-16 is downregulated by TGF-β1, gene trap clone 16 was treated with TGF-β1, or a single inhibitor of p38/RK, PI3K/AKT, ERK, mTOR and NF-κB, or TGF-β1 combined with individual inhibitors for 24 hours. Revealed by LacZ staining and RT-PCR, p38 pathway was demonstrated to be involved in the downregulation of mgt-16 by TGF-β1. Further study showed that p38 pathway was involved in TGF-β1-induced phenotypic conversion of 10T1/2 cells to smooth muscle cells as supported by following evidences: the p38 inhibitor (i.e., SB203580) can suppress the morphological changes caused by TGF-β1; the phosphorylation of p38 MAPK increased after TGF-β1 treatment and the p38 inhibitor (i.e., SB203580) attenuated the phosphorylation of p38 MAPK as determined by Western blot analysis; SB203580 suppressed TGF-β1-induced SMC-specific gene SM22a expression in 10T1/2 cells as confirmed by RT-PCR. Linking above data together, we proposed a hypothesis: TGF-β1 downregulates mgt-16 by activating p38 pathway in 10T1/2 cells to promote smooth muscle gene expression.6. Cloning and molecular characterization of a novel gene, mgt-6, from mutipotent 10T1/2 cells.Gene trap clone 6 exhibited a LacZ staining pattern of pan-cytoplasmic distribution. When treated with 5-azacytidine, the localization ofβ-galactosidase activity in cells of clone 6 translocated from the cytoplasm to the cell nucleus. This result was also demonstrated by overexpressed EGFP fused MGT-6 protein in 10T1/2 cells, which gives the clues that the novel gene mgt-6 may participate in the process of DNA hypomethylation. Additionally, the four splice variants of mgt-6 had different expression patterns in different murine tissues and 10T1/2, C2C12, Lewis cells as shown by RT-PCR. The transcript 4 was the only one expressed in skin, spleen and intestine, and predominantly presented in kidney, heart and skeletal muscle, while the transcript 1 predominantly expressed in liver. All four transcripts expressed in brain and lung. C2C12 predominantly expressed the transcript 4, while all four transcripts expressed in both 10T1/2 and Lewis cells. The transcript 1, 2 and 4 were expressed more strongly in Lewis than in 10T1/2 cells. C2C12 mouse myoblasts and adult murine skeletal muscle predominantly expressed the transcript 4 which endcoded MGT-6L protein, but weakly expressed the transcript 1, 2, 3 which endcoded MGT-6S protein. As for whether MGT-6L or MGT-6S protein is related with the development of skeletal muscle, further study is needed. After gene trap clone 6 or 10T1/2 cells was treated with TGF-β1, p38/RK inhibitor, PI3K/AKT inhibitor or ERK inhibitor, as shown by LacZ staining and RT-PCR, the mgt-6 gene expression was downregulated when treated with TGF-β1 or PD98059 (the ERK inhibitor) but upregulated when treated with LY294002 (the PI3K/AKT inhibitor), which indicated mgt-6 may be involved in the process of SMC differentiation and this differentiation may be positively correlated with TGF-β1-evoked P38 and PI3K but repressed ERK signaling pathway.In conclusion, focusing on the key unsolved problems on the molecular mechanisms of mesenchymal stem cell proliferation and differentiation, the forward genetic approach gene trapping was applied to scan the active genes in MSCs for the first time. Established were a large number of gene trap clones and some differentiation models of 10T1/2 cells, which were valuable of comprehensive application. In addition, we cloned and characterizated two novel genes. These results expand our understanding of TGF-β1 signaling pathway and the molecular mechenisms of smooth muscle cell differention, and provide theoretical support and expremental basis for further research on the committed differentiation of MSCs in order to more effectively improve wound healing by MSCs.