The Immortalization And Related Studies Of Bone Marrow Stromal Stem Cells

Bone defect is a tough problem in orthopaedics. There are many troubles in the repairs of the defects by using autografts, allografts or xenografts. In 1980s, the repairing have entered the era of tissue engineering. Growth factors, seed cells and scaffolds are three elements of tissue engineering. Today, the problem of seed cells is a bottleneck in this field. Recently, the bone marrow stromal cells (BMSCs) have caught scientists' eyes because of its plasticity and easy harvesting. But it is not clear about its isolation, purification, identification and conditions of multi-inducing. Moreover, we can't obtain enough cells for tissue engineering because of the aging of cells in vitro culture. With the development of tissue engineering, people are looking forward to obtaining BMSCs easily as standard cells in basic research and a cells bank in clinical tissue engineering. To satisfy the needs, the best way is to establish a cell line. However, the methods of establishing cell lines were transfection with oncogene and viral genes (for example: SV40 T, HPV18 E6/E7) in last the century. These cell lines have some disadvantages: the exogenous genes interfere with cells' normal physiology, the cells are not ormal, they are transformant, their phenotypes and karyotypes have changed.Human telomerase reverse transcriptase (hTERT) is expressed in embryonicand germinal cells, it is a normal human gene. The aim of the study is to activate the telomerase of BMSCs by transcription of hTERT gene, thereby the BMSCs are immortalized. Because the way of activating the human telomerase is similar to the physiological way in the embryo stem cells, the immortalized cells have normal biological behaviors and the characters of adult stem cell, and this study have testified these opinions.1 Culture of BMSCsFirstly, the study explored the best condition to culture BMSCs, the materials are Hyclone fetal calf serum (FCS), bone marrows from five patients in operation with informed consents. Conclusion: the 10% L-DMEM culture medium was optimization to culture BMSCs, the isolation method using Percoll was better than the method of full bone marrow if we want to obtain more purified cells, but the first day to exchange the culture medium was late to the later, it was on the 6th day.Secondly, after obtaining the optimization way to culture BMSCs, we cultured the primary BMSCs. The bone was barrow obtained from 6-8 months abortion fetals, after digesting the primary cells, we made clone culture by limiting dilution assay and obtained a well-growth clone, named BMSCs-2. We identified the clone cells with flow cytometry, and found the cells expressed adhesion molecule CD29, CD44, CD 106, and CD 14, CD34, CD45, HLA-DR were not detected. The result indicated the clone cells were not hematopoiesis cells.According to the needs of clinics, the study induced BMSCs into osteoblasts, chondrocyte and cadiocyte. The conditional culture mediums, for inducing chondrocyte: TGF-p\ dexamethasone, vitamin C, the final concentration were 5ug/L ,10~-8mol/L, 10mmol/L; for inducing osteoblast: dexamethasone, vitamin C, glycerophophric acid-β the final concentraton were 10~-8 mol/L, 10mmol/L,50mg/L; for inducing cadiocyte: 3wmol/L 5-azacitidine. The BMSCs-2 cells were cultured in the three conditional mediums. At 6, 12, 18, 24d, these cells were tested. The osteoblasts were stained with HE to observe the cells morphology, with immunocytochemical stain to observe the expression of collogen I and osteocalcin, with alizarin red stain for calcium nodes. The chondrocytes were stained with toluidine to observe the secretion of mucopolysaccharides, immunocytochemical stain for collogen II. The cadiocytes were stained with immumocytochemical method to observe the expression of actin. The study used the method RT-PCR to observe the mRNAs expression of collogen I, collogen II, osteocalcin and MLC-2V. The results indicated BMSCs have the character of plasticity and could be induced into osteoblasts, chondrocytes and cadiocytes. We can draw the conclusion that the BMSCs-2 were bone marrow stromal stem cells.2, Establishment of human bone marrow stromal cell lineThe plasmid pCIneo- hTERT was presented by American scholar Weinberg RA. The clone BMSCs were transfected with eukaryotic expressing plasmid pCIneo-hTERT encoded hTERT and neo genes. After selection with G418 for three weeks, twenty-seven anti-G418 cell clones were selected to expand culture, only eight clones could expand culture in flask. After three months continuous culture, only clone 2, clone4, clone5 grew well. The clone 1, clone 6 and clone 7 were dead. The clone 3 and 8 were contaminated. We choose clone 2 for continuous culture, up to now, the cell line have continuous passage with 179PDs, named as MSCxj. We tested MSCxj after stable transfection, and found that hTERT was expressed at mRNA and protein level and telomerase activity turned to be positive in these transfected cells.The biology of MSCxj was similar to that of BMSCs-2. They were fibroblast-like cells, growing and proliferating more actively than BMSCs andbeing able to form cells clone in culture plates. The population double time was 40.08 hours. They also were normal karotype, had nontumorigenic in nude mice, and had no anchorage-independent growth with normal serum dependence and contact inhibition. These indicated that MSCxj is a normal cell line without obvious transformed phenotype.MSCxj had the same cell adhension molecules with BMSCs-2 cells. They expressed adhesion molecule CD29, CD44, CD 106, did not express CD 14, CD34, CD45 and HLA-DR. MSCxj also had the plasticity of adult stem cells and could be induced into osteoblasts, chondrocytes and cadiocytes.3, Ectopic osteogensis of MSCxj and xenograft compoundsIn order to test whether MSCxj could be applied in tissue engineering field, the study designed the experiment of ectopic osteogensis. The materials were 35th and 128th PDs MSCxj cells, xenografts, conditional osteoblast medium. After continuous culture for 18 days, the compounds were implanted in nude mice subcutaneously. At 4th, 8th, 12th week, we observed the osteogensis.The osteogensis could be observed at 8th week, which was obvious at 12th week. The osteogensis between 35th and 128th PDs MSCxj had no difference.4, ConclusionsHuman bone marrow stromal stem cell's optimized culture condition: 10% FCS, L-DMEM medium, first exchange medium at 5-6 days, the isolation method of density gradient centrifugation is better than the full bone marrow in obtaining purified cells, the later is facilitating to the growth of BMSCs. We can obtain clonal BMSCs by using limiting dilution assay. The activity of telomerase can be activated by transfecting hTERT gene, and can be used to make the BMSCs immortalized. The immortalization cells have normal cell biological behaviors and characteristics of adult stem cells which can be used in tissue engineering field.