Experimental Study On Immortalization Of Rat Bone Marrow-derived Mesenchymal Stem Cells

Stem cells are self-renewable populations that can differentiate into single or multiple cell types. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into neural cells in vivo and in vitro and secrete some types of neurotrophins. BMSCs can be used as seed cells for central nervous system transplantation. It has been reported that BMSCs lose proliferating and differentiating capacities in long term culture. This will greatly restrict transplantation treatment because of decrease in qualities and quantities of BMSCs.This study will establish the immortalized rat bone marrow-derived mesenchymal stem cell line by transfection of expression plasmids of human telomerase reverse transcriptase (hTERT) mediated with cationic liposome and then investigate the proliferation and differentiation potential into neural cells of immortalized BMSCs in vitro and initially evaluate the growth variation and application safety of BMSCs. Afterwards we try to discover the long term outcome of allogenic intracerebral transplantation with immortalized BMSCs after fluid percussion brain injury. We hope that our study will prove experimental proofs for clinical usages of immortalized stem cells.Part 1 Isolation, culture and identification of rat BMSCsObjective: To isolate and culture rat BMSCs and to examine their surface antigens and Nestin expression.Methods: Bone marrow cells were flushed out of bone marrow cavity of femurs and tibias of rat with L-DMEM containing 10% FBS and inoculated into 10 cm diameter plastic culture dishes. Then they were cultured under conditions of 37℃, 5% CO₂ and saturated humidity in a incubator. Monolayer adherent cells werepassaged at ratio of 1:2 with 0.25% trypsin digestion solution when they reached 90% confluence. Afterwards the proliferating cells were always passaged at ratio of 1:2 when they were 80⁹0% confluent.P5 cells were digested and collected when they reached 90% confluence. 1x10cells were suspended in lOOul PBS and FITC conjugated anti-CD29, anti-CD31, anti-CD44, anti-CD45, anti-CD90 antibodies were added separately at their proper concentration. Staining reaction lasted for 30 min in dark. CD34 staining need 30min reaction of both anti-CD34 antibody and FITC conjugated second antibody one by one. At last the cells were suspended in 600ul 1% paraformaldehyde and examined by flow cytometry.After treatment with 4% paraformaldehyde, 1% Triton X-100 and 10% goat serum, P5 cells were stained with 1:100 diluted anti-Nestin antibody and FITC conjugated second antibody. Nestin expression was examined in fluorescence microscope.Results: A few cells became adherent 24⁷2 hours after inoculation. They continued to proliferate until many cells reached confluence 10—14 days after inoculation. Then they were passaged at ratio of 1:2 every 4⁵ days. P5 cells became homogenous and had fibroblast-like morphology. During long term culture senescent cells, ultra proliferating cells and multinucleated giant cells were occasionally found. Flow cytometry analysis showed that rat BMSCs expressed CD29(99.83%), CD44(99.77%) and CD90(99.86%), but not CD31(0.83%), CD34(1.78%) or CD45(2.90%). Immunofluorescence examination showed that cytoplasm of rat BMSCs was Nestin positive.Conclusion: Rat BMSCs can be obtained by complete marrow cells culture. They express CD29, CD44 and CD90, but not CD31, CD34 or CD45. They are Nestin positive in cytoplasm. During long term culture senescent cells, ultra proliferating cells and multinucleated giant cells appear occasionally.Part 2 Immortalization of rat BMSCs and examination of the biological features of immortalized rat BMSCs in vitroObjective: To establish immortalized bone marrow-derived mesenchymal stem cell line and investigate proliferation, differentiation to neural cells and other biological features of immortalized rat BMSCs in vitro.Methods: P5 rat BMSCs was inoculated into 6-well plate and transfection of pLXSN, expression plasmids of sense and antisense hTERT was carried out when they reached 90—95% confluence. Transfection system contained 4ug plasmids, lOul Lipofectamine 2000, 2.5ml L-DMEM and rat BMSCs in a single well. Control group was set up. Medium was replaced after 6 hours of transfection. 24 hours later, these cells were passaged at ratio of 1:10. 200ug/ml G418 was added since the following day. After control group cells were all killed, G418 amount was changed to 50ug/ml.Total RNA of rat BMSCs and stable transfected cells during their 10th week of in vitro culture and C6 glioma cells was obtained by use of RNAultra kit. Then the cDNA was synthesized by use of AMV reverse transcriptase and PCR reaction was carried out by use of 2><10 cells/ml. Then the average value of cell numbers of 3 wells was calculated every 24 hours and cell culture curves of 7 days were made.After 24 hours of primary induction with 20% FBS, lOng/ml bFGF and L-DMEM, rat BMSCs and stable transfected cells during their 10th week of in vitro culture were induced with 200mmol/L BHA, 2%DMSO and L-DMEM. Induced cells were stained with 1:100 diluted anti-MAP2,anti-GFAP antibodies.Results: G418 killed all control group cells in 2 weeks and only a few cells transfected with pLXSN, pLXSN-S-hTERT and pLXSN-AS-hTERT survived. After G418 amount was changed to 50u-g/ml, pLXSN and pLXSN-S-hTERT transfected BMSCs continued to proliferate while pLXSN-AS-hTERT transfection inhibited proliferation of BMSCs and caused BMSCs death in 5 to 6 weeks. RT-PCR results showed that rat BMSCs and pLXSN transfected BMSCs expressed internal TERT lower than C6 did but pLXSN-S-hTERT transfected BMSCs expressed hTERT greatly so their total TERT amount was much higher. Immunocytochemical results showed that TERT staining was weak in some nuclei of rat BMSCs and pLXSN transfected BMSCs while pLXSN-S-hTERT transfected BMSCs were obvious TERT staining positive in nuclei and even partly in cytoplasm.Flow cytometry analysis showed that pLXSN-S-hTERT transfected BMSCs expressed CD29(99.67%), CD44(99.93%) and CD90(99.71%), but not CD31(1.56%), CD34(1.71%) or CD45(1.83%). Immunofluorescence examination showed that cytoplasm of pLXSN-S-hTERT transfected BMSCs was Nestin positive.Proliferation level of pLXSN-S-hTERT transfected BMSCs was much higher than that of simple BMSCs and pLXSN transfected BMSCs. Proliferation rate of simple BMSCs and pLXSN transfected BMSCs tended to decrease during long term culture while that of pLXSN-S-hTERT transfected BMSCs always kept in high level. After induction some cells from these 3 groups extended processes and could form networks. Their MAP2 and GFAP staining was positive.Conclusion: Rat BMSCs have differential potential towards neural cells and low level of telomerase. Proliferation rate of rat BMSCs tend to decrease during long term culture. Blockage of activity of telomerase can inhibit proliferation of ratBMSCs while pLXSN-S-hTERT transfection can accelerate proliferation of BMSCs without causing changes of expression of cell surface antigens or Nestin or their differentiation towards neural cells and could be a feasible method to establish immortalized bone marrow-derived mesenchymal stem cell line.Part 3 Long term survival and differentiation of immortalized rat BMSCs after their transplantation into rat brains subjected to fluid percussion injuryObjective: To investigate the long term survival and differentiation of immortalized rat BMSCs after their transplantation into rat brains subjected to fluid percussion injury.Methods: Before moderate fluid percussion injury and transplantation 45 SD rats were divided into 5 groups: sham injury group, injury group, sham transplantation group, BMSCs transplantation group and pLXSN-S-hTERT transfected BMSCs transplantation group. After anesthetization with 10% Chloral Hydrate experimental rat was kept breathing with tracheal intubation and respirator. Then the cranium was exposed after rat was fixed in stereotactic apparatus. 4mm diameter bone window with the center at 1.0mm post to bregma and 2.5mm right to midline was made and dura was kept intact. Fluid percussion injury tube was fixed on the edge of bone window. When the rat became conscious it was injured with maxim impact pressure 1.5-2.0atm and average impact time 23ms.24 hours after moderate fluid percussion injury experimental rat was anesthetized and fixed again. 5ul cell solutions with concentration at l><10 cells/5ul and labeled with lOug/ml Hoechst 33258 were injected into two sports at injury area with the coordinates at 2.0mm post to bregma, 2.0mm right to midline and 1.5mm or 3.5mm below dura. After transplantation it was fed for 8 weeks. Then the rat was decapitated and 8(im coronal continuous frozen sections were made. MAP2,GFAP and Nestin immunofluorescent staining was carried out to the surviving cells after transplantation. All antibodies were 1:100 diluted.Results: Respiratory inhibition, convulsion and lose of autokinetic movementappeared immediately after rat received moderate fluid percussion injury. The dura at injury area swelled up and turned brown. Automatic breath recovered in most rats in half a minute with the help of respirator and only a few died. Surviving rats became conscious 1-3 hours after injury and hemiplegia, movement retardation, balance missing and spirit depression appeared but most of the symptoms recovered in a few days.Pathological examination 8 weeks after transplantation showed that brain surface of injury area depressed slightly and had a different color. Cells labeled with Hoechst 33258 of BMSCs transplantation group and pLXSN-S-hTERT transfected BMSCs transplantation group were found mostly in injection tunnel and only a part of them extended outwards. No tumor was found. There were more pLXSN-S-hTERT transfected BMSCs survived than simple BMSCs in transplantation area. Immunofluorescent results showed that some cells labeled with Hoechst 33258 were MAP2,GFAP or Nestin positive and had blue nuclei and green cytoplasm. MAP2 and Nestin positive transplanted cells were mostly found in hippocampus or close to ventricular wall while GFAP positive cells were mostly found in pallium.Conclusion: Rat BMSCs transplanted into rat brains subjected to fluid percussion injury can survive in a long time and express neural cell markers. Their differentiation was circumstance influenced and exogenous hTERT expression can improve survival of rat BMSCs after transplantation.