Reconstruction of corneal epithelium by human amniotic epithelial cells cultured in vitro

AIM: To investigate the plasticity of human amniotic epithelial cells induced to differentiate into corneal epithelial cells and the feasibility of corneal epithelium reconstruction. METHODS: A piece of amniotic membrane taken from an uncomplicated elective caesarean section was digested by collagenase, trypsin and EDTA. The amniotic epithelial cells were primarily cultured and passaged in Dulbeccos modified Eagles medium containing 10% fetal bovine serum. The HAECs of the 2nd or 3rd generation were continuously cultured on fresh corneal stroma. One or two days later, the human amniotic epithelial cells formed a monolayer, and then the air-lifting cultivation was carried out in an insert culture dish. The cultured cells on corneal stroma were investigated morphologically by hematoxylin-eosin staining,and its ultramicrostructure was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Meanwhile, the expression of cytokeratin (CK)3/12 was detected by immunohistochemistry. RESULTS: Human amniotic epithelial cells were successfully reproduced on corneal stroma in vitro . The monolayer was formed after cultured for 1 or 2 days, and stratification with 4 to 5 layers of epithelial cells was observed 14 days after air-lifting cultivation. A wealth of microvilli on the surface of HAECs were observed by SEM,while desmosomes among HAECs were found by TEM.The stra-tified cells expressed CK3/12. The composed tissue was very similar to normal corneal epithelium. CONCLUSION: Human amniotic epithelial cells can be used as seed cells to reconstruct the corneal epithelium by the technique of tissue engineering.     

Primary study on the differentiation mechanism of embryonic stem cells to epidermal like cells induced by human amnion

AIM: To investigate the mechanism by which human amnion induced mouse embryonic stem (ES) cells to differentiate into epidermal like cells. METHODS: ES- BALB/ c cells were cocultured with human amnionintranswells for 4 - 5 days , andthose cultured alone without amnion were taken as control group. The morphological differentiation were observed . The committed differentiation of EScells into epidermal like cells were detected by integrin-β₁ , CK19 , CK15 andinvolucrin immunohistochemistry , respectively .RESULTS: After 4-5 days of coculture, ES cells differentiated into single layer of epidermal like cells, fitted tightly, with polyhedral in shape. The immunohistochemical staining results showed that, most of the cells were integin-β₁ positive, only a few cells were CK19 and CK15 positively stained. Most of the cells in control group died, the survived ones were different in morphological shapes, and no integrin-β₁, CK19 and CK15 positive cells were found. CONCLUSION: Soluble substances secreted by human amnion may play an important role in inducing the differentiation of mouse ES cells into epidermal like cells.     

Changes of tear film stability after rebuilding ocular surface with corneal stem cells

AIM: To study the physiological function changes of the tears film after rebuilding ocular surface with corneal stem cells, and to discuss the validity and the estimate system of rebuilding ocular surface with the corneal stem cells. METHODS: The male New Zealand rabbits were used to establish the alkali burning model in the right eye. The corneal stem cells of the left eye were cultured on the amniotic membrane in vivo, and then transplanted to the right eye. Furthermore, the physiological function changes of the tear film were examined. RESULTS: Compared to the before alkali burning, the ocular surface cell morphology was similar after rebuilding ocular surface with the corneal stem cells, which were cultured on the amniotic membrane in vivo; The tear film breakup time test showed the a remarkable difference between after and before the alkali burning, but the cell modality after rebuilding had no remarkable difference compared to that before the alkali burning. CONCLUSIONS: It's an effective method to rebuild the ocular surface with the corneal stem cells in vivo, the cell framework and the physiological function of the tears film recover well after rebuilding. It may be a validity estimate system of rebuilding ocular surface to analyze framework and configuration of the ocular surface and test the tear film breakup time.     

Experiment study of fibrin gels as the substrate for cultivating human amniotic epithelial sheets in vitro

AIM: To investigate the feasibility of using human amniotic epithelial cells (HAECs) sheets, which are cultured by using a biodegradable fibrin sealant to re-establish corneal surface layer. METHODS: Human amniotic membrane (AM) harvested at the time of elective caesarean section was digested by collagenase and trypsin respectively to obtain HAECs. The HAECs were planted on Millicell culture dishes coated with biodegradable fibrin glue, and cultured by using the air-lifting cultivation. The HAECs sheets were investigated morphologically by Hematoxylin -Eosin staining, scanning electron microscopy (SEM) and were identified by cytokeratin immunohistochemistry. RESULTS: HAECs sheets were transparent and successfully cultured by using a biodegradable fibrin sealant. Most of the cultured cells were round or polygon and typical slabstone-like appearance. Many microvilli were observed on cell surfaces by SEM. Cytokeratin staining was positive. HAECs had stratificated growth tendency and became corneal-like stratificating epithelial cells. CONCLUSION: A commercially available fibrin sealant was an effective means of tissue engineering to create a carrier-free, transplantable HAECs sheets. The sheets were potential graft to re-establish corneal surface layer.     

Differentiation of rat marrow-derived mesenchymal stem cells into myoid cells in vitro

AIM:To study the differentiation of rat marrow-derived mesenchymal stem cells(MSCs) into myoid cells in vitro.METHODS:The MSCs of SD rat were cultured、passaged、induced and differentiated in vitro used by routine culture technique, and evaluated by FACScan flow cytometer, detected by immunohistochemistry and analyzed by Hitachi H-600 transmission electron microscope(TEM).RESULTS:FACScan shows that cells expressed the antigens of CD29 and CD44, not those of CD11b and CD45;cells show positive response of staining with desmin and myoglobin after processing by two compounds of 5-azacytidine and amphotericin B. There were stripform zone of myofilament without any organells beside the edge of membrane in myoid cell of induction and differentiation checked by TEM.CONCLUSION:The passaged cells were MSCs and the MSCs may have specific gene structures that can differentiate into myoid cells. The demethylation or hypomethylation may conduct by compounds of 5-azacytidine and amphotericin B, which could be involved in activating phenotype-specific genes to differentiate MSCs into myoid cells. There are good outlook on clinical treatment of illness of myatrophy using by MSCs.     

Preliminary investigation on combining autologous marrow stromal cells transplantation with granulocyte colony stimulating factor for improvement of rabbit cardiac performance after actue myocardiac infarction

AIM: To test the hypothesis that autologous marrow stromal cells (MSCs) transplantation combined with granulocyte colony stimulating factor (G-CSF) can enhance cardiac function of ischemic hearts in vivo.METHODS: In order to achieve a safe and persistent effect,we explored the potential of autologous MSCs transplantation.Acute myocardial infarction induced by occlusion of left anterior descending artery,autologous MSCs labeled with BrdU bromodeoxyuridine in vitro were administered intramyocardially into the infarct area of the same donor rabbits and G-CSF was administrated by subcutaneous injection.Four weeks later,the transplanted labeled MSCs were detected by laser scanning confocal microscopy and the cardiac functions were examined by echocardiogram and multichannel physiologic recorder.Myocardial infarct size was measured from mid-transverse sections stained with Massons trichrome.RESULTS: After 4 weeks,transplanted MSCs were demonstrated myogenic differentiation with the expression of α-sarcomeric actin and connexin 43 located in intercalated disk.MSCs combined with G-CSF transplantation improved the left ventricular contractility and reduced myocardial infarct size markedly compared to that without G-CSF tratment.CONCLUSION: Our finding indicates that autologous MSCs combined with G-CSF transplantation may represent a promising therapeutic strategy on ischemic heart disease.     

Autologus marrow stromal cell transplantation improves rabbit cardiac performance after myocardiac infarction

AIM: We tested the hypothesis that marrow stromal cells (MSCs), when implanted into self-myocardium, can undergo milieu-dependent differentiation, express cardiomyogenic phenotypes and enhance angiogenesis and cardiac function of ischemic hearts in vivo. METHODS: In order to achieve a safe and persistent effect, we explored the potential of autologous MSCs transplantation. One week after myocardial infarction induced by occlusion of left anterior descending artery, autologous MSCs labeled with BrdU (bromodeoxyuridine) in vitro was administered intramyocardially into the infarct area of the same donor rabbits. RESULTS: By 1 months, transplanted MSCs demonstrated to be myogenic differentiation with the expression of α-sarcomeric actin (5C5). MSCs implantation significantly increased vascular density in the infarct zone and resulted in markedly improved the left ventricular contractility. CONCLUSION: The finding indicates that autologous MSCs transplantation may represent a promising therapeutic strategy with free of ethical concerns and immune rejection.     

Effect of compressive stress on proliferation and differentiation of human epidermal stem cells cultured in vitro

AIM: To investigate the mechanism of proliferation and differentiation of human epidermal stem cells cultured in vitro under the influence of compressive stress. METHODS: Epidermal stem cells were isolated by adhering to type IV collagen and were cultured with conditioned medium, then were detected by PowervisionTM two-step immunohistochemical method with keratin 19 and cell cycle analysis. The cultured epidermal stem cells transplanted on silica gel membranes, which were put in a new apparatus, was designed to offer cell culture and intermittent compressive stress (4 kPa, 6 kPa, 8 kPa, 10 kPa, 12 kPa) for 2 h, 3 times a day simultaneously. A week later, cells on silica gel membranes were identified with keratin 19 and 10 by PowervisionTM two-step immunohistochemical method. RESULTS: The new apparatus offered cell culture and intermittent compressive stress simultaneously. The isolated and cultured epidermal stem cells were identified with keratin 19 positive and 84.80 percent of them were showed in G1 period with cell cycle analysis. Cells on silica gel membranes had been subjected intermittent compressive stress above 8 kPa for a week. The number of the cells was increased, which was more than that in control group. However, some cells identified by immunohistochemical staining with keratin 10 positive were detected among the disposed epidermal stem cells. CONCLUSION: The intermittent compressive stress above 8 kPa induces and promotes epidermal stem cells to proliferate and differentiate, indicating that epidermal stem cells respond to mechanical stress, probably is one of their major biological features.     

Isolation and multilineage differentiation of mesenchymal stem cells from human umbilical cord vein in vitro

AIM: To investigate the isolation, purification, expansion and multilineage differentiation of mesenchymal stem cells (MSCs) derived from human umbilical cord vein in vitro.METHODS: By 1% collagenase Ⅱ digestion, endothelial cells were isolated from human umbilical cord vein and cultured in IMDM medium.The morphology of the cells was observed by Wright’s staining and electron microscope.Cell cycle and immunophenotype were investigated by flow cytometry.Assays of adipogenic and osteogenic differentiation were performed in vitro.von Kossa staining, Oil Red O staining and mRNA expression of osteopontin and lipoprotein lipase were studied in the induced cells.RESULTS: The cells from the cord vein displayed a fibroblast-like morphology adhering to the culture plate.FACS showed that the cells expressed several MSCs-related antigens such as CD29, CD44 and CD105, while CD13, CD31, CD45, CD34, and HLA-DR were negative.Adipocyte and osteocyte differentiation were induced successfully.CONCLUSION: The morphology, growth characteristics, immunophenotype and pluripotentiality of the MSCs from human umbilical cord vein are similar to the MSCs from bone marrow (BM).They could potentially be an excellent source of MSCs for experiments and clinics.     

Differentiation of embryonic-like stem cells derived from human bone marrow into multinucleated fibers in vitro

AIM: To compare the capacity of in vitro differentiation into multinucleated fibers between embryonic-like stem cells (ELSCs) and mesenchymal stem cells (MSCs) derived from human bone marrow. METHODS: To isolate ELSCs, human bone marrow mononuclear cells were cultured in gelatin-coated flask with serum-free Knockout-DMEM medium designed for the expansion of human embryonic stem cells. MSCs were isolated from the same bone marrow by the traditional method. The morphological characters of both ELSCs and MSCs were observed under inverted phase-contrast microscope, and the expression of their multipotent antigen markers was identified by immunofluorescent staining. ELSCs and MSCs were cultured in myogenic differentiation medium. The protein levels of muscle-specific antigen markers myosin heavy chain (MHC), myogenin and MyoD were detected by the method of immunostaining. The mRNA expression of MHC, myogenin and MyoD was detected by RT-PCR. The capacity of in vitro differentiation into multinucleated fibers was compared between ELSCs and MSCs by calculating the proportion of MHC-positive multinucleated fibers. RESULTS: ELSCs, which weakly expressed the multipotential markers Oct-4, Nanog-3 and Sox-2, were isolated from bone marrow by the method of serum-free medium. ELSCs appeared smaller, slenderer and more homogeneous, and were morphologically different from MSCs derived from the same marrow. No multipotential marker in MSCs was expressed. ELSCs and MSCs were induced into long multinucleated fibers expressing MHC and myogenin at mRNA and protein levels by culturing in the myogenic differentiation medium. However, on the 10th day after induction, the proportion of the MHC-positive fibers in ELSCs was (25.7?4.1)%, and the proportion in MSCs was (15.8?7.6)%.The capacity for differentiation into muscle in ELSCs was significantly higher than that in MSCs (P<0.05). CONCLUSION: Bone marrow ELSCs are induced into multinucleated fibers and have the stronger myogenic differentiation capacity than MSCs derived from the same marrow. ELSCs are a more ideal candidate for muscular disease therapy.     

Treatment of acute tubular necrosis by autologous bone marrow stem cells transplantation through renal artery in the rabbits

AIM: To observe the effects and location of autologous bone marrow stem cells (BMSCs) transplanted through renal artery into ischemic-reperfusion (I/R) injured kidney.METHODS: BMSCs were collected from rabbits after isolated and then labeled with 5-bromo-2-deoxyuridine (BrdU). Twenty-eight rabbits were subjected to clamping renal pedicles for 105 min and divided into the transplantation group and control group randomly. BrdU labeled BMSCs or saline were injected into the kidney by renal artery, respectively. Before and after I/R at the 1st, 3rd, 5th, 7th, 14th, 21th and 28th d, the venous blood was collected to measure serum Cr and BUN. In the same time, renal tissue was collected for pathological and immunohistochemical study.RESULTS: After I/R, serum Cr and BUN levels in the rabbits in two groups became higher, and on the 1st and 3rd d after I/R, reached the highest level. On the 7th d the serum Cr and BUN levels in transplantation group were lower than those in control group. On the 28th d the levels of serum Cr (90.1±11.1) μmol/L and BUN (8.0±1.5) mmol/L in transplantation group were significantly lower than those in control group (135.6±32.5) μmol/L and (10.9±2.5) mmol/L, respectively (P<0.05). Pathological observation of renal tissue showed the degeneration, necrosis and abscission in renal tubular epithelial cells. The BrdU positive staining by immunohistochemical study was found in renal tubular in transplantation group on the 5th and 7th d after I/R, and maintained to the end of experiment, but no detection in control group.CONCLUSION: BMSCs transplantation through renal artery accelerates the repairment of renal functions after acute tubular necrosis by ischemic-reperfusion. Autologous BMSCs transplantation is a safe and valid method to accelerate the repairment after acute tubular necrosis.     

Study on animal intracorneal implantation model with xenogeneic swine corneal stroma

AIM: To study the immunogenicity and biocompatibility of xenogeneic swine corneal stroma as biological carrier for cornea reconstruction and to reconstruct corneal endothelial tissue with this carrier in vitro. METHODS: (1) The lymphocytes from the peripheral blood of F344 rats were immunologically labeled by anti-rat CD25-FITC and anti-rat CD4/CD8-PE, then determined by flow cytometry (FCM) at 12th， 90th day after intracorneal implantation with fresh and dehydrated swine corneal stroma. (2) The fresh and dehydrated grafts made of swine corneal stroma were implanted intralamellarly in corneas of New Zealand rabbits. Clinical examinations were performed monthly and histological examinations were made at 14th， 30th， 60th， 120th and 240th day. (3) The cat corneal endothelial cells were seeded on the Descemet's membrane of the dehydrated swine corneal stroma, then cultured in the medium with epidermal growth factor and laminin for 7 days. The morphology of reconstructed tissue was tested by microscope. RESULTS: (1) Compared to isograft group and negative control, the expression CD4＋CD25＋， CD8＋CD25＋ and CD4＋/CD8＋ of xenograft rat group implantation with swine corneal stroma did not appear significantly different in statistics (P>0.05). (2) In the total 12 rabbits, all the cornea grafts survived without rejection reaction， corneal haze or corneal neovascularization. The fresh grafts got transparent after 2 months, and the dehydrated grafts got transparent after 6 months. Histological study demonstrated both fresh and dehydrated stroma grafts had fused with rabbits'corneal stroma very well without lymphocytes infiltrating. (3) As shown in histological observations, the reconstructed cat corneal endothelial tissue was similar to the nature tissue. Cultured cat endothelial cells connected tightly to each other and attached to the Descemet's membrane closely. CONCLUSION: Swine corneal stroma has low immunogenicity and satisfying biocompatibility,it is an ideal biological carrier for cornea reconstruction in vitro.     

Differentiation of bone marrow mesenchymal stem cells in dilated cardiomyopathy

AIM: To investigate the feasibility of differentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes and vascular endothelial cells in dilated cardiomyopathy (DCM). METHODS: 20 rabbits were randomly divided into two groups. MSCs were isolated from bone marrow and cultured. Adriamycin was applied to the two groups to create rabbit DCM models. At 3 weeks after the creation of DCM models, the experiment group animals received intramyocardial injection of autologous MSCs. 4 weeks after transplantation, the implanted sites were examined to identify the labelled cells and to investigate its differentiation through immunofluorescence. RESULTS: At 4 weeks after the MSCs transplantation, the implanted cells were found in the experiment group and some differentiated into cardiomyocytes and vascular endothelial cells, which was not founded in the control group. CONCLUSION: Autologous bone marrow mesenchymal stem cells can differentiate into cardiomyocytes and vascular endothelial cells in dilated cardiomyopathy.     

Reconstructed corneal endothelial tissue on allo-stroma in vitro

AIM: In order to search carrier material and better tissue culture method, the morphology and structure of the cultured cat corneal endothelium was observed. METHODS: The cat corneal endothelial cells were seeded on the Descemet's membrane of the dehydrated swine corneal stroma, and then cultured in the medium with epidermal growth factor and laminin for 7 days. The morphology and structure of reconstructed tissue was tested by the inverted microscope and the scanning electron microscope. RESULTS: As shown by the morphological observations, the cultured cat corneal endothelial cells formed an integrated membrane, and attached to the Descemet's membrane, which was similar to the nature tissue. The cells connected tightly to each other, and some of them arranged in hexagon approximately. CONCLUSION: The cat corneal endothelial cells could be rebuilt on the carrier of the dehydrated swine corneal stroma successfully, which is similar to the nature tissue.     

Biological characteristics of chronic myelogenous leukemia bone marrow derived mesenchymal stem cells

AIM: To study the biology characteristics of mesenthymal stem cells (MSCs) derived from chronic myelogenous leukemia(CML) and normal adult bone marrow.METHODS: Mononuclear cells from chronic myelogenous leukemia (n=19) and normal adult (n=8) bone marrow were obtained, cultured in expanded medium with low serum concentration.Cell morphology, cell cycle, immunophenotype and in vitro differentiation capacity were investigated.The differentiations of osteocytes and adipocytes were detected by von Kossa staining and Oil-red O staining.The chimeric oncogene BCR/ABL and Ph chromosome, two hall marks of CML, were detected in CML derived MSCs, normal adult MSCs, CML derived hematopoietic cells and K562 cells.RESULTS: CML and normal adult derived MSCs showed similar characteristics in cell morphology, phenotype and growth pattern.A typital fibrablast like morphology was observed.Under suitable conditions, CML and normal adult MSCs had the similar ability to differentiate into adipocytes and osteoblasts in vitro.Moreover, CML and normal adult MSCs did not express BCR/ABL gene products and Ph chromosome was not observed.CONCLUSIONS: We isolated and cultured a population of cells with characteristics of multipotent stem cells from CML bone marrow.There were similar biologic characteristics and differentiation ability between normal adult and CML bone marrow-derived MSCs.     

Transplantation of exogenous mesenchymal stem cells treats post-infarction ventricular remodeling in rats

AIM: This study was performed to investigate the feasibility and efficiency of exogenous mesenchymal stem cells (MSCs) transplantation on post-infarction ventricular remodeling and heart function in rats and compare the effects between adult rat MSCs and neonate rat MSCs transplantation. METHODS: 1-2 hours after left coronary artery ligation, MSCs cultured in ex vivo, marked with BrdU, were injected directly into the border of infarcts in exogenous rats. 6 weeks after transplantation, rat heart function, ventricular remodeling and pathological results were measured. RESULTS: MSCs transplantation decreased LV end-diastolic diameter and end-systolic diameter, limited LV chamber dilatation and reduced collagen content significantly. The numbers of blood vessels and cardiomyocytes were increased. BrdU-labelled MSCs with oval nucleus were widely distributed. There were no significant difference between adult rat MSCs and neonate rat MSCs transplanted groups. CONCLUSION: MSCs can survive and home in exogenous host infarct hearts without addition of any immunosuppressant. MSCs transplantation has benificial effects on remodeling processes and contributes to improvement of cardiac function, which may be related with the reduction of the amount of the collagen, promotion of myogenesis and angiogenesis.     

Effect of human β-NGF gene-modified bone marrow-derived mesenchymal stem cells on rotational behavior of rats with Parkinson disease

AIM：To investigate the effect of human β-nerve growth factor (β-NGF) gene-modified bone marrow-derived mesenchymal stem cells (MSCs) transplantation on the rotational behavior improvement in a rat model of Parkinson disease (PD). METHODS：The rat model of PD was established successfully and the animals were divided into 4 groups:β-NGF-MSCs group (transplanted with 5×105 β-NGF-engineered MSCs), MSCs group (transplanted with 5×105 MSCs), DMEM/F12 group (5 μL transplantation medium was injected in the right striatum of the rats) and PD model group (without transplantation). The rotational scores were assessed 2 weeks, 4 weeks and 6 weeks after transplantation. At different time points after transplantation, the rats were tested for apomorphine (APO)-induced rotational behavior and the brains of the PD model rats were examined by fluorescence microscopy and immunohistochemical staining. RESULTS：Transplantation of human β-NGF gene-modified MSCs effectively improved the behavioral performance in the rats. At the 2nd, 4th and 6th weeks after cell transplantation, the rotational frequencies after injection of APO decreased significantly in β-NGF-MSCs group compared with MSCs group and PD group (P<0.05). Both β-NGF gene-modified MSCs and MSCs survived in the brains of PD model rats, had good compatibility with the host cells, and showed no signs of destroying the host and the glial cicatrisation. The β-NGF gene-modified MSCs expressed β-NGF stablely in the brains of PD model rats, and showed obvious improvement of the rotational behavior in the PD model rats induced by APO compared with MSCs group. CONCLUSION:The behavior of the rats with PD is significantly improved by transplanting β-NGF-modified MSCs in right striatum, and β-NGF gene therapy has potential clinical value．     

Decellularized porcine corneal posterior lamellae as carrier matrix for cultivating human umbilical vein endothelial cells in vitro

AIM：To investigate the feasibility of corneal posterior lamellar reconstruction with human umbilical vein endothelial cells (HUVECs) and porcine cornea acellular matrix in vitro, and to observe the physiological function of the transplantation in vivo. METHODS：HUVECs were isolated, cultured, and labeled with fluorescent dye CM-DiI. Porcine corneas were treated with 100% glycerinum, cut to a thinner structure step by step, and dried on the super-clean bench. Transmission electron microscope were used to observe the histological changes of the porcine cornea acellular matrix. Labeled HUVECs were seeded onto the porcine cornea acellular matrix, and examined by scanning electron microscopy. When the HUVECs and Descemets membrane fusion formed a monolayer, the corneal transplantation in rabbits was performed. Twenty-four New Zealand white rabbits were randomly divided into experimental group and control group (n=12 each), and their left eyes served as recipients. RESULTS：Cultured HUVECs exhibited polygonal shape. More than 90% HUVECs were labeled with CM-DiI and the cell membrane was positive with red fluorescence, which was detectable at least up to 3 generations. The histological examination indicated that porcine cornea cells were clearly extracted, and the collagen fibers were well arranged. A continuous monolayer of HUVECs on the porcine cornea acellular matrix was observed under scanning electron microscopy. The reconstructed corneal posterior lamellae were similar to the normal cornea. The observation of transplantation showed that the cornea in experimental group was substantially transparent. However, that in control group was oedematous and adiaphanous. CONCLUSION：Corneal posterior lamellae can be reconstructed in vitro by cultivating HUVECs on porcine cornea acellular matrix. After xenogeneic transplantation, the graft survives in vivo and expresses normal corneal endothelial cell biological functions. Deep lamellar corneal endothelial transplantation is an effective keratoplasty.     

Ectopic hTERT gene expression in human bone marrow mesenchymal stem cells

AIM: To investigate the effect of transfection of hTERT gene into human mesenchymal stem cells (MSCs) on their telomerase activity and life-span.METHODS: Human MSCs were transfected with a pEGFP-hTERT plasmid by liposome-mediated transfection. Then the hTERT mRNA expression in MSCs was detected by RT-PCR. The activity of telomerase in transfected MSCs was detected by PCR and ELISA. The telomerase-positive MSCs was cultured in vitro and induced into neuron-like cells with EGF and bFGF. Neuron-specific markers (NF-M, MAP₂) were detected by RT-PCR.RESULTS: hTERT fragment was identified in the hTERT-transfeced cells but not in the untransfected human bone marrow MSCs. The untransfected human MSCs remained telomerase-negative but the hTERT-transfected cells showed robust telomerase activity. The telomerase-negative MSCs entered a nondividing state and senesced after about 20 to 25 passages. In test group, however, telomerase-positive MSCs to date had undergone 35 passages. RT-PCR analysis showed that telomerase-positive MSCs expressed neuron-specific markers, such as NF-M or MAP₂ after induced with EGF and bFGF in vitro. CONCLUSION: Ectopic expression of the hTERT gene in human MSCs reconstitutes telomerase activity. The transfection of hTERT gene into human MSCs extends their replicative life span and maintains their multipotent differentiation capacity.