Experimental study on inducing bone mesenchymal stem cells to differentiate into cardiomyocytes in vitro

AIM: To study the differentiation of rat bone mesenchymal stem cells (MSCs) into cardiomyocytes in vitro. METHODS: MSCs were isolated and purified from the bone marrow of rats by density gradient centrifugation and adhering to the plastic culture. The third passage MSCs were treated by 5-azacytidine (5-aza). The induced cells were evaluated by immunocytochemistry staining and RT-PCR analysis. RESULTS: After being induced by 5-aza, some MSCs became bigger and longer. The connection of the cells were formed on day 14.The direction of the cells arraying was similar gradually. The induced cells were stained positively for desmin, α-actin and troponin I. RT-PCR showed that these cells expressed β myosin heavy chain. CONCLUSION: 5-aza can induce MSCs to differentiate into cardiomyocytes in vitro.     

Differentiation of mesenchymal cells derived from mouse fetal liver into skeletal muscle-like cells in vitro

AIM: To study whether mesenchymal stem cells (MSCs) from fetal liver can differentiate into skeletal muscle-like cells. METHODS: MSCs were isolated from C57BL/6J mouse fetal liver and were induced by 5-azacytidine and amphotericin B. Myf5 and myogenin were tested by RT-PCR. Desmin and β-actin was examined by immunocytochemistry. RESULTS: RT-PCR showed that the treated cells expressed Myf5 and myogenin orderly from 6 hours to 72 hours, while the untreated cells did not. Immunocytochemistry confirmed that these cells were positive for desmin and β-actin and the positive rate was higher in 7 days than that in 14 days. CONCLUSION: MSCs derived from fetal liver can be induced into skeletal muscle-like cells in vitro.     

Human mesenchymal stem cells differentiate into osteoblasts

AIM:To investigate the differentiation from human mesenchymal stem cells (hMSC) into osteoblasts. METHODS:MSC were separated from human marrow with Ficoll-Paque reagent and expanded in cuture medium. To detect the surface antigens, The labeled cells were analysed on a FACScan flow cytometer. hMSC were induced to differentiate from mesenchymal stem cells into osteoblasts with dexamethasone, vitamin C, β-GP. Cell morphology、AP activity、calcium deposition and osteopontin were detected. P10 MSC were compared to P3 MSC in the tendency of osteoblastic differentiation. RESULTS:The cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. hMSC showed a strong self-renewal capacity. After primary culture, approximately (5-6)×10⁵ cells were obtained. These expanded attached MSC were uniformaly positive for CD29,CD44,CD59,CD105,CD166 and didn’t express CD11a, CD14, CD33, CD34, CD45, CD38, CD80, CD86, CD117. After osteoblasts induction, the cells changed from spindle-shape to cuboidal and polygonal in cell morphology. The AP activity increased gradually and many scattered calcium nodes were observed. The expression of osteopontin was positive. CONCLUSION:hMSC can be induced to differentiate into osteoblasts.     

Human mesenchymal stem cells differentiate into neuron-like cells with Tanshinone II A

AIM:To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells with Tanshinone II A.METHODS:hMSC were separated from rib marrow with Ficoll-Paque reagent and expanded in culture medium. To detect the surface antigens, the labeled cells were analysed on a FACScan flow cytometer to determine the effect of the capacity of proliferation and differentiation of the mesenchymal stem cells with FGF-2. hMSC were induced to differentiate into neurons with DMEM Tanshinone II A. Neuron-specific enolase(NSE), neurofilament(NF), Nestin, glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry.RESULTS:hMSC were expanded as undifferentiated cells in culture for more than 15 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. These expanded attached MSC were uniformly positive for CD29, CD44, CD90, CD105, CD166 and didn't express CD11a, CD14, CD34, CD38, CD45, CD80, CD86. FGF-2 have special effect on low denisity MSCs. Simple methods with Tanshinone II A induced hMSC to exhibit a neuronal phenotype, expressing NSE, NF-M, Nestin at 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION:hMSC can be induced to differentiate into neurons with Tanshinone II A.     

Study on inducing differentiation of mouse embryonic stem cells into cardiomyocytes in vitro

AIM: To set up a method of inducing mouse embryonic stem cells (mESC) to differentiate into cardiomyocyte after treatment with 5-azacytidine. METHODS: Cytotoxicity of 5-azacytidine was measured by MTT assay. Treatment of mESC with conditioned culture mediums, which were composed of 5-azacytidine alone or combined with retinoic acid, induced the cell differentiation to cardiomyocytes. The cells induced were identified by detecting the expression of cardiac proteins (myosin, desmin, α-actin and α-actinin). Gene MLC-2v, a specific gene of ventricular-like cardiomyocyte, was also detected by RT-PCR. RESULTS: The non-cytotoxic dose of 5-azacytidine was 8 μmol/L, which was able to induce mESC to differentiate into cardiac syncytiums. Cells induced expressed many cardiac proteins and MLC-2v mRNA. However, combined with retinoic acid inhibited mESC differentiation into cardiomyocyte. CONCLUSION: 5-azacytidine is able to promote mESC differentiation into cardiomyocytes. A method of inducing mESC to differentiate into cardiomyocytes in vitro has been established.     

Rat mesenchymal stem cells differentiate into neuron-like cells induced by berberine in vitro

AIM: To investigate whether berberine can induce rat mesenchymal stem cells (MSCs) to differentiate into neuron -like cells in vitro. METHODS: MSCs were separated from young rat femurs marrow and expanded in culture medium. MSCs were induced to differentiate by berberine. The morphological changes of MSCs were evaluated by light microscope.Neuron-spcific enolase (NSE), neurofilament (NF), glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. RESULTS: Induced by berberine for 8 hours,MSCs exhibited neurotype . The expression of NSE and NF in the neuron-like cells was positive, but the glial astrocyte marker GFAP didn't express. CONCLUSION: Berberine may induce adult rat MSCs to differentiate into neuron-like cells in vitro.     

Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro via cardiotrophin 1

AIM: To investigate the effects of cardiotrophin 1 (CT-1) on differentiation of swine bone marrow mesenchymal stem cells (MSCs) into cardiomyocyte-like cells in vitro.METHODS: MSCs were isolated and proliferated from Tibet miniswine. Adipogenic and osteogenic potentials were identified. MSCs were divided into 4 groups for induction: untreated group, 5-azacytidine (5-Aza) group,CT-1 group and 5-Aza combined with CT-1 group. After induction for 4 weeks, the expression of cardiac cell markers including α-actin and cardiac troponin-T (cTnT) was estimated by immunofluorescence staining. Finally, the rates of red fluorescence positive-staining cells were calculated. RESULTS: The expression of α-actin in the 4 groups by red fluorescence staining was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 29.90%±4.76%, significantly higher than that in 5-Aza group (17.73%±2.34%, P<0.01), CT-1 group (6.63%±0.55%, P<0.01) and untreated group (1.62%±0.09%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.05). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01). The expression of cTnT in the 4 groups was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 36.50%±4.09%, significantly higher than that in 5-Aza group (14.37%±1.65%, P<0.01), CT-1 group (7.50%±0.61%, P<0.01) and untreated group (1.12%±0.23%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.01). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01).CONCLUSION: Appropriate concentrations of 5-Aza (10 μmol/L) and CT-1 (0.1 μg/L) induce swine bone marrow MSCs to differentiate into cardiomyocyte-like cells in vitro. CT-1 combined with 5-Aza significantly increases the differentiation rate.     

Macaca irus bone mesenchymal stem cells differentiate into neuron-like cells induced by cryptotanshinone in vitro

AIM: To determine the optimal protocol and condition in which macaca irus mesenchymal stem cells (MSCs) are induced to differentiate into neuron-like cells by cryptotanshinone in vitro. METHODS: MSCs from macaca irus bone marrow were generated in vitro and induced with cryptotanshinone. The morphological changes of MSCs were evaluated by microscope. The positive percentages of neurofilament (NF), neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression were measured by immunocytochemistry with ABC staining. RESULTS: The result showed that MSCs were positive for CD29, CD44, CD105, CD166, and negative for CD34, CD71, CD80 and CD86. After induced with cryptotanshinone, MSCs began to display neuronal morphologies, such as contracted multipolar cell body and formed extensive networks. The percentages of positive NSE, NF expression were 68.3%±3.5%, 70.3%±1.5%， respectively. CONCLUSION: Macaca irus MSCs could be induced to differentiate into neuron-like cells in vitro by cryptotanshinone and might be applied in cell transplantation and gene therapy in nervous system disorders.     

Biological characterics of human fetal cord blood mesenchymal stem cells

AIM: To investigate the biological characterics of human second-trimester fetal cord blood mesenchymal stem cells (MSC) and its application prospects in utero gene transfer/therapy (IUGT). METHODS: Nuclear cells separated from cord blood were cultured in DMEM medium. Surface antigens of the MSC were analyzed by the FACScan flow cytometry. Adipogenic and osteogenic mediums were used to assess the differentiation ability of the cells. Adenovirus vector deliver green fluorescent protein gene (Ad-GFP) was used to transfected the MSC and the expressing of GFP was detected by fluorescent microscope. The MSC were injected into the liver of newborn rat. The immunofluorescence analysis was conducted to determine the presence of double-positive CD105⁺/CD166⁺ cells in different organs of rats. MSC were subcutaneous injected into the human-nonobese diabetes/severe combined immunodeficiency disease (NOD/SCID) mice and carcinogenesises of the MSC in vivo were detected by pathological diagnosis. RESULTS: MSC could be separated from fetal cord blood. These cells were uniformly positive for CD29, CD44, CD59, CD105, CD166 and negative for CD34, CD45, CD80, CD86, HLA-DR. The cells had the abilities to differentiate into adipogenic and osteogenic cells in vitro, expressed the GFP at high levels (56.32%±3.28%). The MSC were located at different organs after injected into the newborn rats and didn't have carcinogenicity in vivo. CONCLUSION: Human second-trimester fetal cord blood MSC is an promising target cells in fetal IUGT.     

Lentivirus-mediated calcitonin gene-related peptide transfection enhances endothelial differentiation of rat bone marrow mesenchymal stem cells

AIM：To study the effect of calcitonin gene-related peptide (CGRP) gene transfection mediated by lentivirus on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to endothelial cells. METHODS：Rat bone marrow MSCs were isolated by density gradient centrifugation combined with adherence method. Recombinant lentivirus vector carrying CGRP gene (Lenti-CGRP) was transfected into the MSCs. The secretion of CGRP in culture supernatants of the transfected MSCs was detected using ELISA method. The cells at passage 3 were divided into three groups: CGRP group (MSCs transfected with Lenti-CGRP), CGRP+CGRP8-37 (an antagonist of CGRP receptor) group and control group (MSCs transfected with PBS). The differentiation of the MSCs was detected by immunocytochemical staining for CD31 and factor Ⅷ-related antigen. The proliferation of the cells was measured by cell counting, and the angiogenic ability of the cells was analyzed using Matrigel assay. RESULTS：The proportion of CD31-and factor Ⅷ-related antigen-positive cells in CGRP and CGRP+CGRP8-37 groups was larger than that in control group (P<0.05). The numbers of the cells in CGRP and CGRP+CGRP8-37 groups were significantly increased compared with control group (P<0.05). Lumen-like structures were observed in CGRP and CGRP+CGRP8-37 groups. The above indexes in CGRP+CGRP8-37 group were reduced compared with CGRP group. CONCLUSION: Transfection with CGRP gene induces rat bone marrow MSCs to differentiate into endothelial cells and enhances their proliferation, suggesting that CGRP may play a role in the regulation of angiogenesis.     

Dexamethason enhanced aorta-derived CD105+ mesenchymal stem cells to differentiate into adipocytes

AIM: To investigate whether aorta-derived CD₁₀₅⁺ cells show characteristics of mesenchymal stem cells, and if dexamethason enhances this kind of CD₁₀₅⁺ cells to differentiate into adipocytes. METHODS: The distribution of CD105 in aorta was assessed by imunohistochemistry. The aorta wall cells were isolated and immunophenotypes were identified by FACS. CD₁₀₅⁺ cells were sorted using MACS CD105 micromagnetic beads. The differentiation of CD₁₀₅⁺ cells into adipocytes and osteoblasts was induced under different conditions and indicated by staining of Oil red O, detecting of alkaline phosphatase activity, calcium accumulation stained with silver nitrate and transmission electron microscope analysis, respectively. RESULTS: The endothelial cells, a part of medial smooth muscle cells and adventital fibrblasts were CD105 positive. The isolated aortic arch cells were positive for CD105, CD106, CD44, CD29, and negative for CD45, CD11a, CD11b and HLADR. The CD₁₀₅⁺ cells differentiated into adipocytes contained Oil-Red-O-positive lipid droplets, the osteocytes with calcium deposition and alkaline phosphatase activity. Ultrastructurally, it was observed that some needle-shaped crystal calcium deposition similar to bone spicules was inside the cytoplasm of induced osteocytes. When the dexamethason was absent in the adipogenic medium, there were no adipocytes with lipid droplets. CONCLUSION: The results demonstrated that CD₁₀₅⁺ cells showed characters of MSCs reside in aortic wall, and was able to differentiate into adipocytes and osteocytes in vitro. Dexamethason enhanced aorta-derived CD₁₀₅⁺ with characters of MSCs to differentiate into adipocytes. These suggested that MSCs might be related with atherosclerosis.     

Biological characteristics and induced differentiation of bone marrow-derived mesenchymal stem cells from sensitized mice in vitro

AIM: To evaluate the biological characteristics and differentiating potentials of bone marrow-derived mesenchymal stem cells (MSCs) from sensitized mice by allogeneic splenocyte transfusion in vitro. METHODS: Adherent culture method was applied for culturing the bone marrow-derived MSCs from sensitized mice. The cell morphology was examined and the surface marker profiles were analyzed by flow cytometry. The differentiating potentials of the MSCs into osteogenic, adipogenic and myogenic lineages were explored. The bone marrow-derived MSCs from the normal mice were collected and served as controls. RESULTS: Both the bone marrow-derived MSCs from sensitized and normal mice were exhibited a homogeneous distinctive morphology and were positive for the surface markers CD29, CD105, CD44 and Sca-1, negative in CD 34 and CD11b. The abilities of both MSCs to differentiate into osteogenic, adipogenic and myogenic pathways in the same condition were also observed. CONCLUSION: There is no difference in the biological characteristics and induced differentiating potentials between the sensitized mouse bone marrow-derived MSCs by allogeneic splenocytes transfusion and the MSCs from normal mice.     

Effect of Zfp521 expression on differentiation of rat bone marrow mesenchymal stem cells into neurons

AIM: To investigate the effect of zinc finger protein 521 (Zfp521) on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons. METHODS: Rat MSCs were cultured by conventional method in vitro and divided into non-transfection group, transfection group (transfected with Rn-Zfp521-siRNA) and negative control group (transfected with negative control siRNA). MSCs were induced by β-mercaptoethanol (β-ME) to differentiate into neurons. The fluorescence expressed by transfected MSCs was observed under inverted fluorescence microscope. The expression of Zfp521 was detected after transfection by RT-PCR. Immunohistochemistry, RT-PCR and Western blotting were used to detect the expression levels of neuron-specific enolase (NSE),microtubule-associated protein 2(MAP-2) and Zfp521 after induction. RESULTS: The fluorescence of MSCs was mostly displayed 72 h after transfection and the efficiency of transfection was up to 84.1%±2.3%. Meanwhile, the mRNA expression of Zfp521 was decreased (P<0.05). MSCs were induced by β-ME to differentiate into neurons. The differentiation efficiency of MSCs transfected with Rn-Zfp521-siRNA was the highest and the expression of NSE and MAP-2 was significantly increased compared with other groups (P<0.05). Zfp521 was detected in all groups, and the expression level of Zfp521 was significantly decreased after induction (P<0.01). CONCLUSION: Zfp521 may be down-regulated during the differentiation. The inhibition of Zfp521 promotes the neural differentiation of MSCs. Zfp521 may play an important role in regulating MSCs differentiation into neurons.     

Calpain regulates signal transduction contributing to cardiomyocyte hypertrophy

AIM: To induce rat bone marrow mesenchymal stem cells (BMSC) into cardiomyocytes and investigate the influence of serum coming from acute myocardial infarction (AMI) rat on the procedure. METHODS: The passage 3 BMSC were divided into six groups: groupⅠwas control group; groupⅡwas induced with 5-azacytidine; group Ⅲ was induced with 5-azacytidine and serum from AMI rat; group Ⅳ was induced with 5-azacytidine and serum from normal rat; group V and group Ⅵ were induced with serum from AMI rat or normal rat. The cardiac troponin T, GATA-4 and desmin were detected 30 days after induction. RESULTS: After inducing by 5-azacytidine, 5-azacytidine and two kinds of serum, some cells in the three groups differentiated into cardiac like cells. The expressions of cardiac troponin T, GATA-4 and desmin were positive in cells differentiated from BMSC. The troponin T expression in control group and group inducing by AMI serum alone were negative but GATA-4 and desmin expressed weakly. Some cells induced with 5-azacytidine and serum were slowly beating 2 weeks after induction, but the cells induced with 5-azacytidine alone was not beating.CONCLUSION: Serum from AMI can not induce BMSC to differentiate into cardiomyocytes, but it promotes BMSC differentiate into cardiomyocytes induced by 5-azacytidine and facilitate the differentiated cells to mature.     

Electrophysiological characteristics of the membrane current of cardiomyocytes-like cells derived from rat bone marrow mesenchymal stem cells

AIM: To study the electrophysiological characteristics of the membrane currents of cardiomyocytes-like cells derived from rat bone marrow mesenchymal stem cells (MSCs).METHODS: MSCs were induced,cultured and identified according to the reference.At the fourth week after treatment with 5-azacytidine(5-aza),cardiomyocytes-like cells were detected for the membrane current with the whole-cell patch-clamp technique and compared with the undifferentiated MSCs.RESULTS: The undifferentiated MSCs only expressed potassium currents.MSCs were stained positive for troponin T after treatment with 5-aza,and two kinds of inward currents and three kinds of outward currents were expressed.They respectively were the fast inward sodium current (I_Na),the L-type calcium current (I_Ca),the transient outward potassium current (I_to),the ultra-rapid delayed rectifier potassium current (I_kur) and the slow delayed rectifier potassium current (I_ks).Compared with the undifferentiated MSCs,the potassium currents of cardiomyocytes-like cells derived from MSCs were mainly made up of I_kur and I_ks.CONCLUSION: After treatment with 5-azacytidine,MSCs are differentiated into cardiomyocytes-like cells,which express the current of I_Na,I_Ca,I_to,I_kur and I_ks.     

Purification and adipogenic differentiation of human yolk sac mesenchymal stem cells

AIM:To purify human yolk sac mesenchymal stem cells (hYS-MSC) and investigate its adipogenic differentiation potential. METHODS:hYS-MSC were separated from yolk sac and purified via passaged culture. Flow cytometric analysis was used to identify the phenotype of hYS-MSC and the alkaline phosphatase(AKP) expression of hYS-MSC was also tested. Adipogenic differentiation of hYS-MSCs was induced by 10 mg/L insulin, 10^-5mol/L indomethacin and 10^-6mol/L dexamethasone. Oil Red O was used for fat staining. RESULTS:hYS-MSCs were purified at passages 2 or 3. Flow cytometric analysis showed the phenotype of purified YS-MSCs was uniformly positive for CD29, CD44, CD105, and CD166, and negative for reactivity to antigens CD34, CD45, or CD86. hYS-MSCs were weakly but clearly positive in AKP. Adipogenic differentiation of YS-MSCs was induced by 10 mg/L insulin, 10^-5mol/L indomethacin and 10^-6mol/L dexamethasone. Accumulation of lipid-rich vacuoles positive in oil red O staining within the cells were appeared and nuclears were pushed to one side of the cells during the period of induction. CONCLUSION:The phenotype of hYS-MSC is coincident with adult human mesenchymal stem cells. hYS-MSC can be induced to differentiate into adipocytes in vitro.     

The correlation between differentiation and apoptosis in the process of differentiation from adult rat mesenchymal stem cells into neuron-like cells

AIM:To supply the theoretic evidences of elongating the lifetime of neuron-like cells differentiated from adult rat mesenchymal stem cells, we investigated the relationship between the differentiation and apoptosis in the process of induction. METHODS: The mesenchymal stem cells(MSCs) were isolated primarily from rat bone marrow, and purified by passage culture. The 5th passage of MSCs was induced by β-mercaptoethanol and all-trans-retinoic acid (ATRA). After 1 h, 3 h and 5 h of induction, the cells were stained immunocytochemically with anti-MAP-2 and anti-GFAP antibodies, respectively. In addition to counting the ratio of neuron-like cells in MSCs, DAPI staining was employed to identify whether the differentiated cells have an apoptotic morphological changes. The ratio of apoptotic cells at 1 h, 3 h and 5 h after induction were detected by flow cytometry (FCM). CONCLUSIONS:1. β-mercaptoethanol and ATRA had the different ability that induced MSCs to differentiate to neuron-like cells. 2. Apoptosis was also initiated in the process of differentiation, and there is positive correlation between the ratio of differentiation and apoptosis.     

Study on the biological characteristics of rat bone marrow mesenchymal stem cells

AIM: To investigate the basic biological characteristics of adult rat bone marrow mesenchymal stem cells(rBMMSCs), and compare to that of human BMMSCs (hBMMSCs). METHODS: rBMMSC and hBMMSCs were separated from bone marrow with the difference of adherence and Ficoll-Paque reagent, and expanded in culture medium in vitro, respectively. The proliferation and growth characteristics of the primary and different passage culture of rBMMSCs and hBMMSCs were analysed. The neural differentiation capacity of rBMMSCs with passages were observed. To detect the surface antigens of rBMMSCs, the labeled cells were analysed on a FACScan flow cytometer. The karyotype of rBMMSCs were detected by blocking cellular fission with colchicines. RESULTS: rBMMSCs and hBMMSCs have a strong self-renewal capacity. Approximately (4-8)×10¹² and (3-4)×10¹² cells were obtained after passage 15 in vitro, respectively. The ability of proliferation, CFU-Fs, and neural differentiation of rBMMSCs and hBMMSCs were decreased gradually with passages, but the ability of proliferation and CFU-Fs of rBMMSCs were higher than that of hBMMSCs at different passage. FACScan result showed rBMMSCs were uniformly positive for CD29 and CD44, and negative for CD11b, CD45, CD61, CD71, CD80, CD86,MHCⅠ and MHCⅡ. rBMMSCs had an normal karyotype, which had an average of 37.0±4.0 to 40.5±2.5 chromosomes. CONCLUSION: Adult rBMMSCs have strong self-renewal and neural differentiation capacity, and have an normal karyotype. So rBMMSCs can be used as the seed cells for tissue engineering.