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.     

The related gene expression in mesenchymal stem cells differentiating to neuron-like cells

AIM: To analyze neuron-related gene expression before and after mesenchymal stem cells (MSCs) differentiating into neuron-like cells. METHODS: MSCs were induced to neuron-like cells with β-mercaptoethanol. Before inducement and at 8 h after inducement, the total RNA was extracted, then the expression of microtubule-associated protein-2 (MAP-2), growth -associated protein -43 (GAP-43), NSE, nestin and neurofilament (NF) mRNA were detected with RT-PCR. RESULTS: NSE mRNA expressed before and after inducement, MAP-2, GAP-43, nestin and NF mRNA only expressed after inducement. CONCLUSION: The differentiation of MSCs into neuron-like cells may be related to MAP-2, GAP-43, nestin and NF expression.     

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.     

Adipose-derived stem cells differentiate to neuron-like cells in vitro

AIM: We isolated and purified adipose-derived stem cells (ASCs) from adipose tissue in order to study their characters and examine their neuron differentiation capacity in vitro. METHODS: ASCs were isolated and cultured. The morphology of ASCs was observed under microscope and their phenotype was examined by flow cytometry. All-trans retinoic acid (ATRA) was used to induce ASCs into neuron-like cells. RT-PCR was used to detect the expression of nestin. Immunohistochemistry and Western blotting were used to detect the expression of neurofilament (NF) and neuron specific enolase (NSE), respectively. RESULTS: ASCs displayed a fibroblast-like morphology adhering to the culture plate. The cells expressed several surface antigens such as CD29 and CD105, while did not express CD31, CD34, CD45 and HLA-DR. Under suitable conditions, various passages of ASCs all had the capacity of neuron differentiation. They expressed nestin, NF and NSE 10 days after adding ATRA in the culture system. CONCLUSION: ASCs isolated from adipose tissue can differentiate into neuron-like cells in vitro. ASCs may be used as an alternative source of cells for neural disease therapy.     

Human mesenchymal stem cells differentiate into neuron-like cells by increase in intracellular cyclic AMP

AIM: To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells by the increase in intracellular cyclic AMP. METHODS: hMSC were separated from human marrow with Ficoll-Paque reagent and expanded in culture medium. hMSC were induced to differentiate into neurons with Forskolin and 3-isobutyl-1-methyl-xanthine (IBMX). Neuron-specific enolase(NSE), neurofilament(NF), glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry. RESULTS: hMSC were expanded as undifferentiated cells in culture for more than10passages. Forskolin/IBMX can induce hMSC to exhibit a neuronal phenotype, expressing NSE and NF-M in 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP. CONCLUSION: hMSC can be induced to differentiate into neurons by increase in the intracellular cAMP.     

Adenoviral-mediated high efficiency expression of enhanced green fluorescence protein gene in ex vivo expanded rat mesenchymal stem cells

AIM:To investigate the feasibility and infection efficiency of MSCs with replication-deficient adenovirus containing delivered gene, and whether enhanced green fluorescence protein (EGFP) gene track the change during rMSCs differentiating neuron-like cells. METHODS: Rat marrow mesenchymal stem cells (rMSCs) were expanded in low density in vitro. Under the control of CMV promoter, pAd-EGFP-Vector was constructed by homologous recombination in E.coil BJ 5183, and the recombinant virus was produced in HEK 293 packaging cell line. rMSCs infected with Ad-EGFP were observed and analyzed with fluorescence microscope. Infection efficiency was assessed by microscopical scoring and flow cytometrics. After withdrawing serum and exposure to β-mercaptoethanol medium, rMSCs infected with Ad-EGFP was induced to differentiate into neuron-like cells. As a control, the plasmid of pTrack-EGFP also was transfected into rMSCs to evaluate transfection efficiency. RESULTS:The results showed that Adenovirus vector (AdVec) delivered EGFP gene with high efficiency to marrow mesenchymal stem cells. Gene expression analysis showed that 36%±2 % of rMSCs infected with recombinant adenovirus expressed the transgene of EGFP at high levels. However, the transfection of plasmid pTrack-EGFP using routine method of lipofectamin mixed with plasmid DNA (pTrack-EGFP) was not easily successful and the transfection efficiency was much lower. rMSCs infected with Ad-EGFP in different passage could differentiate into typical morphology alike neural cells after withdrawing serum and exposure to β-mercaptoethanol medium. Immuno-staining with neuron-specific enolase (NSE), a neuronal marker, was strong positive, which suggested that rMSCs infected with Ad-EGFP had the potential to differentiate into neurons or neuron-like cells. CONCLUSION:The AdVec system can deliver target gene into MSCs and EGFP gene carried by AdVec can track the change during rMSCs differentiating into neuron-like cells.     

Role of caveolin-1 in differentiation of rat bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of caveolin-1 in bone marrow mesenchymal stem cells (MSCs) differentiation into neurons. METHODS: The MSCs used in the experiment were divided into non-transfected group, transfected group (transfected with Rn-caveolin-1 siRNA), positive control group (transfected with Rn-MAPK-1 control siRNA) and negative control group (transfected with negative control siRNA). The MSCs were induced by β-ME to differentiate into neurons. The fluorescence expressed by transfected MSCs was observed under inverted fluorescence microscope. The mRNA expression of caveolin-1 and MAPK-1 was detected by RT-PCR. NSE, NF-M and GFAP, the neural cell specific markers, were detected by immunocytochemistry staining. The survival ratio of MSCs was detected by MTT method. RESULTS: The fluorescence of MSCs was mostly displayed at 72th h after transfection and the efficiency of transfection was up to 81.5%±2.8%. Meanwhile, the mRNA expression of caveolin-1 in transfected MSCs was decreased (P<0.05). No significant difference of the survival MSCs ratio between transfected group and other groups was observed by MTT method. β-ME induced MSCs into neural cells. The differentiation efficiency of MSCs transfected with Rn-caveolin-1 siRNA was the highest and the expression of NSE and NF-M was increased significantly compared to the other groups (P<0.01). The expression of caveolin-1 was increased persistently with time and the highest expression was observed 6 d after induction. Furthermore, there was significant difference between before induction and 6 d after induction. CONCLUSION: Lipid raft labeled with careolin as marker protein has important role in regulating MSCs differentiation into neurons.     

Efficient production of neuron-like cells from embryonic stem cells induced by astrocyte-conditioned medium in vitro

AIM:To investigate whether efficient production of neuron-like cells from embryonic stem cells (ESCs) can be indued by astrocyte-conditioned medium (ACM) in vitro. METHODS:Based on the 4-/4+ protocol established by Bain, two groups were studied:ATRA group, ATRA with ACM group. ESCs were induced into neuron-like cells by means of three-step differentiation in vitro. The totipotency of ESCs was identified by the observation of cells’ morphology and the formations of teratoma in immunocomprised mice. The cell differentiation was evaluated continuously by detection of the cellular specific markers of neural stem cell, neurons and astrocytes such as nestin, NF-200, NSE and GFAP using immuno-histochemistry assay. RESULTS:(1) The ESC-D3 cells kept the ability of differentiation into cellular derivations of all three primary germ layers after continuous passage culture. (2) The ratio of NF-200 and NSE positive cells in the cells induced by ATRA with ACM was higher than that in the cells induced by ATRA only. (3) Finally, the positive rate of the neuron-like cells was up to 73.5% in the group induced by ATRA with ACM. CONCLUSION:The ESCs are induced into neuron-like cells with high purity and efficiency by ATRA with ACM.     

Rat mesenchymal stem cells differentiate into neuron-like cells with adrenaline hormones

AIM: To investigate the differentiation from rat mesenchymal stem cells (rMSC) into neuron-like cells. METHODS:rMSC were separated from femur marrow and expanded in L-DMEM culture medium supplemented with 10% FSC. rMSC were induced to differentiate into neurons with L-DMEM/adrenaline,L-DMEM/noradrenaline and L-DMEM/isoprenaline, respectively. Meanwhile, rMSC were cultured in L-DMEM in control group. Nestin, neuron-specific enclose (NSE), glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry. RESULTS: rMSC were expanded as undifferentiated cells in culture from 5 to 22 passages, indicating their differentiated capacity. Simple method induced rMSC to exhibit a neuronal phenotype, expressing positive NSE,nestin, and GFAP, at 5 hours in all group. The undifferentiating cells (control group 53.1%±4.3%), and differentiating cells (treated group: adrenaline 74.7%±2.6%; noradrenaline 75.9%±2.4%; isoprenaline 72.1%±4.4%), expressed characteristics of various neuronal cells, from 5 hours to 6 days. There were neuron-like cells in rMSC cultured in L-DMEM/10%FBS from 7 to 13 passage(66.5%±6.4%). CONCLUSION: It suggests that rat neural stem cells (rNSC) exist in bone marrow, rMSC can be differentiated into various neural cells with adrenaline hormones in vitro.     

Osteogenic and neurogenic differentiation of human yolk sac mesenchymal stem cells

AIM: To purify human yolk sac mesenchymal stem cells (hYS-MSC) and investigate its osteogenic and neurogenic differentiation potentials. METHODS: hYS-MSC were separated from yolk sac and purified via passage culture. The karyotype of hYS-MSCs was analyzed via G-banded characteristics. Flow cytometric analysis was used to determine the cell cycle and phenotype of hYS-MSC. The AKP expression of hYS-MSC was also tested. Osteogenic differentiation of hYS-MSCs was induced by 10-8mol/L dexamethasone, 10 mmol/L β-glycerophosphate and 50 mg/L vitamin C. Alizarin red S stain was used for identification of mineralization. β-mecaptoethanol or salviae miltiorrhizae were used to induce neurogenic differentiation of hYS-MSCs. The expressions of NSE, NF and GFAP were identified by immunohistochemical method. RESULTS: hYS-MSCs could be purified at passages 2 or 3. The cell cycle analysis suggested that hYS-MSCs showed strong proliferational potentials by which the cells kept normal diploid karyotype during the in vitro culture. Flow cytometry showed the phenotype of purified hYS-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. Osteogenic differentiation was appeared after induction of osteogenic differentiation. hYS-MSCs, which were of spindle shape, uniform in size, were induced to pleomorphism osteoblast-like cells which expressed high level of AKP. Aggregates or nodules were formed at day 7 and calcium accumulation was detected by alizarin red S staining on day 10 or day 14. Neurogenic differentiation of hYS-MSCs was induced by β-mecaptoethanol or salviae miltiorrhizae. NSE, NF or GFAP positive cells were detected by immunohistochemical staining. CONCLUSIONS: hYS-MSCs have strong proliferation potential and the normal diploid karyotype is kept during the in vitro culture. The phenotype of hYS-MSCs is coincident with adult hMSCs. hYS-MSCs could be induced to differentiate into osteogenic or neurogenic cells.     

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.     

Differentiation of mesenchymal stem cells derived from human umbilical cord

AIM: To explore an ideal method to induce the differen-tiation of human umbilical cord mesenchymal stem cells(hUCMSCs) into neuron-like cells and to provide some evidence for the transplantation of hUCMSCs for spinal cord injury. METHODS: The hUCMSCs were isolated from human umbilical cord digested with collagenase Ⅱ. The hUCMSCs was verified by flow cytometry analysis. The passage 5 cells were randomly divided into 4 groups. The differentiation of hUCMSCs was induced by bFGF in group A, bFGF and BDNF in group B, or BHA, bFGF and BDNF in group C, while the cells in group D served as a control group cultured with DMEM-F12 and 10% FBS. Two weeks later, the expression of nestin, neurofilament protein H(NEFH) and glial fibrillary acidic protein(GFAP) was detected by real-time PCR and immunocytochemistry. The morphological changes of cells were observed under an atomic force microscope. RESULTS: Mesenchymal stem cells were isolated and cultured from human umbilical cord by enzyme digestion. hUCMSCs expressed CD29, CD44 and CD105, but no CD34, CD45 or HLA-DR. After cultured with inducing medium for 2 weeks, the cells were successfully induced into neuron-like cells. The appearance of the cells had great change. The induced hUCMSCs developed round cell bodies with multiple neurite-like extensions observed under an atomic force microscope. The result of real-time PCR showed that nestin was positive in A, B and C groups, and NEFH was positive in A and B groups, but GFAP was negative in 4 groups. The difference of nestin and NEFH expression among the induced groups was significant(P<0.05). CONCLUSION: Mesenchymal stem cells were isolated and cultured from human umbilical cord by enzyme digestion in vitro, and all the hUCMACs presented stable biological properties. Moreover, hUCMSCs were induced to differentiate into neuron-like cells in vitro via bFGF combined with BDNF.     

Correlation between the expression of neuron-specific protein and apoptosis in the process of differentiation from rat bone marrow stromal cells into neuron with BDNF

AIM: To investigate the correlation between the expression of neuron-specific protein and apoptosis in the process of differentiation from rat bone marrow stromal cells into neuron with brain-derived neurotrophic factor (BDNF). METHODS: The 5th passage MSCs were induced by BDNF and 2-mercaptoethanol (β-ME), respectively. At 1 h, 6 h, 12 h and 24 h, nestin, neuron specific enolase (NSE), microtubulease associated protein (MAP)-2 and glail fibrillary acidic protein (GFAP) were detected by Western blotting. Cell cycle and apoptosis were examined by flow cytometry. RESULTS: Nestin and NSE of neuron-like cells induced by BDNF and β-ME were all positive by Western blotting. At 12 h, nestin and NSE turned to negative and apoptosis was detected in β-ME group, nestin and NSE still positive and apoptosis wasn't detected in BDNF group. Till 24 h, nestin and NSE in BDNF group were negative but apoptosis still not detected. Notably, GFAP (glial astrocyte marker) was detected and MAP-2 wasn't detected in the two induced groups. CONCLUSION: The down-expression of neuron-specific protein correspondingly with apoptosis in the process of differentiation from MSCs into neuron with β-ME shows that apoptosis may be one of the causes of induced cell death. BDNF induction was not the cause of apoptosis. Other factors may include for the cell death in the presence of neuron-specific protein expression induced by BDNF.     

Adult rat and human bone marrow mesenchymal stem cells differentiate into neurons with Musk's polypeptide

AIM: To investigate the differentiation from adult rat and human bone marrow mesenchymal stem cells (BMMSCs) into neuron with musk polypeptide (Mu-P).METHODS: Adult rat and human BMMSCs were induced with Mu-P.Neuron-specific enolase (NSE),neurofilament (NF),Nestin,glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry.RESULTS: Simple methods with Mu-P induced adult rat and human BMMSCs exhibiting a neuronal phenotype,expressing Nestin at 3 hours to 5 hours,and expressing NE and NF at 5 hours to 7 days.But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION: Adult rat and human BMMSCs can be induced to differentiate into neurons with Mu-P.     

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.     

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.     

Apoptotic mechanism of neuron-like cells differentiated from adult rat mesenchymal stem cells in vitro

AIM: To investigate the mechanism of apoptosis during the process of adult rat mesenchymal stem cells (MSCs) differentiating into neuron-like cells in vitro. METHODS: The MSCs were isolated primarily from adult rats bone marrow by density gradient and then expanded in medium as undifferentiated cells for 3-5 generations. The MSCs were divided into three groups at random. The control group was induced with β-mercaptoethanol (β-ME). Meanwhile, the U0126 group was given β-ME and U0126 (10 μmol/L) added at the beginning of induction. The PMA groups were treated with β-ME and different concentrations of PMA since pre-induction. The effects of U0126 and PMA on the activity of neuron-like cells were observed by MTT. The effects of U0126 and PMA on the expression of neuron specific marker nestin and expression of apoptosis-related protein caspase, Bcl-2, Bax in neuron-like cells were detected by using immunocytochemistry method. TUNEL technique was used to detect apoptosis index. RESULTS: Compared to control group, neuron viability, nestin and Bcl-2 increased and neuron apoptosis decreased in U0126 group (P<0.05). The activity of neuron-like cells, the expression of nestin and Bcl-2 in experiment group treated with 300 nmol/L PMA decreased (P<0.05), while Bax protein expression and apoptosis index increased (P<0.05). CONCLUSION: Both MAPK and PKC signal pathways may be involved in the differentiation of MSCs into neuron-like cells as well as the apoptotic process in differentiated neuron-like cells.     

Effect of lentiviral vector of microRNA-9-1 on differentiation of mouse bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of microRNA-9 in inducing bone marrow mesenchymal stem cell(MSCs) differentiation into neurons.METHODS: The lentiviral vector of microRNA-9-1(microRNA-9-1-LV) was constructed and transfected into mouse MSCs. The cells were divided into non-transfected group, transfected group(transfected with microRNA-9-1-LV) and negative control group(transfected with FU-RNAi-NC-LV). MSCs were treated with β-mercaptoethanol(β-ME) as an inducer for triggering the cells to differentiate into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The mRNA expression of microtublin-associated protein 2(MAP-2) was detected by RT-PCR. The expression of neuron-specific markers,neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP), were measured by immunocytochemical method. The viability of MSCs was determined by MTT method. RESULTS: The results of PCR confirmed successful construction of mouse microRNA-9-1-LV. The virus titer was 1×10¹² TU/L(TU, transduction unit). The best transfection efficiency(up to 91.3%±4.2%) and survival rate appeared when multiply of infection(MOI)was 20 and on 4th day. β-ME induced MSCs to differentiate into neurons and the best efficiency of the induction was observed in transfected group. The expression levels of NSE and MAP-2 in transfected cells were higher than those in the cells of other group(P<0.05).CONCLUSION: MicroRNA-9-1-LV has high transfection efficiency in mouse MSCs. Higher differentiation rate from mouse MSCs to neurons is induced by β-ME after the cells are transfected with microRNA-9-1-LV.