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.     

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.     

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.     

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.     

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.     

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.     

Transplantation of neuron-like cells induced from rat bone marrow mesenchymal stem cells improves motor function in a MCAO rat model

AIM:To observe the amelioration of motor function in a media cerebral artery occlusion (MCAO) rat model after transplantation of neuron-like cells induced from rat bone marrow mesenchymal stem cells. METHODS:Transplant neuron-like cells derived from rat bone marrow mesenchymal stem cells, which were isolated, cultivated, predicated and induced in vitro, were introduced into infracted cerebral cortex. L-DMEM were injected in control group. Screen test, beam test, prehensile traction test and Morris maze test were conducted at 2 and 8 weeks alternately after transplantation. Rat brain tissues were stained with TTC and percentage of infracted volume was analyzed. RESULTS:There was significant difference between control group and test group in the time and length for rat finding aim and in the grade and time of screen test, beam test, Prehensile traction test (P<0.05). CONCLUSION:Transplantation of neuron-like cells induced from rat bone marrow mesenchymal stem cells improves the motor function of MCAO rats.     

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.     

Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro

AIM:To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro.METHODS:BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo.RESULTS:We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION:Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.     

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.     

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.     

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.     

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.     

Direct differentiation of embryonic stem cells into neural cells without embryonic body culture period in vitro

AIM:To investigate the feasibility and effect of directly differentiation of embryonic stem cells (ESC) into neural cells induced by retinoid acid (RA) without embryonic body (EB) culture period in vitro. METHODS:ESC were digested and divided into 4 groups:group A and B were undergone EB culturing. After that, cells in group A were induced by RA, cells in group B were differentiated spontaneously, cells in group C were committedly induced by RA directly without EB culturing, and cells in group D were differentiated spontaneously without EB period. Morphologic changes were observed under inverted microscope and scanning electron microscope. MAP-2 and GFAP were detected by immunocytochemistry and flow cytometry after differentiated for 9 days. RESULTS:In groups A or C, neuron-like cells increased gradually, forming neural network. At the 9th day, a large part of cells in these groups were MAP-2 positive cells, and the positive rate was higher than that in groups B or D (P<0.01). Groups B and D were almost epithelial-like cells. At the 9th day, GFAP positive cells were predominant. The rate of GFAP staining cells in groups A or C was significantly lower than that in groups B or D (P<0.01). There was no significant difference of MAP-2 or GFAP positive rates between group A and C, nor between group B and D (P>0.05). CONCLUSION:ESC was directly induced into neural cells by RA without EB culture period in vitro. This modified method has the same effect as the traditional RA 4－/4＋ assay and can replace the traditional method.      

Differentiation of neurons from mouse induced pluripotent stem cells in vitro

AIM: To study the induction method of mouse induced pluripotent stem cells (iPSCs) that differentiate into neurons in vitro. METHODS: Mouse iPSCs were cultured in non-adherent culture dishes for 2 d to form embryoid bodies (EBs). The EBs were cultured for consecutive 2 d in the presence of retinoic acid (RA), and then were plated in the serum-free medium for adherent culture. Seven days later, Pasteur pipette was used to detach the differentiated cells around adherent EBs into “fragment” cell colonies with the help of dissecting microscopes, and these “fragments” were transferred to culture dishes with neural stem cell medium. Another 7 days later, the cells were plated onto the culture dishes using differentiation medium containing fetal bovine serum (FBS) and RA. The morphological changes of the cells were observed under inverted microscope. The iPSCs markers Oct4, Sox2 and SSEA1, the neural stem cell (NSC) marker nestin, the neuronal marker microtubule-associated protein 2 (MAP-2), the astrocyte marker glial fibrillary acidic protein (GFAP) and oligodendrocyte marker myelin basic protein (MBP) were detected by immunofluorescence method. The mRNA expression of GFAP, nestin, β3-tubulin, MAP-2 and MBP was detected by RT-RCR. MAP-2 gene sequence was identified. The proportions of NSCs differentiated from iPSCs and neurons from NSCs were detected by flow cytometry. RESULTS: Mouse iPSCs strongly expressed Oct4, Sox2 and SSEA1, and formed spherical EBs by suspended culture. The EBs were induced by RA and serum-free medium in adherent culture for 2 d, and rosette structure was observed under the microscope. “Fragments” separated by Pasteur pipette from the rosette structure formed neurosphere-like colonies. After the colonies were cultured in adherent condition for 5 d to 7 d in the presence of RA and FBS, the typical neurite was observed under the microscope. The neurospheres expressed nestin and their differentiated derivatives expressed MAP-2, GFAP and MBP, respectively. RT-PCR analysis and gene sequencing showed that the neurons were induced successfully. The results of flow cytometry demonstrated that 63.93%±1.47% of iPSCs differentiated into NSCs and 21.4%±1.70% of NSCs differentiated into neurons. CONCLUSION: Mouse iPSCs proliferate stably and differentiate into neurons in vitro, which provide a reliable source for the treatment of spinal cord injury.     

Lithium chloride promotes neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy

AIM: To study the influence of lithium chloride (LiCl) on the neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to explore whether autophagy was involved in this process. METHODS: MSCs were isolated and cultured in vitro. The cells were divided into LiCl group and control group. MSCs were treated with β-mercaptoethanol as an inducer for triggering the cells to differentiate into neurons. The expression of neuronal markers-neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2), and autophagic marker-microtubule-associated protein 1 light chain 3 (LC3) were measured by immunofluorescence method and Western blot. An autophagy activator rapamycin and autophagy inhibitor 3-methyladenine (3-MA) were applied to modulate the autophagy in the LiCl treated-cells. The protein expression of NSE and MAP-2 were determined by Western blot. RESULTS: After induction, the expression of NSE and MAP-2 were increased. The percentage of NSE-and MAP-2-positive cells and the expression of NSE and MAP-2 in the LiCl group were greater than those in control group (P<0.05). After induction, the number of LC3-positive dots and the expression of LC3-Ⅱ in LiCl group were greater than those in control group (P<0.05). The expression of NSE and MAP-2 increased when the autophagy was modulated by rapamycin in LiCl treated-cells, and on the contrary, the expression of NSE and MAP-2 were inhibited as autophagy was modulated by 3-MA. CONCLUSION: Lithium chloride may promote the neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy.     

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.     

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.