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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

In vitro hematopoietic stem cells from embryonic stem cells reconstruct hematopoiesis in mice

AIM: To investigate the potential of hematopoietic stem cells (HSCs) derived from mouse embryonic stem cells (ESCs) to reconstruct hematopoiesis in vivo. METHODS: Using a three-step method, a mice embryonic stem cell line, E14.1 was induced into hematopoietic stem cells. The cell markers with CD34+/ Sca-1+ were identified by flow cytometry analysis, then HSCs (1×109 cells/L) from third-step were injected into SCID mice for observing teratoma formation. To validate function of HSCs, colonogenic cell assay was conducted and the hematopoiesis in lethally irradiated mice was reconstituted. RESULTS: The method of three-step differentiation, combined to use more hematopoietic stimulating factor promoted the E14.1 cell differentiation into HSCs with highest percent of CD34+/Sca-1+ cells (as high as 58.64%±4.20%) with more CFU-E, CFU-GM and CFU-GEMM populations. The cells showed the character of hematopoietic progenitors by Wright-Giemsa staining. Positive selected CD34+/Sca-1+ cells by magnetic sorting from third-step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 d after transplantation, with 71.4% (5/7) successful engraftment rate. Three recipients showed that the cell population of the peripheral blood leukocytes, red blood cells and hemoglobin approached to normal index at 40 d after transplantation, but followed relative slow renew in platelet count. Survival rate in transplant group was 43%, compared to 100% mortality in control mice. Karyotyping assays confirmed the female mice with XY. CONCLUSION: The three-step differentiation and the culture conditions described here support the differentiation of mouse ESCs into HSCs. HSCs derived from mouse ESCs can reconstruct hematopoiesis.     

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.     

NT-3 genetically modified Schwann cells promote neural stem cells to differentiate into neuron-like cells

AIM: To explore the effects of neurotrophin-3 (NT-3)-genetically modified Schwann cells (NT-3-SCs) on differentiation of neural stem cells (NSCs) into the neuron-like cells. METHODS: The NSCs were co-cultured with NT-3-SCs. Report gene LacZ genetically modified Schwann cells (LacZ-SCs) and normal SCs respectively in vitro. 7 d later, the differentiation of NSCs was studied by immunohistochemistry, and the percentage of neuron-like cells was calculated. RESULTS: NSCs differentiated to the GFAP-positive cells (glial-like cells) and NF-positive cells (neuron-like cells) in vitro. Compared to the normal SCs, NT-3-SCs more efficiently promoted NSCs to differentiate into the neuron-like cells. The effect of LacZ-SCs was as the same to the normal SCs. CONCLUSION: NT-3-SCs promote NSCs to differentiate into the neuron-like cells.     

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.     

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.     

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.     

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.