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OBJECTIVE@#To evaluate the capacity and efficiency of human umbilical cord mesenchymal stem cells (HUCMSCs) to differentiate into neuron- like cells after induction with B27- supplemented serum- free medium.@*METHODS@#HUCMSCs at passage 4 were cultured for 14 days with serum-containing medium (SCM) (group A), SCM supplemented with 20 ng/mL nerve growth factor (NGF) and 10 ng/mL basic fibroblast growth factor (bFGF) (group B), serum-free medium (SFM) (group C), or SFM supplemented with 20 ng/mL NGF and 10 ng/mL bFGF. The culture medium were changed every 3 days and the growth of the neurospheres was observed using an inverted microscope. The cell markers were analyzed with flow cytometry and the expressions of nestin, neuron- specific enolase (NSE), neurofilament heavy polypeptide (NEFH), and glial fibrillary acidic protein (GFAP) were quantified by quantitative real-time PCR (qRT-PCR) and Western blotting.@*RESULTS@#Before induction, HUCMSCs expressed abundant mesenchymal stem cell surface markers including CD29 (99.5%), CD44 (49.6%) and CD105 (77.7%). Neuron-like cells were observed in the cultures on days 7, 10, and 14, and the cell differentiation was the best in group D, followed by groups C, B and A. In all the 4 groups, the cellular expressions of nestin and GFAP gradually lowered while those of NEFH and NSE increased progressively. The expressions of GFAP, NEFH, nestin and NSE were significantly different between group A and the other 3 groups ( < 0.001 or 0.05).@*CONCLUSIONS@#B27-supplemented SFM effectively induces the differentiation of HUCMSCs into neuron- like cells, and the supplementation with cytokines (NGF and bFGF) strongly promotes the cell differentiation.
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AIM:To explore an ideal method to induce the differen-tiation of human umbilical cord mesenchy-mal stem cells (hUCMSCs) into neuron-like cells and to provide some evidence for the transplantation of hUCMSCs for spi-nal 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 hUC-MSCs developed round cell bodies with multiple neurite-like extensions observed under an atomic force microscope .The re-sult 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 signifi -cant (P<0.05).CONCLUSION:Mesenchymal stem cells were isolated and cultured from human umbilical cord by en-zyme 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 .
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Objective Bone marrow mesenchymal stem cells (BMSCs) were induced to differentiate to the special histological types of neurons in vitro.The morphological change of cells and positive expression of specific antigen on membrane were studied,and the function of connection between the induced BMSCs was also detected.The feasibility of BMSCs differentiate to the special histological types of neurons was investigated.Methods BMSCs were divided into group Ⅰ (induced with bFGF+GDNF),group Ⅱ (induced with bFGF+GDNF+WHI-P131 +Shh),and control group (no revulsive).The morphologic change of cells was observed,and the positive rate of neuron specific surface antigen and the content of dopamine were detected.Formation of mature synaptic structure was detected by immunohistochemical assay of postsynaptic density protein 95 (PSD-95) expression,and synaptic loop was shown by FM1-43 stain synaptic vesicles.Results By immunohistochemical staining,the positive rates of dopamine transporter (DAT) and tyrosine hydroxylase (TH) in group Ⅱ were significantly higher than those in group Ⅰ,and dopamine can been detected in cell culture supematant of group Ⅱ.After BMSCs was induced into dopamine neuron-like cells,number and length of cell protrusions,positive rate of PSD-95 and fluorescence intensity of FM1-43 in group Ⅱ were significantly higher than those of group Ⅰ.Conclusions There were no significant change in positive rate of neuron-specific surface markers,rate of cell survival and differentiation rate after BMSCs differentiated to dopaminergic neuron-like cells.The number and length of cell protrusions,content of dopamine in cell culture supematant,positive rate of dopaminergic neuron-specific surface antigen (DAT and TH),synaptic function index (positive rate of PSD-95 and fluorescence intensity of synaptic loop) of group Ⅱ were all significantly higher than that of group Ⅰ.
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PURPOSE: Mesenchymal stem cells (MSCs) are multipotent and give rise to distinctly differentiated cells from all three germ layers. Neuronal differentiation of MSC has great potential for cellular therapy. We examined whether the cluster of mechanically made, not neurosphere, could be differentiated into neuron-like cells by growth factors, such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: BMSCs grown confluent were mechanically separated with cell scrapers and masses of separated cells were cultured to form cluster BMSCs. As described here cluster of BMSCs were differentiated into neuron-like cells by EGF, HGF, and VEGF. Differentiated cells were analyzed by means of phase-contrast inverted microscopy, reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, and immunocytochemistry to identify the expression of neural specific markers. RESULTS: For the group with growth factors, the shapes of neuron-like cells was observable a week later, and two weeks later, most cells were similar in shape to neuron-like cells. Particularly, in the group with chemical addition, various shapes of filament structures were seen among the cells. These culture conditions induced MSCs to exhibit a neural cell phenotype, expressing several neuro-glial specific markers. CONCLUSION: bone marrow-derived mesenchymal stem cells (BMSCs) could be easily induced to form clusters using mechanical scraping, not neurospheres, which in turn could differentiate further into neuron-like cells and might open an attractive possibility for clinical cell therapy for neurodegenerative diseases. In the future, we consider that neuron-like cells differentiated from clusters of BMSCs are needed to be compared and analyzed on a physiological and molecular biological level with preexisting neuronal cells, and studies on the possibility of their transplantation and differentiation capability in animal models are further required.
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Adult , Humans , Blotting, Western , Bone Marrow Cells/cytology , Cell Culture Techniques , Cell Differentiation , Cells, Cultured , Epidermal Growth Factor/pharmacology , Hepatocyte Growth Factor/pharmacology , Immunohistochemistry , Mesenchymal Stem Cells/cytology , Neurons/cytology , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Objective To approach a new method induce the differentiation of mesenchymal stems cells（MSC）into neuron-like cells.Methods We co-cultured Schwann cells with MSC to induce the differentiation of rat MSC into neuron-like cells.Immunohistochemistry was employed to observe the expression of NSE and Brdu,and Western blot to examine the expression of NeuN by in transdifferenciated MSC（Neuron-like cells）.Additionally,we analyzed the differentiating rate of MSC.Results The co-culture of MSC with Schwann cells could induce the expression of NSE and NeuN.They have been differentiated into neuron-like cells.The differentiating rate of MSC was（80.51±5.65）%.Conclusion The co-culture with Schwann′s cells could differentiate rat bone marrow derived MSC into Neuron-like cells.
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AIM: To investigate the process of human bone marrow stromal cells (hBMSCs) differentiation into neural-like cells and to determine the role of 26S proteasome in neuronal differentiation. METHODS: Purified hBMSCs were treated with β-mercaptoethanol (β-ME) for 1 day and retinoic acid (RA) for 3 days, followed by growth factor (10 μg/L bFGF or 20 μg/L NGF) for another 3 days. Immunofluorescence was performed to detect the expression of nestin (a neural precursor cells marker), Tuj1 (a premature neuronal marker), and neurofilament (NF, a mature neuronal marker) at all stages of induced differentiation. Immunostaining and RT-PCR were used to analyze the expression of 26S proteasome during neuronal differentiation of hBMSCs. To further confirm the role of 26S proteasome in hBMSCs differentiation, cells were treated with β-ME/RA and then followed by protesome inhibitor MG132 and growth factor. Immunostaining was performed to detect NF-positive cells. RESULTS: Quantification results showed that the untreated cells were almost never positive for nestin, Tuj1 and NF. After treated with β-ME/RA, the numbers of nestin-positive cells (34.41%±1.27%) and Tuj1-positive cells (27.79%±1.27%) were increased. Notably, the numbers of NF-positive cells were significantly increased to 56.72%±2.4% after induction with β-ME/RA/GF. Immunofluorescence analysis showed that undifferentiated hBMSCs cells were weakly stained by antibody against 26S proteasome, but the numbers of cells with high-intensity of 26S proteasome were increased after treated with β-ME/RA. The RT-PCR result of 26S proteasome further confirmed that the mRNA level of the cells differentiated by β-ME/RA (1.33), as well as by β-ME/RA/GF (1.77), was significantly increased compared to the undifferentiated cells. Moreover, hBMSCs incubated with protesome inhibitor MG132 significantly decreased the numbers of NF-positive cells (37.59%±1.52%). CONCLUSION: After induction with β-ME/RA/GF, hBMSCs can be differentiated into neural-like cells, which is concomitant with the increase in 26S proteasome expression. Inhibitor of 26S protesome prevents hBMSCs differentiation, suggesting that 26S proteasome may be involved in the differentiation of hBMSCs into neural-like cells.
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g revealed nestin (+) or NF (+), and GFAP (-). It was concluded that hMSCs were successfully cultured and induced to differentiate into neuron-like cells.
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@#Objective To investigate the possibility that bone marrow mesenchymal stem cells (BMSCs) differentiated into neuron-like cells induced by ganglioside in vitro.Methods BMSCs were separated, cultured and differentiate into neuron-like cells induced by ganglioside. Neuro-specific enolase (NSE) and neurofilament (NF) on the surface of differentiated and induced BMSCs were detected by immunocytochemistry.Results BMSCs were induced to differentiate into the cells with a typical neuronal morphology. The induced neuron-like cells expressed NSE and NF.Conclusion BMSCs can be differentiated into neuron-like cells by induction of ganglioside in vitro.
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@#ObjectiveTo study the mechanism of differentiation of mesenchymal stem cells(MSCs) into neuron-like cells in vitro.MethodsMSCs of Wistar rats were separated and cultured, and then induced with DMSO and BHA in vitro. The specific marking proteins of neurons, glia and neural stem cells were detected before preinduction, at 24h after preinduction, at 6h, 24h, and 48h after neuronal induction.ResultsAfter the inducement, many MSCs turned into bipolar,multipolar and taper,and then intersected as network structure. Nestin was strong positive at 6h after neuronal induction, and decreased at 24h, 48h after the induction. NeuN was present at 6 h after neuronal induction, and increased at 24h, 48h after the induction.ConclusionMSCs can be induced into neural stem cells(NSCs) at first, and then differentiate into neuron-like cells in vitro.
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Objective:To culture mesenchymal progenitor cells(MPC)from compact bone fragments in C57 mice in vitro and to study the feasibility of inducing directed differentiation of MPC into neuron-like cells in vitro. Methods:Bones of hind limbs of C57 mice were sheared into bone fragments and digested by collagenase type Ⅱ. Then,MPC were cultured in vitro and analyzed by flow cytometry for identification of its immunology phenotype. MPC of P3 in good growth were induced directionally by the supernatant cultured with primary neuron,and then detected the expression of neuronal specific markers neuron specific enolase(NSE)and neurofilament protein(NF)by immunocytochemical staining. Results:The primary MPC were cultured successfully and they grewwell after passage. Compared with that of the control group,the positive rate of CD29 and CD44 of MPC of antibody group had significant difference (P
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Objective To explore the effects of Neurotrophin-3(NT-3) genetically modified Schwann cells(SCs) on grafted neural stem cell(NSC) differentiation into the neuron-like cells in the transected spinal cord. Methods The NSCs were single-transplanted or co-engrafted with NT-3 modified SCs,report gene LacZ modified SCs,and unmodified SCs respectively into the transected spinal cord 67d later,the differentiation of NSCs was studied by immunohistochemisty,and the percentage of neuron-like cells was calculated. Results NSCs could differentiate into the neuroglial-like cells and neuron-like cells in the injured spinal cord.Compared to the unmodified SCs,NT-3 modified SCs could more efficiently promote NSCs differentiate into the neuron-like cells.The effect of LacZ modified SCs was the same to the unmodified SCs.Conclusion NT-3 modified SCs could promote NSCs differentiate into the neuron-like cells in the injured spinal cord.
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Objective To explore the differentiation of human bone marrow measenchymal stem cells (hMSCs) into neuron-like and dopaminergic neuron-like cells in vitro.Methods The hMSCs were isolated from adult human bone marrow and expanded on the flask undifferentiated state for 2 passages. After pretreatment with WHI-P131 for 48 h, the hMSCs were cultured at the medium containing 10 ng/ml basic fibroblast growth factor for 24 h, then incubated with all-trans-retinoic acid and glial-derived neurotrophic factor in serum-free media for 5 h. The surface markers of differentiated neuron were detected by immunocytochemical method and the transdifferentiation process was observed under light microscope.Results Under induction conditions, hMSCs progressively resumed typical neuronal morphological characteristics. After hMSCs were incubated in induction medium for 5 h, the percentage of NSE, nestin, GFAP, TH and DAT positive cells were (77.0?5.7)%, (54.2?3.7)%,(8.8?2.4)%, (36.5?15.8)% and (26.0?14.2)%, respectively. There were no positive expressions in the control group.Conclusion The hMSCs are able to differentiate into neuron-like and dopaminergic neuron-like cells in vitro.
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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. [
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Objective To explore the potential of bone marrow mesenchymal stem cells(MSCs) to be modified by neurotrophin-3(NT-3) gene and pretreated with retinoic acid(RA) to differentiate into neuron-like cells in the transplanted site of the spinal cord injury. Methods MSCs,RA-induced MSCs,LacZ gene modified MSCs,NT-3 gene modified MSCs,and MSCs both modified by NT-3 gene and pretreated with RA were immediately transplanted respectively into the completely transected site(T_(10) spinal segment) of spinal cord.On the 67th day after the operation,the spinal cord segment was removed and frozenly sectioned.The differentiation potential of MSCs was examined by immunofluorescence histochemistry and the percentage was calculated of neuron-like cells that were differentiated from MSCs among all the transplanted cells groups. Results Transplanted MSCs could differentiate into neural stem cells(nestin-positive), neuroglial cells(GFAP-positive) and neuron-like cells(NF and MAP2-positive) in the injured spinal cord.Some of them also differentiated into the neuronlike cells which contained some neurotransmitters(ChAT and 5-HT positive) or had the potential to form a synapse(PSD95-positive).The percentage of neuron-like cells differentiated from MSCs modified by NT-3 gene and pretreated with RA was the highest among all the transplanted cell groups.Conclusion MSCs modified by NT-3 gene and pretreated with RA could better to differentiate into neuron-like cells in the injured spinal cord.