Preliminary study on induction of mouse embryonic stem cells into neural stem cells in vitro

AIM:To explore the feasibility of inducing mouse embryonic stem cells into neural stem cells in vitro. METHODS:Embryonic body induced by retinoic acid and retinal müller cells were selected in neural stem cell-defined medium for 7 days, and the morphological changes were observed. The selected cells were stained immunocytochemically with anti-nestin, anti-BrdU antibodies, and their ability of expansion and differentiation were analyzed. RESULTS:Large amounts of neurospheres were derived from embryonic body in the selected medium on the 7th day, which could be passaged and differentiated, stained positive with nestin and BrdU, and expressed nestin, glutaminase and Brn-3 genes. CONCLUSION:Neural stem cells could be derived from embryonic body induced by RA and müller cells in the selected medium, which would offer an alternative to treat neuropathy such as glaucoma and retinal degeneration in the future.     

Trichostatin A promotes mouse bone marrow mesenchymal stem cells to differentiate into insulin-secreting cells

AIM：To investigate whether trichostatin A (TSA), a new revulsant,can induce mouse mesenchymal stem cells to differentiate into insulin-secreting cells and to explore the appropriate concentration of TSA. METHODS：The mesenchymal stem cell line from C57BL/6 mice was cultured in vitro and divided into 5 groups before treated with different concentrations of TSA, (group A: DMSO; group B~E: treated with 25 nmol/L, 50 nmol/L, 100 nmol/L and 200 nmol/L of TSA, respectively). After exposed to different cultured media for 10 d during the 2 stages, the cells were detected by the following methods: the insulin-secreting cells in each group were identified by dithizone staining and the results were calculated with immunohistochemical half quantitative analysis. The insulin secreted by insulin-secreting cells in each group was identified by immunofluorescence, and the mean fluorescence intensity of insulin was compared. The content of insulin in each group was quantified by ELISA. The appropriate concentration of TSA was determined according to the above results. RESULTS：TSA treatment for 10 d promoted the mouse bone marrow mesenchymal stem cells to differentiate into insulin-secreting cells which produced insulin. The immunohistochemistry and immunofluorescence imaging analysis of insulin-secreting cells showed that the insulin staining positive area, positive ratio, total density of insulin expression and mean fluorescence intensity of insulin in group B were significantly higher than those in the other TSA-treated groups. When the concentrations of TSA gradually increased, the content of insulin reduced accordingly. The content of insulin in group B was significantly higher than that in the other TSA-treated groups. CONCLUSION：TSA treatment for 10 d promotes bone marrow mesenchymal stem cells from C57BL/6 mice to differentiate into insulin-secreting cells and the appropriate concentration of TSA is 25 nmol/L.     

Sox2 induce mouse embryonic fibroblasts direct reprogramming into neural stem cells

AIM: To study the direct reprogramming method of mouse embryonic fibroblasts (MEFs) converted into induced neural stem cells (iNSCs). METHODS: Sox2-infected MEFs were cultured in NSCs culture medium for 10 d. Subsequently, repeated suspension and adherent culture were performed for 3 times for the purification of iNSCs. The iNSCs were cultured in suspension medium. Real-time PCR was used to detect the expression of neural stem cell marker genes and pluripotent marker gene. In vivo, iNSCs were microinjected into the mouse cerebral cortex. Immunofluorescence was performed to detect the expression of neural stem cell, neuron, oligodendrocyte and astrocyte markers in vitro and vivo. RESULTS: A variety of neural stem cell marker gene expression was significantly increased in iNSCs detected by real-time PCR. Immunofluorescence confirmed that iNSCs expressed nestin and differentiated into neurons, oligodendrocytes and astrocytes in vitro and vivo. CONCLUSION: Sox2 is sufficient to trigger the direct reprogramming from MEFs to iNSCs. iNSCs have the ability of self-renew and 3 differentiation potentials in vivo and vitro. iNSCs are the suitable seed cells of SCI.     

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

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

Sodium butyrate induces mouse embryonic stem cells to differentiate into hepatocytes in vitro

AIM:To explore mechanism by which sodium butyrate induces mouse embryonic stem cells (ES) to differentiate into hepatocytes in vitro. METHODS:E14 mouse ES cells were cultivated in a routine way, and then cultivated in suspension to form embryonic bodies (EBs). EBs were transferred into 6-well culture dishes and 3 mmol/L sodium butyrate was added into the culture medium. Morphological changes were investigated by phase contrast microscopy. α-fetoprotein (AFP), albumin (ALB) and cytokeratin 18 (CK18) were examined by immunofluorescence staining. AFP, ALB, α1-antitrypsin (AAT) and TTR mRNA were assayed by RT-PCR. Proportion of ALB positive cells was analyzed by flow cytometry. Periodic acid Schiff (PAS) reaction and indocyanine green (ICG) uptake assay were performed to assess the characteristic hepatocyte function of the differentiated cells. RESULTS:In the presence of sodium butyrate, parts of ES cells differentiated into a population with epithelial morphology similar to mouse hepatocytes. AFP and TTR mRNA expression were observed at 7 d, and ALB and AAT mRNA expressed at 14 d. Hepatocytes specific markers, ALB, AFP and CK18 were positive expression in immunofluorescence staining at 14 d. PAS reaction and ICG uptake were positive for the hepatocyte-like cells. CONCLUSION:Mouse ES cells can be induced into hepatocyte-like cells by sodium butyrate efficiently, and these ES cells-derived hepatocytes possess characteristic hepatocytic function.     

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.     

miRNA-122 promotes mouse embryonic stem cell-derived hepatic precursor cells to differentiate into hepatocytes

AIM：To investigate whether miRNA-122 (miR-122) promotes the differentiation of mouse embryonic stem cell (ESC)-derived hepatic precursor cells (HPCs) into hepatocytes. METHODS：Mouse ESCs were initially induced to differentiate into HPCs by stimulating with fibroblast growth factor 4 (FGF-4), sodium butyrate and dexamethasone (Dex) sequentially. Then a recombinant adenovirus expressing vector pAV.Ex1d-CMV>miR-122/IRES/eGFP was constructed by Gateway technology and transfected into the mouse ESC-derived HPCs 9 d after induction, so as to gain the cells with stable high expression of miR-122. The morphological changes of transfected cells were observed under inverted phase-contrast microscope. The liver-specific gene expression levels were detected by real-time RT-PCR. The liver-specific protein expression levels were also detected by immunofluorescence method. The liver functions were assessed by indocyanine green (ICG) uptake experiment, glycogen staining and urea synthesis function test. RESULTS：The mouse ESCs were successfully induced into HPCs by stimulating with FGF-4, sodium butyrate and Dex sequentially. At 6 d after transfection of miR-122, the morphology of the cells was closer to the mature hepatocytes. The mRNA levels of liver-specific genes such as albumin (ALB), transthyretion, α1-antitrypsin, glucose-6-phosphatase, cytokeration 8, cholesterol 7α-hydroxylase and cytochrome P450 3A4 were up-regulated. The expression levels of liver-specific proteins such as ALB and cytokeratin 18 were increased, while alpha-fetoprotein was decreased. The results of ICG uptake experiment, glycogen staining and urea synthesis function test indicated that the hepatocyte functions were strengthened as compared with control group. CONCLUSION：The combination of FGF-4, sodium butyrate and Dex successfully induces the mouse ESCs into HPCs. Over-expression of miR-122 effectively promotes the differentiation and maturation of mouse HPCs.     

Embryonic tissue extracts modulate cell-fate determination in human epidermal stem cells

AIM: To investigate the phenotypic changes and cell-fate determination of keratinocyte stem cells in vitro. METHODS: Improved collagen Ⅳ-coated adhesion method was used to isolate and culture the keratinocyte stem cells after neutron-protease selectively digested the dermo-epidermal junctions. Morphological features and identification of these immature cells were studied by immunochemistry and confocal microscopy analysis. After treated with extracts from 14-day-old mouse fetuses at the concentration of 10% (W/V), phenotypic changes and the cell-fate determination of keratinocyte stem cells (KSCs) were detected by flow FACS analysis and RT-PCR assays. RESULTS: Immunocytochemical analysis proved that KSCs and their subunits were positive for α₆ and β₁ integrin, keratin 19 and 14, p63, nestin and PCNA, yet negative for keratin 10 expressions. Double staining immunofluorescence demonstrated that markers such as α₆ integrin and CD71 was co-expressed in KSCs, and there were important regional differences in the distribution of α6 integrin and CD71 in KSCs and transit amplifying cells (TA cells). Meanwhile, two-color flow cytometric analysis of α₆ integrin and CD71 consistently revealed that after treated with an extract from mouse fetuses, the percent of α+6 CD71+ and CD71+ fraction increased and reached to 68.43% and 4.51% of the total isolated cells, respectively. However, the percent of α+6 CD71- fraction reduced from 22.49% to 10.92% determined by flow cytometry. Moreover, the expression levels of β₁ integrin and some putative biological markers in human epidermal cells were analyses by RT-PCR after fetus extract treatment. The relative gene expressions of β₁ integrin, K19 and K10 increased in KSCs, whereas the expression of K14 in keratinocyte stem cells was observed to be significantly suppressed when compared to the appropriate controls. CONCLUSION: The keratinocyte stem cells isolated by collagen Ⅳ-coated adhesion method have some characteristics of adult stem cells and perhaps the potentiality to differentiate and cross-differentiate. The regional differences in the distribution of α6 integrin and CD71 are one of the transition markers to discriminate the KSCs and TA cells. Furthermore, fetus extracts contribute to not only the proliferation and self-renewal of KSCs but also the dedifferentiation of TA cell subunits.     

A method of inducing mouse embryonic stem cells cultured in vitro to differentiate into cardiomyocytes

AIM:To evaluate the method of inducing mouse embryonic stem (ES) cells in vitro to differentiate into cardiomyocytes without using any chemical reagents.METHODS:BRL conditioned medium was used to promote the growth of ES cells and maintain them in an undifferentiated state before the experiment of differentiation. Then a three-step method including ES cell culture in hanging drops and in suspending was used to induce the differentiation of ES cells. RESULTS:Rhythmically contracting cells were observed among differentiated cells, which were proved to be cardiomyocytes with electron microscope and immunocytochemistry. CONCLUSION:A simple and economical method was established to induce mouse ES cells cultured in vitro to differentiate into cardiomyocytes without using any chemical reagents.     

Comparison of the effects on differentiation of mouse embryonic stem cells into insulin-secreting cells among three cell culture protocols

AIM：To compare the effects of three different cell culture protocols：embryonic body (EB) formation，EB formation-monolayer and monolayer on differentiation of mouse embryonic stem (ES) cells into insulin-secreting cells.METHODS：E14.1 mouse ES cells were treated with GLP-1,betacellulin，activin A,bFGF and nicotinamide by using EB formation,EB formation-monolayer and monolayer culture protocol respectively for 30 days,then insulin expression was examined by RT-PCR,DTZ-staining and immunohistochemistry.The percentage of insulin-secreting cells was evaluated by flow cytometry.RESULTS：DTZ-staining positive cells and insulin immunohistochemical staining positive cells were observed in the differentiated cells for all the three groups.mRNAs of insulin and some other islet-related genes were detected,insulin expression was the strongest in EB formation-monolayer,and the weakest was in monolayer.The percentage of insulin-positive cells of the differentiated cells in the EB formation-monolayer group was higher than that in the EB formation group (P＜0.01),the latter was higher than that in the monolayer group (P＜0.01).CONCLUSION：Among the three cell culture protocols,EB formation-monolayer is the most effective approach in the induction of mouse ES cells to differentiate into insulin-secreting cells.     

Human mesenchymal stem cells differentiate into osteoblasts

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

Orthotopic transplant fetal rat pancreatic stem cells for treating diabetes mellitus

AIM: To observe the possibility of differentiation of fetal rat pancreatic stem cells into islet-like cell cluster by transplantation of fetal rat pancreatic stem cells into pancreatic parenchyma in diabetic SD rats. METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16. SRY DNA was examined to discriminate gender by fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry. Adult SD rats were divided into three groups including 10 pancreatic parenchymal orthotopic transplantation, 10 experimental controls and 10 normal controls. In orthotopic transplantation group, 1×10⁶ male fetal pancreatic stem cells per rat were injected into diabetic rat pancreatic parenchyma while in experimental control group equivalent volume of PBS was injected into diabetic rat pancreatic parenchyma. Glucose and insulin level in serum were monitored periodically. 8 weeks after transplantation pancreata were excised for histological and morphometric analysis. SRY DNA was detected by FISH. Nestin, PDX-1 and insulin mRNA expression in pancreata were detected by RT-PCR, insulin and PDX-1 protein contents were assessed by Western blotting. RESULTS: 5 of 12 fetal rats were male according to FISH. After passaged 3 generations, the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry with 74.1% of PSCs expressed nestin. The orthotopic transplantation of PSCs led to stable reduction in hyperglycemia and increase in insulin level in serum (3 weeks after transplantation), culminating (5 weeks post-transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse. Exogenous islet-like cell clusters were found and expressed SRY DNA in the orthotopic transplanted recipients pancreata 8 weeks post-transplantation. The expression levels of insulin mRNA and protein in the orthotopic transplanted recipients pancreata were higher than those in experimental control (P<0.05), and the expressions of PDX-1 mRNA and protein were also higher than those in normal control (P<0.05). CONCLUSION: When orthotopic transplant into pancreatic parenchyma PSCs from fetal rat differentiates into islet-like cell cluster, gains comparable function with normal islets and reverses experimental diabetes.     

In vitro differentiation of exocrine pancreatic cells from mouse embryonic stem cells

AIM: To explore the effects of sodium butyrate, activin A and dexamthasone on inducing mouse embryonic stem (ES) cells to differentiate into exocrine pancreatic cells in vitro. METHODS: E14 mouse ES cells were cultured in suspension to form embryonic bodies (EBs). The EBs were cultured with differentiating medium containing different concentrations of sodium butyrate, and the spontaneously differentiated ES cells were used as control. Exocrine pancreatic genes such as amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected by RT-PCR at different time points to determine the optimal concentration and exposure time of sodium butyrate. Furthermore, activin A or dexamthasone was also used to explore the effects on exocrine differentiation. After that, the combination of sodium butyrate, activin A and dexamthasone was used to promote the differentiation of exocrine pancreatic cells from ES cells. During the differentiation course, the gene expressions of amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected by RT-PCR. Morphological changes were investigated by phase contrast microscopy. Amylase expression was examined by immunofluorescence staining. RESULTS: Exocrine pancreatic gene expressions such as amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected in spontaneously differentiated EBs. A relatively lower concentration of sodium butyrate with a shorter exposure time significantly promoted those above gene expressions as compared to that of spontaneously differentiated EBs. Activin A and dexamethasone induced upregulation of exocrine gene expression. The combination of activin A, sodium butyrate and dexamethasone significantly enhanced the mRNA levels of amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase. Under the treatment of activin A, sodium butyrate and dexamethasone, differentiated cells were polygonal in shape with large, round, and center-situated nuclei. According to the observation of immunofluorescence staining, amylase was positive expressed at the final stage. CONCLUSION: These data indicate that exocrine pancreatic differentiation of ES cells is induced by sodium butyrate, activin A and dexamethasone. The combination of pancreatic inducing factors improves the differentiating efficiency.     

Supportive effects of human aorta-gonad-mesonephero stromal cells on the directed differentiation of embryonic stem cells into hematopoietic stem cells

AIM: To direct embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) in vitro by simulating the hematogenic microenvironment in human early embryonic aorta-gonad-mesonephero (AGM) region.METHODS: Murine E14 embronic stem cell line was used for two-step differentiation.In the first step of primary differentiation,E14 ESCs were seeded into semisolid methylcellulose-based medium containing bone morphogenesis protein 4 (BMP4) and vascular endothelial growth factor (VEGF) for embryoid body (EB) formation.On days 3,6,9,12 and 15,single EB cells were analyzed for Flk-1+ cells amount through flow cytometry.In the second step,single cell from EB containing most Flk-1+ cells was further co-cultured with human AGM stromal cells in non-contact system.On co-culture days of 3,6,9 and 12 days,cells were collected for cell count,flow cytometry for Sca-1+c-kit+ cells analysis,and colony forming cell assay.RESULTS: During the EB formation,BMP4+VEGF promoted Flk-1+ cell genesis on day 9 at peak pencentage value of 27.53%±2.84％,which was statistically higher than that in control group as 8.77±1.10 (P<0.05).Collagenase-disassociated single cell from day 9 EB was co-cultured with human AGM stromal cells of hAGMS3 or hAGMS4 for further hematopoietic differentiation.On day 6 Sca-1+c-kit+ cells got to peak value as 7.31%±1.21％ ［(2.57±0.48) folds］ and 7.62%±1.52％ ［(2.35±0.36) folds］ in hAGMS3 and hAGMS4 feeder systems,respectively,both of which were greater than those values of no-stroma groups at the same culture duration (P<0.05).Colonogenic cell assay showed that these Sca-1+c-kit+ cells had ability of forming multiple lineage hematopoietic colonies.CONCLUSION: BMP4 in combination with VEGF promotes Flk-1+ cell genesis during EB formation in vitro.Stromal cells from early human embryonic AGM region further enhance the directed differentiation of these primitive cells into HSCs.This two-step induction differentiation model can be used for molecular mechanism study of ESCs hematopoietic differentiation.     

Differentiation of bone marrow mesenchymal stem cells into chondrocytes

AIM: To investigate the differentiation of human bone marrow mesenchymal stem cells (MSC) into chondrocytes in vitro and determine factors involving in the differentiation process. METHODS: MSC were separated from iliac bone marrow with lymphocyte separating medium using density centrifugation. Cells were cultured and expanded in medium until reaching required number. MSC was induced to differentiate into chondrocytes by adopting high cell density, supplying growth factor and using micromass culture. Cells were observed by HE staining. Matrix of cartilage was detected by alcian blue and toludine blue and cartilage specific collagen II was detected by immunohistochemistry. RESULTS: The structure of the micromass assumed that of cellular cartilage, alcian blue staining were uniformly positive and toludine blue detected diffuse metachromasia substance, cells uniformly expressed collagen Ⅱ. CONCLUSION: High cell density, growth factor and appropriate culture conditions are critical to induce differentiation of MSC into chondrocytes.     

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.     

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.     

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.     

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.     

Expression and function of calcium-sensing receptor in mouse embryonic stem cells

AIM: To observe the functional expression of calcium-sensing receptor (CaSR) in the mouse embryonic stem cells (mESCs). METHODS: The expression and distribution of CaSR were detected by Western blotting and immunofluorescence observation in 129 mouse ES-D3 cells. The intracellular concentration of free calcium (［Ca2+］i) was determined by confocal laser scanning microscopy. The cell viability was analyzed by MTT assay and flow cytometry. RESULTS: CaSR protein was expressed in mESCs. Extracellular calcium or neomycin significantly increased the expression of CaSR and ［Ca2+］i. Neomycin increased the cell viability, up-regulated the protein expression of p-ERK2. These effects of neomycin were inhibited by NPS2390. CONCLUSION: CaSR is expressed in mESCs. The activation of CaSR is involved in the proliferation of mESCs.