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1.
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.  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
AIM: To explore the effects of metanephric cell microenvironment on inducing embryonic stem cells (ESCs) to differentiate toward renal cells.METHODS: Embryoid bodies (EBs) of D3 mouse embryonic stem cells were prepared by hanging drop culture, and the EBs were co-cultured indirectly with metanephric cells derived from E12.5 d mouse embryo. The EBs cell with spontaneous differentiation was used as the control. The proteins of Pax2 and WT-1 were analyzed by immunofluorescence assay. The mRNA expression of Pax2, WT-1, Lim1, Sall1, Emx2, GDNF, Wnt4, BMP7, Nephl, Nephrin, KSP and CD24 genes was detected by RT- PCR.RESULTS: The genes related to kidney development were expressed in the EBs cells after co-culture on day 3, and the mRNA expression of Pax2, WT-1, Emx2, GDNF, Nephl, Nephrin, KSP and CD24 was stronger than those in control group. Pax2 positive cells were found on day 3 in the co-cultured EBs cells, and the positive cells increased on day 5 and day 7. WT-1 protein positive cells were found in the co-cultured EBs cells on day 5. No Pax2 or WT-1 positive cell was observed in control group.CONCLUSION: Metanephric cell microenvironment promotes ESCs differentiation toward renal cells.  相似文献   

5.
AIM: To evaluate the different conditions inducing mouse embryonic stem cells (ESC) in vitro to differentiate into cardiomyocytes. METHODS: BRL conditioned medium was used to promote the growth of ESC and maintain them in an undifferentiated state. During the inducing process, retinoic acid (RA), DMSO, activin-A and TGF-β1 were used as inducing reagents, and made up six kinds of differentiating medium. Then a three-step method inducing ESC cultured in hanging drops, in suspension and in plating was used to induce the differentiation of ESC. RESULTS: ESC were induced in vitro to differentiate into cardiomyocytes. Of all groups, the highest differentiating rate was observed in the group induced by activin-A (20 μg/L) and TGF-β1 (2 μg/L). CONCLUSION: The inducing conditions including activin-A (20 μg/L) and TGF-β1 (2 μg/L) is very valuable in inducing ESC differentiation into cardiomyocytes.  相似文献   

6.
AIM:To analyze genome-wide DNA differential methylation site-related genes and genomic diffe-rential methylation levels involved in embryonic mouse cleft palate formation. METHODS:MethylRAD-Seq was performed on the gestational day 13.5 (GD13.5), GD14.5 and GD16.5 in experimental group and control group, which is a method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes. A comparison was made to process the different location of the methylation site and the methylation level differential genes among genome-wide different functional components (3'-untranslated region, 5'-untranslated region, TSS2000, intron, exon, and intergenic region). The genome-wide DNA differential methylation site-related genes and methylation differential genes were analyzed using GO function enrichment and KEGG pathway enrichment. RESULTS:(1) The peak number and peak value of methylation sites in different components of DNA did not change significantly in the control group, and the methylation level of the experimental group decreased gradually over time. (2) GO function enrichment analysis and KEGG pathway enrichment ana-lysis were performed to process methylation level differential genes and genome-wide DNA differential methylation site-rela-ted genes, among which there were 6 genes (Fgf16, Jarid2, Kdm6a, Nr3c1, Tbx22 and Trp53) involved in embryonic mouse cleft palate formation. (3) MAPK, Wnt, TGF-β and other signaling pathways were found to participate in the formation of cleft palate. CONCLUSION:DNA methylation plays an important regulatory role in the process of cleft palate formation.  相似文献   

7.
AIM: To investigate the differentiation of murine embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) by the supportive effects of human aorta-gonad-mesonephros (AGM) region and fetal liver (FL) stromal cells.METHODS: E14 ESCs were induced into embryoid body (EB) first. Then the cells from EB were further co-cultured with human AGM region and FL stromal cells in non-contact system. On day 6, the cells derived from EB were collected for Sca-1+c-Kit+ cell analysis by flow cytometry, colony forming unit (CFU) assay and teratoma formation checking. BALB/c female mice conditioned with lethal dose of γ-ray irradiation were transplanted with EB cells from different culture systems. The survival rates, engraftment of donor cells, reconstitution of hematopoietic were monitored.RESULTS: Sca-1+c-Kit+ cells in EB cells co-cultured with human AGM region and FL stromal cells had the value of (21.96±2.54)%, and the total CFU was as (520±52)/105 cells, which were statistically greater than those in EB cells only cultured with human AGM region stromal cells (P<0.05). No teratoma was found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region and FL stromal cells. In BALB/c female mice transplanted of EB cells co-cultured with human AGM region and FL stromal cells, the survival rate was 77.8%, and the peripheral blood cell count was obviously improved on day 14. PCR results showed the recipients all had sry gene copies from donor in bone marrow. The recipient mice transplanted with EB cells only cultured with human AGM region stromal cells all died within 15 days.CONCLUSION: Stromal cells from human AGM region and FL enhance the directed differentiation of ESCs into HSCs which can reconstruct hematopoiesis in vivo.  相似文献   

8.
AIM: To investigate the mechanism by which human amnion induced mouse embryonic stem (ES) cells to differentiate into epidermal like cells. METHODS: ES- BALB/ c cells were cocultured with human amnionintranswells for 4 - 5 days , andthose cultured alone without amnion were taken as control group. The morphological differentiation were observed . The committed differentiation of EScells into epidermal like cells were detected by integrin-β1 , CK19 , CK15 andinvolucrin immunohistochemistry , respectively .RESULTS: After 4-5 days of coculture, ES cells differentiated into single layer of epidermal like cells, fitted tightly, with polyhedral in shape. The immunohistochemical staining results showed that, most of the cells were integin-β1 positive, only a few cells were CK19 and CK15 positively stained. Most of the cells in control group died, the survived ones were different in morphological shapes, and no integrin-β1, CK19 and CK15 positive cells were found. CONCLUSION: Soluble substances secreted by human amnion may play an important role in inducing the differentiation of mouse ES cells into epidermal like cells.  相似文献   

9.
AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.  相似文献   

10.
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12.
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.  相似文献   

13.
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.  相似文献   

14.
AIM: To study the proliferation, differentiation and the capacity of forming teratomas of ESC-derived hepatic stem cells in mouse pre-treated with retrorsine and 70% partial hepatotomy. METHODS: The ESC-derived hepatic stem cells, labelled with CFDA SE, were transplanted into BALB/c mouse liver. The distribution, incorperation and proliferation of transplanted cells were observed under fluorescent microscopy. Hepatic function was assayed by detecting albumin level in serum. The situation of forming teratomas in vivo was also evaluated.RESULTS: 1 week post-transplantation, some scattered region was green under fluorescent microscopy. The aera of green region increased apparently in 2 weeks, and cord-like structure was observed. Immunofluorescent staining of albumin demonstrated some positve cells, but there was no significant difference for albumin level in serum (P>0.05). No teratoma was formed in the experimental group, while a large teratoma was observed in control group in 6 weeks post-transplantation.CONCLUSION: The ESC-derived hepatic stem cells are normally incorporated into mouse liver parenchymal structure, proliferate and differentiate further in vivo and possess some hepatic functions without forming teratomas.  相似文献   

15.
Pluripotent stem cells are characterized by the properties of self-renewal and the ability to differentiate into multiple cell types. Reactive oxygen species (ROS) are highly reactive metabolites. High levels of ROS are toxic and involved in stem cell senescence and apoptosis. However, regulation of ROS has an important role in maintaining “stemness” and differentiation of the stem cells. The role of ROS in the stem cells varies among different stem cell types. NADPH oxidase is one of the major sources of ROS in stem cells. Excessive amounts of ROS are produced in various pathophysiological states such as atherosclerosis, heart failure, hypertension, diabetes, and aging. Induced pluripotent stem cells have the potential to be used in modeling of ROS-associated diseases.Understanding the molecular mechanisms how ROS regulate the functions of stem cells will greatly enhance their translational applications. In this review, we summarize the recent progress regarding the roles of ROS in regulating the functions of embryonic and induced pluripotent stem cells.  相似文献   

16.
AIM:To examine DNA methylation at CpG sites in the promoter region of tumor necrosis factor-alpha (TNF-α) gene in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMC).
METHODS:DNA methylation in the promoter region of TNF-α gene was measured by bisulfite sequencing PCR.
RESULTS:The promoter region of TNF-α gene was from -294 bp to +58 bp, including 11 CpG sites. The PCR products identified by aga-rose gel electrophoresis were consistent with the theoretical size. Two sites were methylated at 0 h and 6 h and 6 sites were methylated at 12 h in TNF-α gene promoter region in DENV2-infected PBMC. The average methylation rates were 103%, 121% and 255% at 0 h, 6 h and 12 h, respectively. Significant differences between 0 h and 12 h and between 6 h and 12 h were observed.
CONCLUSION:The DNA methylation in the promoter region of TNF-α gene is increased in DENV2-infected PBMCs.  相似文献   

17.
LI Ping  ZHONG Xue-yun 《园艺学报》2009,25(8):1652-1655
The tumor stem cell theory supposed that tumor stem cells are the origin of tumor abnormal proliferation, invasion, metastasis, drug resistance and recurrence. As the theory is put forward, it redefines the functions of classic stem cells in tumorigenesis. It is a great event for recent studies on glioma initiating cells as brain tumor stem cells were identified and isolated successfully. A lot of evidence from experiments in vivo and in vitro demonstrates that brain tumor stem cells might play an important role in glioma tumorigenesis. In this review, we discuss the relationship between tumor stem cells and tumorigenesis, and the research on the correlation between brain tumor stem cells and glioma genesis.  相似文献   

18.
AIM:To induce lymphoid stem cells and/or T-cell precursors to differentiate into functional mature T lymphocyte, and to increase the surface marker of T lymphocytes such as CD3+, while embryonic stem(ES) cells differentiated into hematopoietic stem/progenitor cells(HSPCs) in vitro. When they were injected into lethally irradiated mice, these differentiated cells had the advantage in immune reconstitution. METHODS:Embryonic stem cells formed embryoid bodies(EBs) in the medium containing methycellulose, hematopoietic growth factors(HGFs) was added to the culture system on the 6th day, thymopeptide was added at the same time. Flow cytometry were performed to detect the surface marker CD34+ and CD3+ of the differentiated cells. Finally the differentiated cells were injected into lethally irradiated mice, 60 days later, the incidence rate of graft versus host disease(GVHD) was taken as the mark of cell mediated immunity, PCR was performed to detect the sex determining region of the Y-chromosome(Sry) in bone marrow cells and spleen cells of the survival host female mice. RESULTS:The percentage of CD3+ T lymphocytes was 10.52% and the incidence rate of GVHD was 0% on the 13th day, but they respectively rose up to 22.93% and 100% if thymopeptide was added in the procedure of inducing ES cells to differentiate into HSPC in vitro. CONCLUSION:The quantity of CD3+ T lymphocytes increased in medium containing thymopeptide when ES cells differentiated into CD34+ HSPC.  相似文献   

19.
AIM To isolate and identify quiescent and activated neural stem cells from mouse embryonic cerebral cortex. METHODS Two cell clusters derived from mouse cerebral cortex on embryonic day 14.5 were separated by flow cytometry. The expression of stem cell marker Pax6 and proliferation marker Ki67 was examined by immunofluorescence. The mRNA expression of stem cell marker genes Pax6, Oct4, Sox2 and Nanog were detected by RT-qPCR. Cell cycle was analyzed by flow cytometry and RT-qPCR. Proliferation ability was investigated by in vitro cell culture. RESULTS In both 2 groups, the cells expressed Pax6. Immunofluorescence staining of Ki67 in the big cell group was positive, while that in small cell group was negative. Cell cycle assay showed that the proportion of G0/G1 phase in the small cells was higher than that in the big cells, the G2/M phase proportion was 0, and the expression of cyclin A and cyclin B was lower than that in the big cells (P<0.05). When cultured in vitro, the number of microspheres formed by the small cells was smaller and the formation speed was slower than those of the big cells. After digestion of microspheres, Pax6 and Ki67 staining of both big and small cells was positive, and the positive rates were not different (P>0.05), indicating that the quiescent neural stem cells were activated. CONCLUSION The 2 cell clusters are quiescent and activated state of neural stem cells. The activated stem cells have strong abilities of self-renewing and proliferation, while these abilities of quiescent stem cells are poor. The quiescent stem cells can translate into activated ones when cultured in vitro for a period.  相似文献   

20.
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.  相似文献   

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