Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

AIM To construct the mouse embryonic stem cell （ESC） line with stable pancreatic and duodenal homeobox 1 （Pdx1） expression by Tet-On system, which may lay a foundation for further research on the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells. METHODS The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC. The cells were divided into 3 groups： blank control group （ESC group）, empty lentivirus control group （PDX1^- ESC group） and Pdx1 lentivirus transfection group （PDX1⁺ ESC group）. Flow cytometry was used to detect the transfected cells after screening by doxycycline （DOX）. The function of Tet-On system and the expression of Pdx1 gene were detected. The transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were sorted by flow cytometry, and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified. RESULTS （1） The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were 90.72% and 94.01% after screening by DOX, respectively. The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group was 97.84% and 98.13% after sorting by flow cytometry, respectively. （2） With DOX, green fluorescence was observed in PDX1^- ESC group and PDX1⁺ ESC group. The mRNA and protein expression of Pdx1 was significantly increased in PDX1⁺ ESC group （P<0.05）. Without DOX, no green fluorescence was observed in the cells of the 3 groups, and no significant difference in the mRNA and protein expression of Pdx1 was observed （P>0.05）. （3） After 3 months of cryopreservation, the cell lines still survived in resuscitation culture and were regulated by DOX. CONCLUSION Using Tet-On system, the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells.     

Functional hepatic differentiation from mouse induced pluripotent stem cells in vitro

AIM: To directly differentiate the induced pluripotent stem cells (IPS cells) into hepatocytes in chemically-defined, monolayer conditions in vitro. METHODS: Based on the knowledge of mammalian hepatic development, we differentiated the IPS cells into hepatic lineage efficiently. The present study involves stepwise transition of IPS cells through a series of media designs to first specify cells towards anterior definitive endoderm, and secondly, to induce hepatic maturation. RESULTS: The IPS cells recapitulated the development of liver in vivo at RNA and protein level. Real-time PCR analysis was performed on cell preparations at various times during the culture process to determine the time course and degree to which the IPS cells differentiated toward a hepatocyte phenotype. Analysis included markers for endoderm-specific gene expression (Sox17; FoxA2, CXCR4), hepatocyte-specific gene expression (albumin and AFP), and markers for undifferentiated IPS cells (OCT4 and Nanog). The endoderm-specific gene expression increased early while the expression of OCT4 and Nanog decreased and hepatocyte-specific gene expression progressively increased over the course of the differentiation program. The resultant hepatocyte-like cells(HLCs) showed phenotypic similarities with adult hepatocytes (expressing AFP and albumin) and additionally showed functionality expected to well-differentiated hepatocytes such as indocyanine green(ICG) uptake and release. The P450 activity of cytochromsome was also positive. CONCLUSION: We successfully construct the IPS cells which efficiently differentiate into hepatocytes and recapitulate the hepatocytic development in vivo. The reproducible and efficient generation of HLCs from IPS cells represents a critical step towards the ultimate goal of producing functional HLCs for clinical use including cell transplantation or bioartificial liver support. Moreover, our system provide a valid way to study the liver development since it is monolayer, serum-free and mimic the in vivo liver development.     

Comparison of different conditions inducing embryonic stem cells

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.     

EP2A promotes human CD34+ cell homing in vitro but not proliferation

AIM: To investigate the role of prostaglandin E₂ receptor 2 agonist (EP₂A) in proliferation and homing of human CD34⁺ cells. METHODS: Bone marrow fluid and peripheral blood containing stem cells were collected from healthy donors mobilized by granulocyte colony-stimulating factor in our department. Human CD34⁺ cells were isolated by the method of magnetic-activated cell sorting microbeads. Bone marrow mononuclear cells were isolated by Ficoll-Paque centrifugation, and the bone marrow mesenchymal stem cells (BMMSC) were cultured with L-DMEM. Human CD34⁺ cells and BMMSC were divided into 4 groups, and treated with PGE₂ (as positive control), DMSO (as negative control), EP₂A and EP₂A+prostaglandin E₂ receptor 2 antagonist (EP₂AA), respectively. After exposed to the reagents, human CD34⁺ cell viability was measured by CCK-8 assay, the number of colonies was evaluated by colony-formation assay, the cell cycle distribution was analyzed by flow cytometry, and the protein expression of survivin, β-catenin and CXC chemokine receptor 4 (CXCR4) was detrmined by Western blot. Moreover, the concentration of stromal cell-derived factor-1α (SDF-1α) in the BMMSC was detected by ELISA. RESULTS: The cell viability and the colony number of human CD34⁺ cells in EP₂A group were not higher than those in negative control group. Furthermore, the proportion of human CD34⁺ cells treated with EP₂A in G₂/M phase was not elevated compared with negative control group. The protein expression of survivin and β-catenin did not up-regulated in human CD34⁺ cells exposed to EP₂A, but the protein expression of CXCR4 in human CD34⁺ cells and the concentration of SDF-1α in BMMSC were elevated. CONCLUSION: EP₂A promotes human CD34⁺ cell homing in vitro but not proliferation.     

Adipose-derived stem cells mediate immunosuppression of Th17 in multiple sclerosis

AIM: To investigate how human adipose-derived stem cells (hASCs) regulates the differentiation of Th17 cells in multiple sclerosis. METHODS: hASCs were isolated from the adipose tissues. Magnetic-activated cell sorting (MACS) kit was used to isolate CD4⁺ T cells from peripheral blood mononuclear cells (PBMCs) which were isolated by density gradient centrifugation. The percentage of CD4⁺ T cells was detected by flow cytometry. The activated CD4⁺ T cells were co-cultured with hASCs for about 4 d at different ratios of hASCs to CD4⁺ T cells (1:4 and 1:10) in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor (LIF) antibody was set up. Th17 cell proportion of the CD4⁺ T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt (RORγt), interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), LIF and leukemia inhibitory factor receptor (LIFR) was detected by real-time PCR. RESULTS: The result of flow cytometry suggested there were mainly hASCs, and the percentage of CD4⁺ T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1:4 and 1:10 co-cultured groups in a dose-dependent manner. The mRNA expression of IL-6R, IL-23R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system, the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added, the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION: hASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF/IL-6 by secreting LIF.     

Conditionally replicating adenovirus activated by CXCR4 promoter in lung cancer

AIM: To construct a conditionally replicating adenovirus vector activated by CXCR4 promoter and to evaluate its ability of lysing the lung cancer cells specifically. METHODS: Human CXCR4-E1A gene amplified by PCR was cloned into the shuttle plasmid pDC316-GFP to construct the recombinant shuttle plasmid pDC316-CXCR4-GFP. The recombinat shuttle plasmid and adenovirus genomic plasmid pBHG-lox-E1, 3Cre were transfected into 293 cells to construct the recombinant adenovirus CRAd-CXCR4-GFP. PCR was used to detect the target gene fragments, and the viral titer was determined. A549 cells with the highest mRNA expression of CXCR4 were screened out from 5 kinds of lung cancer cell lines by real-time PCR. CXCR4 promoter activity and adenovirus replication numbers were detected in A549 cells after transfection of CRAd-CXCR4-GFP and Ad-NULL. CRAd-CXCR4-GFP and Ad-NULL were transfected into A549 cells and 16HBE cells, the apoptotic rates were detected by flow cytometry and the viability was analyzed by CCK-8 assay. RESULTS: The recombinant plasmid pDC316-CXCR4-GFP was constructed successfully. Green fluorescence was observed in 293 cells under fluorescent microscope after co-transfection of pDC316-CXCR4-GFP and pBHG-lox-E1, 3Cre at 11 d. Green fluorescence was observed in 293 cells after infection of amplified 3rd generational adenovirus. PCR showed that the purpose gene was successfully integrated in recombinant adenovirus genome. The virus in the supernatant reached a titer of 1×10¹³ PFU/L. The mRNA expression of E1A and E4 in the A549 cells after transfection of CRAd-CXCR4-GFP was markedly increased compared with Ad-NULL group. Compared with Ad-NULL group and empty control group, the apoptotic rate and the viability of A549 cells in CRAd-CXCR4-GFP group had no significant difference in the first 4 d, the apoptotic rate increased significantly at 5 d, and the cell viability declined significantly at 5 d, but the apoptotic rate and the viability of 16HBE cells in each group had no significant difference within 5 days. CONCLUSION: The conditionally replicating adenovirus vector CRAd-CXCR4-GFP has been successfully constructed, which has the ability of lysing lung cancer cells specifically.     

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.     

IL-1β promotes differentiation of naïve T cells into Th22 cells and its expression in non-small cell lung cancer

AIM:To investigate the mechanism of interleukin-1β (IL-1β) promoting the transformation of naïve T cells into Th22 cells and the correlation of its peripheral blood expression in non-small cell lung cancer patients. METHODS:CD4⁺ naïve T cell magnetic bead sorting kit was used to isolate the peripheral blood mononuclear T cells from healthy people. Transforming growth factor-β (TGF-β) and IL-2 were added to promote differentiation and proliferation. IL-1β was used to induce differentiation into Th22 cells. The proportion of CD4⁺ IL-22⁺ T cells was analyzed by flow cytometry, and the expression of IL-22 was detected by ELISA. We selected 60 cases of non-small cell lung cancer patients in our hospital, including 18 in I phase, 20 in Ⅱ phase, 13 in Ⅲ phase and 9 in IV phase, as well as 25 healthy persons. The proportion of Th22 (CD4⁺ IL-22⁺) cells in peripheral blood was detected by flow cytometry, and the serum levels of IL-1β and IL-22 were measured by ELISA. RESULTS:IL-1β induced the transformation of naïve T cells into Th22 cells and promoted the secretion of IL-22 (P<0.05). The proportion of Th22 cells and the IL-22 and IL-1β levels in peripheral blood of the patients with non-small cell lung cancer were higher than those in healthy subjects, and correlated with the clinical stage. CONCLUSION:IL-1β induces the differentiation of Th22 cells and the expression of IL-22. The levels of IL-1β and IL-22 are related to the progression of non-small cell lung cancer, which may be involved in immunosuppression and promote the occurrence of non-small cell lung cancer.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

Effect of AMD3100 on dengue virus type 2-induced apoptosis in Eahy926 cells

AIM: To investigate the role of AMD3100 (an inhibitor of CXCR4) in dengue virus type 2 (DV2)-induced apoptosis in human umbilical vein endothelial cell line Eahy926. METHODS: The expression of factor Ⅷ in Eahy926 cells was examined by immunohistochemistry staining. The cells were divided into untreated group, DV2 infection group and DV2+AMD3100 group. Flow cytometric analysis was used to detect the expression of CXCR4 in Eahy926 cells 24 h, 36 h, 48 h and 60 h after DV2 infection. In addition, the percentage of apoptotic cells was also analyzed by flow cytometry. Immunofluorescence was performed to detect the phosphatidylserine (PS) on the surface of Eahy926 cells.RESULTS: Eahy926 cells were factor Ⅷ-positive. Compared with untreated group, the expression of CXCR4 increased in DV2 infection group, most markedly 48 h after infection (66.13%±10.30%, P<0.05). The percentage of apoptotic Eahy926 cells after DV2 infection was the highest at 36 h (29.85%±15.78%, P<0.05). The percentage of DV2-induced apoptotic cells in DV2+AMD3100 group was higher than that in DV2 infection group. The green fluorescence-labeled cells in DV2 infection group and DV2+AMD3100 group were more than those in untreated group. CONCLUSION: DV2 infection induces apoptosis and increases the expression of CXCR4 in Eahy926 cells. AMD3100, the inhibitor of CXCR4, may be a promoter of apoptosis in Eahy926 cells after DV2 infection.     

Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

Supportive and expansive effects of aorta-gonad-mesonephros (AGM)-derived stromal cells on hematopoietic stem in vitro

AIM: To explore the supportive and expansive effects of aorta-gonad-mesonephros(AGM) region derived stromal cells on hematopoietic stem cells (HSC) in vitro.METHODS: The murine stromal cells were separated and cultured from AGM region of a 11 day postcoitum (dpc) mouse embryo and 6 week mouse. After identification by Wrights staining and flow cytometry, the stromal cells were co-cultured with the embryonic stem cell(ESC)-derived, cytokine-induced HSCs, and the maintenance and expansion of HSCs were evaluated by detecting CD34+，CD34+Sca-1+cells with flow cytometry.Blast colony-forming cell (BL-CFC) and high proliferative potential colony-forming cells(HPP-CFC) were determined by semi-solid medium clonal culture.RESULTS: AGM-derived and bone marrow(BM)-derived stromal cells were similar in morphology and phenotype, and had common character of stromal cells. Supported by AGM stromal cells or by BM stromal cells, more primitive progenitor cells HPP-CFC were expanded, but BL-CFC expansion was only detected in AGM-derived stromal cells. In the supporting of BM stromal cells CD34+ hematopoietic stem/progenitor cells were expanded 3-4 times, but no significant expansion in CD34+Sca-1+ cells was observed. While in the supporting of AGM stromal cells, both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded significantly from 4 to 5 times, respectively (P<0.05).CONCLUSION: AGM-derived stromal cells significantly support the expansion of HSC, and also maintain the self-renewal activity and multi-lineage differentiation of HSC in vitro.     

Butylated hydroxyanisole promotes expression of neural specific genes in mouse fetal liver cells through extracellular signal-regulated kinase signaling pathways

AIM: To study the mechanism of butylated hydroxyanisole-induced neural differentiation of fetal liver Sca-1+ cells in vitro. METHODS: Sca-1+ cells from 14.5-day-old mouse fetal liver were isolated with a magnetic cell sorting kit. Cultured cells pretreated with or without extracellular signal-regulated kinase (MEK1) inhibitor, PD98059, were induced by 200 μmol/L butylated hydroxyanisole (BHA) for 24 hours, and then incubated in serum-free medium. Expression of genes in treated or untreated cells were assayed by Western blotting and RT-PCR. RESULTS: There was low level of neuronfilament-L (NF-L) and brain factor-1 (BF-1) in fetal liver Sca-1+ cells, but no neuronfilament-H (NF-H) and tyrosine hydroxylase (TH) was observed. BHA significantly promoted the expression of neuron-specific NF-L, NF-H, BF-1, and TH in fetal liver Sca-1+ cells. NF-L, NF-H, BF-1 and TH increased by 6.32 fold, 2.73 fold, 3.37 fold and 2.68 fold, respectively (all P<0.01 vs untreated cells). However, the expression of BHA-induced genes were inhibited by ERK kinase inhibitor PD98059 (25 μmol/L). CONCLUSION: BHA induces neural differentiation of fetal liver cells through ERK.     

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.     

Relationship between Th17 cells infiltrated in lungs and pulmonary vascular reconstruction under hypobarric hypoxia

AIM: To investigate the changes of retinoid-related orphan receptor γt(RORγt) mRNA and interleukin-17(IL-17) protein in the lung tissue under hypobaric hypoxia, and the relationship between Th17 cells and hypoxic pulmonary vascular reconstruction. METHODS: Male BALB/c mice(n=50) were randomly divided into control group and 3 d, 7 d, 14 d and 28 d hypobaric hypoxia groups. The mice in hypobaric hypoxia groups were housed in a hypobaric hypoxia chamber(simulated altitude of 6 000 m) for 3 d, 7 d, 14 d or 28 d. The mice in control group were housed in normal pressure and oxygen environment. The hemodynamic data were recorded by cardiac catheterization. The hypertrophy of right ventricle was evaluated by the ratio of weight of the right ventricle to the weight of the left ventricle plus interventri-cular septum, and the right ventricular weight over body weight. The spleen was collected and the proportions of the Th17(CD4⁺IL-17⁺RORγt⁺) cells were detected by flow cytometry. The serum levels of IL-4, IL-6 and IL-17 and the change of IL-17 in the lung tissue were measured by ELISA. The mRNA expression of RORγt in the spleen and lung tissues was measured by RT-qPCR. RESULTS: Compared with control group, the mouse right ventricular systolic pressure, the hypertrophy index of right ventricle and the serum IL-17 level were significantly elevated in hypoxia groups, which was consistent with the results of flow cytometry. The mRNA expression of RORγt in the lung tissue was also significantly increased in 7 d, 14 d and 28 d hypoxia groups. The expression of IL-17 in the lung tissue was significantly increased in 14 d and 28 d hypoxia groups. CONCLUSION: Hypoxia promotes differentiation of Th0 cells to Th17 cells in the spleen. The Th17 cells infiltrated in the lung tissue under hypobarric hypoxia are involved in pulmonary vascular reconstruction.     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

Differentiation of embryonic-like stem cells derived from human bone marrow into multinucleated fibers in vitro

AIM: To compare the capacity of in vitro differentiation into multinucleated fibers between embryonic-like stem cells (ELSCs) and mesenchymal stem cells (MSCs) derived from human bone marrow. METHODS: To isolate ELSCs, human bone marrow mononuclear cells were cultured in gelatin-coated flask with serum-free Knockout-DMEM medium designed for the expansion of human embryonic stem cells. MSCs were isolated from the same bone marrow by the traditional method. The morphological characters of both ELSCs and MSCs were observed under inverted phase-contrast microscope, and the expression of their multipotent antigen markers was identified by immunofluorescent staining. ELSCs and MSCs were cultured in myogenic differentiation medium. The protein levels of muscle-specific antigen markers myosin heavy chain (MHC), myogenin and MyoD were detected by the method of immunostaining. The mRNA expression of MHC, myogenin and MyoD was detected by RT-PCR. The capacity of in vitro differentiation into multinucleated fibers was compared between ELSCs and MSCs by calculating the proportion of MHC-positive multinucleated fibers. RESULTS: ELSCs, which weakly expressed the multipotential markers Oct-4, Nanog-3 and Sox-2, were isolated from bone marrow by the method of serum-free medium. ELSCs appeared smaller, slenderer and more homogeneous, and were morphologically different from MSCs derived from the same marrow. No multipotential marker in MSCs was expressed. ELSCs and MSCs were induced into long multinucleated fibers expressing MHC and myogenin at mRNA and protein levels by culturing in the myogenic differentiation medium. However, on the 10th day after induction, the proportion of the MHC-positive fibers in ELSCs was (25.7?4.1)%, and the proportion in MSCs was (15.8?7.6)%.The capacity for differentiation into muscle in ELSCs was significantly higher than that in MSCs (P<0.05). CONCLUSION: Bone marrow ELSCs are induced into multinucleated fibers and have the stronger myogenic differentiation capacity than MSCs derived from the same marrow. ELSCs are a more ideal candidate for muscular disease therapy.