Mixed culture of human iPSCs with rabbit corneal endothelial cells induces differentiation of iPSCs

AIM:To investigate the morphology and protein expression of human induced pluripotent stem cells(iPSCs) in the mixed-culture environment with rabbit corneal endothelial cells(CECs) and to provide the experimental basis and the mechanism of iPSC differentiation into CECs. METHODS:Primary rabbit CECs were isolated with trypsin and subcultured. The human iPSCs were cultured and amplified by a feeder-free method and their characteristics were evaluated by Western blotting. iPSCs labeled with quantum dots of appropriate concentration were used to establish mixed-culture model with rabbit CECs. The morphology of iPSCs was evaluated by atomic force microscopy(AFM) and inverted microscopy. The protein expression of CD31, CD34, CD133 and aquaporin 1(AQP1) in iPSCs was tested by the method of immunofluorescence. RESULTS:The rabbit CECs were hexagonal and showed a typical cobblestone appearance. iPSCs grew in the cloning form, and 3 pluripotent proteins Oct4, Nanog and Sox2 were expressed positively. 1/4 suspension of iPSCs labeled with 10 nmol/L quantum dots and 60% confluence of rabbit CECs made best mixed culture for each other. Under AFM and inverted microscope, the volume of iPSC became bigger and the nuclear-cytoplasm ratio was decreased after 7 days of mixed culture. Some granular protrusions of the membrane were observed and the surface roughness of the cell membrane increased. The protein expression of CD31, CD34, CD133 and AQP1 in iPSCs was negative, while AQP1 was detected after mixed culture for 2 weeks. CONCLUSION:iPSCs morphologically change to endothelial-like cells after mixed culture with rabbit CECs and express the marker protein AQP1 of CECs at the same time.     

Generation,identification and differentiation of primary ovarian insufficiency patient-derived iPSCs

AIM: To generate and identify primary ovarian insufficiency (POI) patient-derived induced pluripotent stem cells (iPSCs) and to explore their differentiation potential to primordial germ cells. METHODS: Plasmid pEB-C5 expressing reprogramming factors Oct4, Sox2, c-Myc, Klf4 and Lin28, and plasmid pEB-Tg expressing SV40 T antigen, were transfected into peripheral blood mononuclear cells (PBMCs) derived from POI patients at the same time. PBMCs were reprogrammed into iPSCs, and the pluripotency of the cells was identified. After supplementation of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4 (BMP4), the mRNA and protein expression of primordial germ cell markers was detected by real-time PCR and Western blot, respectively. RESULTS: iPSCs derived from the PBMCs of POI patient differentiated into 3-germ layer cells and maintained pluripotency by the detection of alkaline phosphatase staining, immunofluorescence, embryoid body and teratoma formation. After addition of TGF-β1 and BMP4, the primordial germ cell markers, including stem cell growth factor receptor (c-Kit), developmental pluripotency-associated 3 (STELLA/DPPA3) and DEAD box polypeptide 4 (VASA/DDX4) were increased at mRNA level (P<0.05), and VASA/DDX4 was also up-regulated at protein level in induced group. CONCLUSION: PBMCs of POI patient are reprogrammed into integration-free iPSCs in vitro and maintain pluripotency. They differentiate into primordial germ cells by adding TGF-β1 and BMP4.     

Effects of Waller degenerative sciatic nerve segments on differentiation of BMSCs into SCs in rats

AIM:To observe the effects of Waller degenerative sciatic nerve segments on the ability of proliferation and secretion in rat bone marrow mesenchymal stem cells (BMSCs) as well as the differentiation of BMSCs into Schwann cells (SCs) under co-culture condition. METHODS:BMSCs were isolated from SD rats and identified by immunofluorescence. In Transwell co-culture system, the degenerative rat sciatic nerve segments and BMSCs were co-cultured in the Transwell inserts and well plates, respectively. The experiments were divided into 3 groups: group A, degenerative sciatic nerve segments co-cultured with BMSCs; group B, normal sciatic nerve segments co-cultured with BMSCs; group C, BMSCs cultured alone. The morphological changes of co-cultured BMSCs were observed under inverted phase-contrast microscope. The expression of S-100 in the BMSCs was tested by immunofluorescence staining after 7 d of co-culture. At 0, 1, 4, 7, 11 and 14 d after co-culture, BMSCs growth curve in each group were drawn by the method of cell counting, the expression of nerve growth factor (NGF) was detected by ELISA, and the mRNA expression of S-100 in the BMSCs was determined by real-time PCR. RESULTS:BMSCs were isolated and cultured successfully. The identification of BMSCs showed positive expression of CD29, CD44, and CD90. At 7 d after co-culture, the BMSCs in group A began retraction, became tapered with the processes and had SCs-like morphology, but most BMSCs in groups B and C showed no obvious morphological changes under inverted phase contrast microscope. The positive expression rates of S-100 in groups A, B, and C were 31.1%±2.9%, 16.2%±1.7%, and 0.42%±0.07%, respectively. There were significant differences of S-100 positive expression rates among 3 groups. The BMSCs growth curve of each group was similar to an "S" shape. From 4 d after co-culture, the BMSCs proliferation in group A were significantly faster than that in group B and group C. NGF in the supernatant of group A was increased in a time-dependent manner and reached the peak at 7 d after co-culture, then decreased gradually. NGF was also also increased in group B and group C, but the content of NGF was lower than that in group A. At 4, 7, 11 and 14 d of co-culture, NGF content was higher in group A than that in group B and group C, and a significant difference was also observed between group B and group C. The S-100 mRNA expression was significantly higher in group A than that in group B and group C at 4, 7, 11 and 14 d after co-culture (P<0.05). CONCLUSION: Waller degenerative sciatic nerve segments can effectively promote rat BMSCs proliferation and induce BMSCs to differentiate into SCs in vitro, and the growth factors secreted by degenerative sciatic nerve segments may be involved in the regulation of inducing BMSCs to differentiate into SCs.     

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.     

Generation of induced pluripotent stem cells and neural cells from urine-derived cells of Alzheimer disease patients

AIM: In this study, we aim to obtain the induced pluripotent stem cells (iPSCs) from the patients with sporadic Alzheimer disease (AD). METHODS: Three typical Alzheimer's patients were chosen, and the epithelial cells were isolated from their urine. We reprogrammed these cells into induced pluripotent stem cells by transfection of 4 factors (Oct4, Sox2, Klf4 and SV40LT) with the technique of electro-transfection. After getting these iPSCs, we continue to differentiate them into neural cells by a specific method—dual inhibition of Smad signaling. RESULTS: The primary cells from 3 AD patients were successfully reprogrammed to iPSCs, and these patients-derived iPSCs were differentiated into neural cells. There was no significant difference, during iPSCs reprogramming and neural differentiation, between cells from AD patients and normal people. CONCLUSION: The urine cells from AD patients were able to transfer to iPSCs, functional neurons and neurogliocytes.     

Metanephric cell microenvironment induces embryonic stem cells to differentiate toward renal cells

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.     

Effect of Ruanmaijiangzhi mixture intervention on endothelial cell function after artery bypass graft

AIM： To observe the effect of Ruanmaijiangzhi mixture on the function of endothelial cells in rabbits after artery bypass graft. METHODS：A New Zealand white rabbit model of right common carotid artery bypass graft was established. The rabbits were randomly divided into 4 groups： model group, aspirin control group, middle dose of Ruanmaijiangzhi group and high dose of Ruanmaijiangzhi group. Eight weeks after treatment, circulating endothelial cells (CECs) in anticoagulated blood were detected by flow cytometry. The serum levels of P-selectin and intercellular adhesion molecule 1 (ICAM-1) were measured by ELISA. RESULTS：Compared with model group, the relative ratio of CECs, and the levels of P-selectin and ICAM-1 in medication administration groups were significantly decreased. The effect in Ruanmaijiangzhi groups was better than that in aspirin control group. Ruanmaijiangzhi treatment showed a positive dose-related correlation. CONCLUSION：Chinese medicine Ruanmaijiangzhi mixture decreases CECs, P-selectin and ICAM-1 to improve the endothelial cell function after coronary artery bypass graft.     

Bone marrow-derived mesenchymal stem cells protect against hypoxia-induced injury and apoptosis of PC12 cells via up-regulation of erythropoietin expression

AIM： To investigate the effects of bone marrow-derived mesenchymal stem cells (BM-MSCs) on cobalt chloride (CoCl2)-induced injury and apoptosis of PC12 cells. METHODS：PC12 cells were divided into control group, CoCl2 group, BM-MSCs-siCTL+CoCl2 group and BM-MSCs-siEPO+CoCl2 group. The viability of the PC12 cells was measured by MTT assay. Flow cytometry and Hoechst 33258 staining were used to determine the apoptotic rate and the changes of chromatin distribution in PC12 cells. The expression of erythropoietin (EPO) in BM-MSCs was measured by RT-PCR and Western blotting. The mRNA expression of Bcl-2 and Bax in PC12 cells was detected by RT-PCR. Caspase-9 and caspase-3 assay kits were used to detect the activity of caspase-9 and caspase-3. RESULTS：The viability of PC12 cells treated with CoCl2 for 24 h and 48 h decreased to （43.0±6.4）% and （33.8±5.7）%, respectively, while 1∶15 ratio of BM-MSCs co-culture increased the cell viability to （77.9±3.8）% and （75.2±9.7）%,respectively. The expression of EPO in BM-MSCs was up-regulated after treated with 0.6 mmol/L CoCl2 for 24 h and 48 h, while EPO siRNA significantly abrogated the EPO expression in BM-MSCs. BM-MSCs-siCTL co-culture significantly inhibited the apoptosis of PC12 cells induced by CoCl2. However, EPO siRNA the protective effect of BM-MSCs. Compared with CoCl2 treatment group, BM-MSCs co-culture induced remarkable increase in the expression of Bcl-2 and decrease in the expression of Bax in PC12 cells, which was reversed by EPO siRNA. BM-MSCs-siCTL co-culture remarkably abrogated the CoCl2 induced up-regulation of caspase-9 and -3, while BM-MSCs-siEPO co-culture significantly reversed the down-regulation of caspase-9 and -3 induced by BM-MSCs-siCTL co-culture. CONCLUSION：BM-MSCs protect PC12 cells from apoptosis induced by CoCl2. The protective effect of BM-MSCs might be executed by up-regulating the expression of EPO.     

Effect of CD151 on biological characteristics of human umbilical cord mesenchymal stem cells

AIM：To study the effect of CD151 on the biological characteristics of human umbilical cord mesenchymal stem cells (hUC-MSCs). METHODS：CD151 expression on hUC-MSCs was interfered by siRNA. The cells were divided into siRNA-CD151 group and negative control group (treated with siRNA-NC). The efficiency of interference after 72 h and the changes of other surface markers were detected by flow cytometry. The ability of differentiation was assessed by oil red O and von Kossa staining. The cell cycle was analyzed by flow cytometry. The mRNA expression of CD151, hepatocyte growth factor (HGF), transforming growth factor β1 (TGF-β1), cyclooxygenase 2 (COX-2) and indoleamine 2, 3-dioxygenase (IDO) in hUC-MSCs was detected by real-time PCR. The secretion of HGF by hUC-MSCs was measured by ELISA. RESULTS：The results of flow cytometry showed that the expression of CD151 (11.97±2.63 vs 95.66±1.56, P<0.01) and CD105 (93.66±0.21 vs 83.37±0.71, P<0.05) on hUC-MSCs in siRNA-CD151 group was lower than that in negative control group. The consistent results were also achieved by using the method of real-time PCR. Treatment with siRNA-CD151 down-regulated the progress of the cell cycle as the G1 phase increased and the S phase decreased. The mRNA expression levels of HGF and TGF-β1 in hUC-MSCs in siRNA-CD151 group were lower than those in negative control group, and opposite result of COX-2 mRNA expression was observed. The IDO mRNA in hUC-MSCs was unchanged with IFN-γ stimulation for 24 h. HGF concentration in siRNA-CD151 group was decreased as compared with negative control group. CONCLUSION：Interfering CD151 expression on hUC-MSCs doesn’t change other surface markers except CD105, and maintains the capacity of adipogenic differentiation. However, it changes the osteogenic differentiation, proliferation and the expression of immunomodulatory cytokines.     

Human umbilical cord mesenchymal stem cells migrate to Raji cells and promote their proliferation

AIM：To investigate the interaction between human Burkitt lymphoma cell line Raji and human umbilical cord mesenchymal stem cells (hUC-MSC). METHODS：The direct and indirect effects of MSC on Raji cells were investigated. The viability of Raji cells was tested by CCK-8 assay, and the cell cycle was determined by flow cytometry. The importance of vascular endothelial growth factor (VEGF) in the migration of MSC to Raji cells was analyzed by blocking VEGF expression in Raji cells with small interfering RNA (siRNA). VEGF level in the supernatant was detected by ELISA, and the mRNA expression of VEGF was measured by quantitative real-time PCR (qRT-PCR). RESULTS：Both MSC and MSC-conditioned medium (MSC-CM) promoted the growth of Raji cells. The viability of Raji cells co-cultured with MSC-CM was 0.99±0.05 at 72 h, and that in control group was 0.71±0.07. Both direct co-culture with MSC and MSC-CM turned the Raji cells from G0/G1 phase to S phase. The number of Raji cells co-cultured with MSC-CM in S phase was increased from 16.33±1.37 to 28.50±1.41, and the number in G0/G1 phase was decreased from 77.70±1.57 to 54.40±1.57. The expression of VEGF was down-regulated either at mRNA or protein level after transfection with siRNA. The ability of MSC migrated to Raji cells was significantly declined (96.00±5.28 vs 143.00±7.20). CONCLUSION：Raji cells recruit MSC by secreting VEGF, and MSC promote the proliferation of Raji cells by turning the cells from G0/G1 phase to S phase.     

Effects of basic fibroblast growth factor on proliferation and collagen production in human umbilical cord mesenchymal stem cells

AIM：To observe the growth pattern and surface markers of human umbilical cord mesenchymal stem cells(hUCMSCs) and to explore the influence of basic fibroblast growth factor (bFGF) on the proliferation and collagen production in hUCMSCs cultured in vitro. METHODS：hUCMSCs were isolated by enzyme digestion method and adherent culture. The surface markers CD45, CD34, CD105, CD29 and HLA-DR of the cells were analyzed by flow cytometry. The osteogenic ability and adipogenic differentiation were confirmed with oil red O and alizarin red staining. The optimal concentration of bFGF to promote the proliferation of hUCMSCs was 20 μg/L. The cells in control group were cultured in the growth medium consisting of DMEM/F12 and 10% volume fraction of fetal bovine serum. The cells in experiment group were cultured under the same condition of control group but plus 20 μg/L bFGF. The proliferation of hUCMSCs was analyzed by MTT assay. The expression of type I and III collagens at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS：The growth curves indicated that bFGF promoted the proliferation of hUCMSCs. The hUCMSCs expressed CD29, but did not express CD34, CD45 or HLA-DR in the presence or absence of bFGF unanimously. The cells were alizarin red staining-positive and oil red O staining-positive. Compared with control group, the expression of type I and III collagens significantly decreased at mRNA and protein levels in experiment group. CONCLUSION：bFGF promotes the proliferation of hUCMSCs and does not change the expression of the surface markers. bFGF inhibits the expression of type I and III collagens at mRNA and protein levels, indicating that bFGF enhances the healing of wound without inducing scar hyperplasia.     

Effect of SCF and G-CSF pretreatment on the proliferation and the differentiation of bone mesenchymal stem cells

AIM: To investigate the effect of pretreatment of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) on the proliferation and the differentiation of mesenchymal stem cells (MSCs) into cardiomyogenic cells. METHODS: The MSCs, isolated primarily from bone marrow, and purified by passage culture, were obtained from the adult rats of four groups: the rats were pretreated by 5 daily injections of SCF; the rats were pretreated with G-CSF; the rats were pretreated with SCF and G-CSF; the rats were treated without any intervention. The 4th passage of MSCs was labeled by DAPI and cellular cycle analysis was conducted by flow cytometry before co-culture. The neonatal rat cardiomyocytes cultured for 3 days were co-cultured with DAPI-MSCs. The percentage of the differentiation of MSCs into cardiomyogenic cells during the five co-culture days was analyzed. The morphologic changes of MSCs and the proteins expression of cardiac myosin heavy chain (MHC) and troponin T (TnT) were recorded respectively with digital microscope camera system and immunofluorescence technique. The percentage of the differentiation of MSCs into cardiomyogenic cells was also calculated. RESULTS: The percentage of MSCs in G₀/G₁ phase in SCF/G-CSF group was significantly lower than that in SCF group, G-CSF group and the control group. The percentage of MHC protein-positive MSCs in SCF/G-CSF group was markedly higher than that in SCF group, G-CSF group and the control group, and that in SCF group and G-CSF group was significantly higher than control group. The percentage of TnT protein-positive MSCs in SCF/G-CSF group, SCF group and G-CSF group was significantly higher than that in control group.CONCLUSION: SCF and G-CSF show the ability to stimulate the proliferation of MSCs and induce MSCs to differentiate into cardiomyocytes. The combination of using SCF and G-CSF is more effective than using only SCF or G-CSF.     

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.     

Effects of marrow stromal cell-mediated microenvironment on human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the effects of marrow stromal HS-5 cells on hepatocellular carcinoma SMMC-7721 cells in the tumor microenvironment. METHODS: The effects of HS-5 cell-conditioned medium (HS-5-CM) on the proliferation, migration and invasion abilities of SMMC-7721 cells were detected by MTT, wound-healing and Transwell assays. After co-culture of SMMC-7721 cells with HS-5 cells in the Transwell chamber, the expression of chemokine CCL5 and its receptor CCR5 at mRNA and protein levels in SMMC-7721 cells was examined by quantitative real-time PCR (qRT-PCR), ELISA or Western blotting. Akt and p-Akt473 protein levels in SMMC-7721 cells treated with PI3K inhibitor LY294002 were observed by Western blotting. RESULTS: HS-5-CM promoted the proliferation, migration and invasion abilities of SMMC-7721 cells. The expression of CCL5 and CCR5 at mRNA and protein levels in SMMC-7721 cells was increased after co-cultured with HS-5 cells. PI3K inhibitor LY294002 inhibited the activation of PI3K-Akt signaling pathway and the secretion of CCL5 in SMMC-7721 cells after co-cultured with HS-5 cells. CONCLUSION: HS-5 cells significantly promote the proliferation, migration and invasion abilities of SMMC-7721 cells. Co-culture of SMMC-7721 cells with HS-5 cells activates PI3K-Akt signaling pathway to increase the secretion of CCL5 in SMMC-7721 cells.     

Anti-inflammatory effect of huperzine A on protection of rat neural stem cells in vitro

AIM：To explore the anti-inflammatory effect of huperzine A (HupA) and its neuroprotective effect on rat neural stem cells (NSCs). METHODS：The microglia and NSCs were isolated from neonatal rat hippocampal tissues and co-cultured in a Transwell system. The cells were divided into 3 groups：control group, amyloid beta-peptide (Aβ) group and HupA group. The microglia layer in Aβ group was treated with Aβ1-42 (10 μmol/L), while that in HupA group was pretreated with HupA (1 μmol/L) before Aβ1-42 stimulation. The culture supernatant levels of inflammatory mediators, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and macrophage inflammatory protein 1α (MIP-1α), were detected by LiquiChip technique. The apoptosis of NSCs was determined by flow cytometry and Western blotting. RESULTS：The microglia secreted a large number of inflammatory mediators with the stimulation of Aβ. In Aβ group, the levels of IL-6, TNF-α and MIP-1α were significantly higher than those in control group at 72 h (P<0.01), and the apoptotic rate of NSCs was 25.46% (P<0.01). In HupA group, the concentrations of IL-6, TNF-α and MIP-1α decreased significantly as compared with Aβ group (P<0.01), and the apoptotic rate of NSCs was only 8.05% (P<0.01). The Bcl-2/Bax ratio in HupA group was higher than that in Aβ group (P<0.05). CONCLUSION：Huperzine A reduces the secretion of cytokines and chemokines, and attenuates microglia-mediated neuroinflammation, thus protecting NSCs against inflammation-induced apoptosis.     

Differentiation of mouse iPSCs into insulin-producing cells and experimental study on treating diabetes

AIM: To induce mouse induced pluripotent stem cells (iPSCs) to differentiate into insulin-producing cells (IPCs) by a new 3-step method, and to detect the efficiency and maturity for the treatment of diabetic mice. METHODS: We constructed iPSCs from mouse embryonic fibroblasts of male C57/C mouse by piggyBac transposon, then induced the iPSCs into IPCs by a 3-step method. The cell morphological change was traced by microscopy during the process of differentiation. The expression of mRNA and protein associated with islet β cell development was determined by real-time PCR and immunofluorescence staining. Flow cytometry was used to analysis the efficiency of differentiation. Insulin and C-peptide secretions of IPCs in response to glucose at high (25 mmol/L) or low (5.5 mmol/L) level were measured by ELISA. The IPCs were transplanted into the capsul of left kidney in the male C57/C diabetic mouse model. Blood glucose was continuously monitored for 28 day, serum insulin was tested by ELISA in different stages. The glucose tolerance test was performed on the 28th day, and the left kidney was excised. RESULTS: IPCs were obtained from mouse iPSCs by the 3-step method. The cells expressed the marker genes (Pdx1, Ngn3, Pax6 and Ins2) and proteins (Pdx1, Nkx6.1 and insulin) of β cells. The glucose stimulation induced the secretion of insulin and C-peptide. The efficiency of differentiation was 28% detected by flow cytometry. After transplantation of IPCs to the diabetic mice, the blood glucose was decreased to normal level on the 3rd day,and serum insulin level and the ability of regulating glucose were improved. IPCs were still alive after 28 d of transplantation by pathological observation. CONCLUSION: iPSCs is efficiently induced into IPCs by a 3-step method , and the induction time is shortened significantly. The hyperglycemia of diabetes mice is reversed after transplanting IPCs to same sex inbred strain mice.     

Effect of advanced glycosylation end products on function of human adipose tissue-derived stem cells in vitro

AIM：To explore the effect of advanced glycosylation end products (AGEs) on the function of human adipose-derived stem cells (hADSCs) in promoting wound healing. METHODS：hADSCs were isolated by conventional method in vitro and divided into control bovine serum albumin (BSA) group, low-dose AGE-BSA group and high-dose AGE-BSA group. The proliferation and migration of hADSCs with different treatments were determined by WST-8 assay and Transwell assay, respectively. In addition, the expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1(IGF-1) at mRNA and protein levels was determined by real-time PCR and ELISA analysis. RESULTS：Compared with control group, the proliferation and migration abilities were significantly inhibited in the hADSCs of AGE-BSA group. The mRNA expression of VEGF, HGF and IGF-1 in AGE-BSA group was obviously lower than that in control group. The contents of VEGF, HGF and IGF-1 in hADSCs-conditioned me-dium in AGE-BSA group were also obviously lower than those in control group. CONCLUSION：AGEs alter the intrinsic properties of hADSCs and impair their functions in promoting wound healing, thus affecting the therapeutic potential of hADSCs in the treatment of diabetic ulcers.     

Effects of airway epithelial cells on phenotype and phagocytosis of macrophages and roles of HIF-1α

AIM: To investigate the effects of airway epithelial cells on the phenotype and phagocytosis of macrophages and the roles of hypoxia-inducible factor-1α (HIF-1α).METHODS: Human bronchial epithelial (HBE) cells treated with CoCl₂ (0, 100, 200, 400 and 800 μmol/L) or transfected with HIF-1α siRNA were co-cultured with the macrophages differentiated from human monocyte line THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The mRNA expression of HIF-1α in the HBE cells was detected by RT-qPCR. The expression of macrophage surface markers and the phagocytosis rate of E.coli by macrophages were analyzed by flow cytometry.RESULTS: CoCl₂ upregulated the mRNA expression of HIF-1α in the HBE cells in a concentration-dependent manner and peaked at 8 h. HBE cells treated with CoCl₂ increased the fluorescence intensity ratio of CCL3, CD163, CD206 and CCL18 in co-cultured macrophages, and the strongest effect was seen in the macrophages co-cultured with HBE cells treated with CoCl₂ at 800 μmol/L. The fluorescence intensity ratio of CCL3 in co-cultured macrophages increased most obviously at 8 h and 12 h, while the fluorescence intensity ratio of CD163, CD206 and CCL18 increased more prominently in the macrophages co-cultured for 24 h. The stimulating effects of the HBE cells transfected with HIF-1α-Homo-488 siRNA on CCL3, CD163, CD206 and CCL18 in the macrophages were significantly attenuated. The phagocytosis rate of E.coli by macrophages co-cultured with HBE cells treated with different concentrations of CoCl₂ for 24 h initially increased (up to 60 min), and then it gradually decreased. Compared with normal HBE co-culture group, the phagocytosis rate in 400 and 800 μmol/L stimulation groups decreased at each time point, and that in 800 μmol/L stimulation group was the most.CONCLUSION: In hypoxia environment, airway epithe-lial cells initially transform macrophages predominantly to an M1-phenotype. However, the long-term hypoxia-stimulated airway epithelial cells inhibit the phagocytosis of macrophages and convert them to M2 superiority. HIF-1α may be an important mediator in these processes.     

Alteration of circulating endothelial cells from acute promyelocytic leukemia patients before and after treatment and its influential factors

AIM: To determine the biological feature of circulating endothelial cells (CECs) in acute promyelocytic leukemia (APL) patients before and after treatment, and to analyze the relationship between CECs and the clinical characteristics. METHODS: The CECs were sorted from peripheral blood by magnetic-activated cell sorting and then counted by 3-color flow cytometry. The cells were identified by immunofluorescence staining for the expression of CD146, CD31, CD144, VEGFR-2, CD45 and CD133. The CECs were cultured in vitro, and the tube formation and colony-forming rate were determined. RESULTS: Increased quantity of CECs was observed in CD34 positive group and group with WBC>10×10⁹/L (P<0.05). The quantity of CECs had a significant difference among low risk, medium risk and high risk groups (P<0.05). The positive rate of CD133 and quantity of CECs significantly reduced in 32 APL patients when they gain complete remission after treatment (P<0.05). The amount of tube formation and colony-forming rate were significantly reduced after treatment (P<0.05). The ratio of CECs quantity from APL patients after treatment to that before treatment had a negative correlation with arsenic concentration in urine on day 7 during As₂O₃ treatment (P<0.05). CONCLUSION: Accurately counting CECs may be helpful for evaluating prognosis and designing treatment strategy.