Effect of co-culture with amniotic epithelial cells on biological characteristics of amniotic mesenchymal stem cells / 中国肿瘤生物治疗杂志

@# Objective: The aim of this study was to investigate the effect of co-culture with AEC (amniotic epithelial cell) on the biological characteristics of AMSC (amniotic mesenchymal stem cell), and to investigate the roles of SDF-1/CXCR4 axis in the homing and migration of AMSC. Methods: AMSC andAEC were isolated from human amnion, and then cultured, amplified and identified, respectively. TheAMSC were divided into three groups:AEC co-cultured group, serum-free cultured group and serum cultured group.After culture for 24 h, 48 h, and 72 h, the proliferation viability ofAMSC was measured by CCK-8 assay and trypan blue staining; the expression of CXCR4 mRNAwas analyzed by flow cytometry and Real-time RT-PCR, and the migration ability ofAMSC in vitro was observed by migration assay. Results: Cell viability (48 h and 72 h) and survival rate in the co-culture and serum groups were higher than those in the serum-free group (all P<0.05). The mRNA and protein expressions of CXCR4 in AMSC of the co-culture and serum-free groups were significantly higher than those of the serum group (P<0.05). The migration ability of AMSC in the co-culture and serumfree groups, which increase with the SDF-1 (stromal cell derived factor-1) concentration gradient, were higher than that in the serum group (P<0.05). Conclusion: AMSC co-cultured with AEC still have the basic biological characteristics of MSC, and showed good growth activity. Co-culture withAEC can up-regulate CXCR4 onAMSC surfaces and enhance the migration ability ofAMSC in vitro.

Ureaplasma urealyticum-derived lipid-associated membrane proteins introduce IL-6, IL-8, and TNF-α cytokines into human amniotic epithelial cells via Toll-like receptor 2 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVE@#The purpose of this study was to determine the role of Ureaplasma urealyticum-derived lipid-associated membrane proteins (LAMPs) in the host innate immune system, specifically their effect on Toll-like receptors (TLRs).@*METHODS@#LAMPs were derived from U. urealyticum strains, and human amniotic epithelial cells (HAECs) were isolated from healthy full-term placentas. Cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and TLR2 mRNA by real-time PCR. Expression of TLR2 was confirmed by Western blotting and immunohistochemistry.@*RESULTS@#LAMPs induced HAECs to produce inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α. Cytokine production was reduced after blocking TLR2 using TLR2 inhibitor (anti-hTLR2-IgA).@*CONCLUSIONS@#LAMPs isolated from U. urealyticum induced TLR2-dependent up-regulation of inflammatory genes and cytokines in HAECs.

Effects of conditioned media from human amniotic epithelial cells on corneal alkali injuries in rabbits

This study was performed to evaluate the effects of conditioned media (CM) from human amniotic epithelial cells (HAECs) on the corneal wound healing process. Eighteen rabbits (36 eyes) were used and randomly assigned to three groups according treatment: CM from HAECs (group 1), vehicle alone (group 2), and saline (group 3). Corneal alkali injuries were induced with 1 N sodium hydroxide. Each reagent used for treatment evaluation was injected into the dorsal bulbar subconjunctiva and the area of the corneal epithelial defect was measured every other day. Two animals from each group were euthanized at a time on days 3, 7, and 15, and the cornea was removed for histological examination. The sum of the epithelial defect areas measured on day 0 to day 6 as well as day 0 to day 14 in group 1 was significantly smaller than those of other groups. Histological examination revealed that the group 1 corneas had less inflammatory cell infiltration and showed more intact epithelial features compared to the other groups. These results suggest that CM from HAECs promote corneal wound healing in rabbits.

Suppression of corneal neovascularization by culture supernatant of human amniotic epithelial cells in vitro / 中华实验眼科杂志

BackgroundCorneal neovascularization (CNV) is a common eye disease.The researches on the pathogenesis and treatment of CNV are focus in Ophthalmology.ObjectiveThis study was to investigate the effects of culture supernatant from human amniotic epithelial cells (AECs) on CNV in vitro and its mechanism.MethodsHuman AECs were obtained from a placenta and cultured in serum-free medium for 48 hours,and the supernatant was collected.The levels of interleukin-1 receptor antagonist (IL-1 Ra) and pigment epithelium-derived factor (PEDF) in the human AECs culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA).Rabbit corneal epithelial cells (CECs) were obtained and cultured in different concentrations of human AECs culture supernatant for 48 hours,serum-free medium was used as the control group.The expression of vascular endothelial growth factor (VEGF) mRNA and basic fibroblast growth factor (bFGF) mRNA in rabbit CECs were measured by quantitative real-time PCR (QRT-PCR).Human umbilical cord vein endothelial cells (UVECs) were cultured in the three mediums above,and the proliferation of human UVECs (absorbance value,A value) was tested by the cell counting kit 8 ( CCK8 ).Migration assay was performed by the wound healing method for the human UVECs.The membrane ultra-structure of human UVECs was examined under the atomic force microscope (AFM).ResultsCultured and passaged human AECs showed a positive response for keratin.The expression of VEGF mRNA (1.00±0.22) and bFGF mRNA (1.00±0.36) in rabbit CECs was suppressed by the human AECs culture supernatant,with a significant reduction in comparison with the serum-free DMEM group (2.98±0.46,2.55±0.48 )(P=0.001,0.002).The A value was significantly lowered in the human AECs culture group for 72 hours compared with the serum-free DMEM group ( 1.941 ± 0.036 versus 2.144 ± 0.059 ) ( P =0.000 ),and the bFGF-induced migration rate of human UVECs was strongly inhibited by the culture supernatant of human AECs in comparison with serum-free DMEM.The plasma membrane of human UVECs cultured with the human AECs culture supernatant was full of bumps,and decreased intercellular connection and cellular pseudopodia were found on the AFM image.The concentration of IL-1Ra was (153.56±0.36)ng/L and that of PEDF was (70.41 ±0.68 )μ,g/L in the human AECs culture supernatant.Nothing was deteched in serum-free DMEM group.Conclusions Human AECs culture supernatant suppressed the expression of VEGF and bFGF in CECs as well as the migration and proliferation of vascular endothelial cells.The effect may be associated with IL-1Ra and PEDF secreted by human AECs.These results suggest that human AECs may be a potential therapy for the inhibition of CNV.

Transversal inducing differentiation of human amniotic epithelial cells into hepatocyte-like cells / 中南大学学报(医学版)

Objective To evaluate the in vitro differentiation of human amniotic epithelial cells (hAECs ) into hepatocyte-like cells. Methods Combined approach of dexamethasone, HGF, IGF and other cytokines were used to induce the differentiation of hAECs into hepatocyte-like cells. The induction lasted 2 weeks. During the induction, the expression of albumin ALB, CYP1A1, CYP1A2, IGFR, c-met and key functional genes related to liver cells as well as transcription factors HNF3, HNF4 and C/EBPa were monitored by RT-PCR. Time dependent changes of the surface marker colony ALB, AFP and CK18 were analyzed by cell flow cytometry. Results After the 2 week induction, the expressions of liver hepatocyte-like cell functional genes such as albumin, CYP1A1, CYP1A2, c-met, and transcription factors such as HNF3, HNF4, C/EBPa and HNF1 were observed. Six days after the induction, hAECs mainly were stained AFP+, and the positive rate was (15.1±2.1)%. While 10 days after the induction, part of the hAECs showed AFP+/ALB+ (6.5±1.4)%; and on 14th day, hAECs only showed ALB+, and the rate was (13.9±2.3)%. ALB+ cell increase indicated a gradual functional maturation from the hAECs to hepatocyte-like cells. Similaritly, the number of CK18+ cells in the whole population was also increased: On 10th day, the rate was (16.1±1.2)%; on 14th day, that was (21.3±4.6)%, which proved the above hypothesis of the trandifferentiation. By extending the induction time, the expression of functional genes increased gradually, and a maturing process of hAECs was detected by cell surface markers. Conclusion The differentiation of hAECs induced in vitro has the characteristics of hepatocyte-like cells.

A comparative study on rabbit bone mesenchymal stem cells and human amniotic epithelial cells transplantation for rabbit limbal stem cell deficiency / 中华实验眼科杂志

BackgroundLimbal stem cell deficiency usually leads to blindness, and traditional therapy is limited. Recent research demonstrated that bone mesenchymal stem cells ( BMSCs ) and human amniotic epithelial cells(AECs) could differentiate into many kinds of cells including corneal epithelial cells, but the outcome and effect of these cells on corneal stem cell deficiency are still unclear. ObjectiveThis study aimed to observe and compare the effects of rabbit BMSCs and human AECs transplantation for rabbit limbal stem cell deficiency. Methods Eighteen clean New Zealand rabbits were randomly divided into the amniotic stroma(AS) group, rabbit BMSCs group and human AECs group with 6 rabbits for each group. Limbal stem cell deficiency models were established by putting a piece of filter paper that had been soaked in a NaOH solution at the corneal center. Rabbit BMSCs were isolated and purified by density gradient centrifugation combined with the attachment culture method, and human AECs were collected by a sequential trypsin digestion technique,and the third generation rabbit BMSCs and the first generation human AECs were identified with RT-PCR. After that,cells were inoculated onto the denuded AS and grafted to the corneal surface of the experimental animals. Twenty-eight days after cell transplantation, the therapeutic effects were evaluated based on the corneal neovascularization and opacity scores. Corneal histopathological examination and immunohistochemistry were performed to evaluate and compare the effectiveness among AS,rabbit BMSCs and human AECs on corneal stem cell deficiency. The procedure complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. ResultsThe third generation of rabbit BMSCs grew well after 12 hours, and the first generation of human AECs formed a membrane-like monolayer after 48 hours of incubation on AS. Immunohistochemistry staining showed that, 28 days after transplantation, the surface cells of the cornea were positive for cytokeratin 3 in both the rabbit BMSCs group and human AECs group.Compared with the AS group,the corneal neovascularization and opacity grades were significantly decreased in the rabbit BMSCs group( Z=-2. 983, P =0. 003 ; Z =-2. 844, P =0. 004 ) and human AECs group ( Z =-2. 817, P =0. 005 ; Z =-2.041, P =0. 041 ). Histopathological analysis exhibited that stratified corneal epithelial-like cells formed on the corneal stroms 28 days after grafting and no signs of goblet cells and neovascularization were found. Less inflammatory cells and regular collagen fiber could be seen in the rabbit BMSCs group and human AECs group. In addition,clinical observation also revealed that the corneas were much clearer in the rabbit BMSCs group than the human A ECs group( Z =-2. 091 , P=0. 037 ), but the corneal neovascularization score was similar between them (Z = -0. 267,P=-0. 789). ConclusionsRabbit BMSCs and human AECs can differentiate into corneal epithelial-like cells onthedamagedcornealsurfaceandfurtherdemonstrateremarkableinhibitoryeffectsoncornealneovascularization and inflammatory cells. The more dominant and prominent effect is the role played by rabbit BMSCsin the improvement of corneal transparency.

Primary culture and hepatocelluar specific protein expression in human amniotic epithelial cells / 上海第二医科大学学报

Objective To optimize the method of primary culture for human amniotic epithelial cells (AECs) and detect the expression of hepatocelluar specific proteins in AECs. Methods AECs were obtained by collagenase and trypsin digestion method. 10,20,40 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic fibroblast growth factor were added separately to the culture media,inverted microscope was used to observe the cell growth and proliferation,and the optimal condition for primary culture of AECs was obtained. The primary cells cultured without growth factors were served as controls. The expression of hepatocelluar specific proteins such as albumin,cytokeratin-18 (CK-18),alpha-1 antitrypsin (AAT) and alpha-1 foetoprotein (AFP) in cultured cells and histological sections were detected by immunohistochemical staining. Results The AECs available were relatively pure,with only a few mesenchymal cells. EGF of 10 ng/mL was chosen as an ingredient of culture medium as the growth and proliferation of AECs significantly accelerated with 10 ng/mL EGF. The expression of albumin,CK-18,AAT and AFP was detected in amnion tissues and AECs cultured in vitro. Conclusion Collagenase and trypsin digestion method and culture medium with 10 ng/mL EGF are favourable conditions for primary culture of AECs. Hepatocelluar specific proteins are expressed in human amnion tissues and AECs cultured in vitro,indicating that AECs have some characteristics of hepatocytes.