OBJECTIVETo investigate the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts.

METHODS(1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post scratch hour (PSH) 0 (immediately after scratch), 12, 24, 48, and 72, the migration distance of cells was observed and measured with inverted phase contrast microscope. (6) Human fibroblasts were grouped and treated as in (5), with 3 battles in each group, and apoptosis rate of cells was detected by flow cytometer. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, one-way analysis of variance, LSD test, and t test.

RESULTS(1) On culture day 3, most hAMSCs were in large form, and spindle-shaped with much prominences like fibroblasts or in flat polygonal shape. hAMSCs of the third passage were spindle-shaped. The expression of vimentin of hAMSCs of the third passage was strongly positive, and the expressions of surface markers CD90, CD73, and CD105 of the cells were positive, while the expression of CD45 of the cells was negative. (2) The content of IGF-Ⅰ, VEGF, EGF, and bFGF in hAMSCs-CS were respectively (11.7±1.0), (316±68), (6.1±0.4), and (1.49±0.05) pg/mL. (3) At culture hour 12-72, the proliferation activity of human fibroblasts in each hAMSCs-CS group was significantly higher than that in blank control group (with P values below 0.01), and the proliferation activity of human fibroblasts in 50% hAMSCs-CS group was the highest. (4) The width of scratch in two groups was nearly the same at PSH 0. The migration distance of cells in 50% hAMSCs-CS group was significantly longer than that in blank control group at PSH 12-72 (with P values below 0.01). (5) The apoptosis rate of human fibroblasts in blank control group was (16.2±2.4)%, which was significantly higher than that in 50% hAMSCs-CS group [(7.4±3.6)%, t=6.710, P<0.01].

CONCLUSIONShAMSCs-CS can promote proliferation and migration of human fibroblasts and inhibit the apoptosis of human fibroblasts.

Amnion ; cytology ; Apoptosis ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; Enzyme-Linked Immunosorbent Assay ; Epidermal Growth Factor ; metabolism ; Female ; Fibroblast Growth Factor 2 ; metabolism ; Fibroblasts ; cytology ; drug effects ; Flow Cytometry ; Humans ; Insulin-Like Growth Factor I ; metabolism ; Male ; Mesenchymal Stromal Cells ; chemistry ; Pregnancy ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo explore the role of Compound Danshen Injection (CDI) in regulating the expression of aquaporin 3 (AQP3) in human amnion epithelium cells (hAECs), and to study the relation between c-Jun N-terminal kinase (JNK) signal pathway and AQP3.

METHODShAECs were isolated and primarily cultured from term pregnancy with normal amniotic fluid volume and from term pregnancy with oligohydramnios, and then hAECs were further divided into four groups, i.e., the blank control group (A), the SP600125 group (B), the CDI group (C), and the SP600125 +CDI group (D). The cell viability was measured by cell counting kit-8 assay (CCK-8). The expression of total JNK, phosphorylated JNK, and AQP3 were determined by Western blot.

RESULTS(1) In hAECs with normal AFV or with oligohydramnios: There was no statistical difference in the cell viability or the expression of total JNK among the 4 groups (P > 0.05). But there was statistical difference in the expression of p-JNK (P < 0.05). Compared with A group, the expression of p-JNK was obviously down-regulated in B group, but obviously up-regulated in C group (P < 0.05). The expression of p-JNK was significantly lower in D group than in C group, but higher than that in A group or B group (P < 0.05).The AQP3 expression in the hAECs with normal amniotic fluid volume of C group and D group were higher than that in the A group (P < 0.05). However, there was no statistical difference in the AQP3 expression between C group and D group (P > 0.05). In hAECs with oligohydramnios, the expression of AQP3 obviously decreased in B group, but up-regulated in C group (both P < 0.05). The expression of AQP3 was lower in D group than in C group, but higher than in B group (P < 0.05).

CONCLUSIONCDI could regulate the AQP3 expression in hAECs with oligohydramnios via activating the JNK signal pathway.

Amnion ; cytology ; drug effects ; Aquaporin 3 ; metabolism ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; drug effects ; metabolism ; Female ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; MAP Kinase Signaling System ; physiology

OBJECTIVETo study the effects of Compound Salvia miltiorrhiza Injection (CSI) on aquaporin 3 (AQP3) expression in human amniotic epithelial cells (hAECs), and to explore its mechanisms for treating oligohydramnios.

METHODSThe hAECs selected from 8 human term pregnancies with oligohydramnios and no other complications (as the test group)and 8 human term pregnancies with normal amniotic fluid volume (as the control group) were primarily cultured. The mRNA and protein expressions of AQP3 in hAECs were detected using reverse transcription-polymerase chain reaction and Western blot with various concentrations of CSI (0.000, 0.001, 0.010, 0.020, 0.060, and 0.100 mg/mL, respectively) at different time points (0, 6, 12,24, and 48 h, respectively).

RESULTS(1) Compared with the control group, the AQP3 expression was down-regulated in the test group (P < 0.05). (2) The AQP3 expression in the two groups reached the peak when the concentration of CSI was 0.010 mg/mL, showing statistical difference when compared with other concentrations (P < 0.05). (3) The AQP3 expression reached the peak when 0.010 mg/mL CSI acted for 12 h, showing statistical difference when compared with other concentrations (P < 0.05). (4) The AQP3 expression was up-regulated in the two groups when 0.010 mg/mL CSI acted for 12 h. But the up-regulated AQP3 expression was more obvious in the test group than in the control group (P < 0.05).

CONCLUSIONSCSI could regulate the AQP3 expression in hAECs. CSI showed more obvious effects on the AQP3 expression in hAECs of oligohydramnios human term pregnancies.

Amnion ; cytology ; Aquaporin 3 ; metabolism ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; drug effects ; metabolism ; Female ; Humans ; Salvia miltiorrhiza

OBJECTIVETo investigate the role of mitogen-activated protein kinases (MAPKs)-extracellular signal regulated kinase1/2 (ERK1/2) signal pathway in the regulation of Compound Danshen Injection (CDI) induced AQP3 expression in the human amniotic epithelial cells (hAECs).

METHODShAECs of term pregnancy with normal amniotic fluid volume (AFV) or isolated oligohydramnios were primarily cultured. And the cells were equally divided into four groups, i.e., the vehicle control group, the U0126 group, the CDI group, the CDI + U0126 group. The expressions of phosphorylated ERK1/2 (p-ERK1/2) and AQP3 in hAECs were detected using Western blot analysis.

RESULTS(1) When compared with the control group, the expression level of p-ERK1/2 in hAECs in those with normal AFV and oligohydramnios obviously decreased in the U0126 group (P < 0.05). The expression level of p-ERK1/2 could be elevated in the CDI group (P < 0.05). The expression level of p-ERK1/2 in hAECs was higher in the CDI +U0126 group than in the U0126 group, but lower in the CDI + U0126 group than in the CDI group (P < 0.05). (2) There was no obvious change in AQP3 expression in hAECs with normal AFV between the U0126 group and the vehicle control group (P > 0.05). There was no statistical difference in the expression level of AQP3 between the CDI group and the U0126 +CDI group (P > 0.05), but they were higher than those in the vehicle control group (P < 0.05). (3) Compared with the vehicle control group, the expression level of AQP3 in hAECs with oligohydramnios significantly decreased in the U0126 group and increased in the CDI group (P < 0.05). The expression level of AQP3 was lower in the U0126 + CDI group than in the CDI group, but higher in the U0126 +CDI group than in the U0126 group (P < 0.05).

CONCLUSIONCDI could regulate AQP3 expression level in hAECs with oligohydramnios via activating the MAPK-ERK1/2 signal transduction pathway.

Amnion ; cytology ; Aquaporin 3 ; metabolism ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; drug effects ; metabolism ; Female ; Humans ; MAP Kinase Signaling System ; Phenanthrolines ; pharmacology

The aim of our study was to investigate the differential effects of dexamethasone (DXM) and hydrocortisone (HCS) on somatic growth and postnatal lung development in a rat model of bronchopulmonary dysplasia (BPD). A rat model of BPD was induced by administering intra-amniotic lipopolysaccharide (LPS) and postnatal hyperoxia. The rats were treated with a 6-day (D1-D6) tapering course of DXM (starting dose 0.5 mg/kg/day), HCS (starting dose 2 mg/kg/day), or an equivalent volume of normal saline. DXM treatment in a rat model of BPD induced by LPS and hyperoxia was also associated with a more profound weight loss compared to control and LPS + O2 groups not exposed to corticosteroid, whereas HCS treatment affected body weight only slightly. Examination of lung morphology showed worse mean cord length in both LPS + O2 + DXM and LPS + O2 + HCS groups as compared to the LPS + O2 alone group, and the LPS + O2 + DXM group had thicker alveolar walls than the LPS + O2 group at day 14. The HCS treatment was not significantly associated with aberrant alveolar wall thickening and retarded somatic growth. The use of postnatal DXM or HCS in a rat model of BPD induced by intra-amniotic LPS and postnatal hyperoxia appeared detrimental to lung growth, but there was less effect in the case of HCS. These findings suggest that effect of HCS on somatic growth and pulmonary outcome may be better tolerated in neonates for preventing and/or treating BPD.
Amnion/drug effects ; Animals ; Animals, Newborn ; Anti-Inflammatory Agents/*pharmacology ; Dexamethasone/*pharmacology ; Disease Models, Animal ; Female ; Hydrocortisone/*pharmacology ; *Hyperoxia ; Lipopolysaccharides/toxicity ; Lung Diseases/*pathology ; Oxygen/metabolism ; Pulmonary Alveoli/*drug effects/growth & development/pathology ; Rats ; Rats, Sprague-Dawley

The aim of the study was to investigate the effect of astragalus on differentiation of human amniotic epithelial cell line WISH into neurons, the expression of Notch1 gene and cell viability. WISH were randomly divided into astragalus group (4 subgroups), alltransretinoic acid (RA) group and control group. Astragalus group and RA group were induced to differentiate into neurocytes by using chemical inducer RA and astragalus, respectively. The expression of neuron-specific enolase (NSE), microtubule associated protein 2 (MAP-2), Nestin and GFAP of induced cells in three groups were detected using immunocytochemical method. RT- PCR was further used to detect the expression of Oct4, Notch1, Hes1, Nestin and NSE. The cell viability was measured by methyl thiazolyl tetrazolium methods. Under the convert microscope it was observed that WISH cells started to change their shape, and there were several axon or dendrite-like processes out from the cell body induced by astragalus for 24 h or RA for 12 h. The positive cell rates of NSE and MAP-2 in 100 μL/mL astragalus-induced group were less than those in RA-induced group at 48 h (P < 0.05), but higher than those in control group. Cell viability in astragalus group was higher than that of RA group (P < 0.05). While the positive cell rates of Nestin and GFAP in 100 μL/mL astragalus-induced group were higher than those in RA-induced group at 48 h (P < 0.05). The positive cell rates of Nestin in the two induced groups were lower than those in control group. RT-PCR showed that the expressions of Oct4, Notch1 and Hes1 in RA and astragalus (100 μL/mL) groups were less than those in control group, but the expression of NSE was higher than that in control group. These results suggest that astragalus (especially at 100 μL/mL, 48 h) and RA can both induce human amniotic epithelial cell line WISH cells into neuron-like cells, but astragalus induction has a higher cell survival rate than RA induction, and the expression of Notch1 signal molecules is inhibited during the induction.
Amnion ; cytology ; Astragalus Plant ; chemistry ; Cell Differentiation ; drug effects ; Cell Line ; Cell Survival ; Epithelial Cells ; cytology ; Humans ; Neurons ; cytology ; Receptor, Notch1 ; metabolism ; Tretinoin ; pharmacology

OBJECTIVETo explore the immunoregulatory effects of human amniotic mesenchymal cells (hAMCs) on allogeneic peripheral blood lymphocytes.

METHODSThe hAMCs were isolated from abandoned human amnion. Peripheral blood mononuclear lymphocytes (PBMLs) were separated from healthy donors by density gradient centrifugation. Then, PBMLs were treated with phytohemagglutinin (PHA) and different concentrations of hAMCs. Proliferation effect of PBMLs was tested using MTS assay, and production of IFN-gamma and TNF-alpha by PBMLs was detected by ELISA.

RESULTShAMCs could remarkably inhibit the lymphocytes proliferation. When the ratios of hAMCs to PBMLs were 0.05: 1, 0.10 :1, 0.20: 1, the inhibitory rates of PBMLs proliferation were 16.91%, 20.83% and 28.19%, respectively. HAMCs also decreased the production of IFN-gamma and TNF-alpha by PBMLs in a dose-dependent manner (P<0.01).

CONCLUSIONSHAMCs could inhibit the proliferation of allogeneic lymphocytes and reduce secretion of IFN-gamma and TNF-alpha, which might be one of the mechanism for prevention and remission of transplant rejection.

Amnion ; cytology ; Cell Proliferation ; Humans ; Immune Tolerance ; Interferon-gamma ; biosynthesis ; Lymphocyte Activation ; immunology ; Lymphocytes ; cytology ; drug effects ; immunology ; Mesoderm ; cytology ; Phytohemagglutinins ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis

OBJECTIVETo explore the mechanism via which the epidermal growth factor (EGF) affects the migration of human amnion-derived mesenchymal stem cells (hAMSCs).

METHODSIn vitro cultured hAMSCs were divided into control (untreated), EGF group, inhibitor AG1478 + EGF group, inhibitor LY294002 + EGF group, and inhibitor U0126 + EGF group. The migration ability of hAMSCs in each group was measured using Transwell chamber. The expressions of phosphorylated EGFR (P-EGFR), phosphorylated AKT (P-AKT), and phosphorylated ERK1/2 (P-ERK1/2) as well as the expressions of metalloproteinase (MMP) -2 and MMP-9 were detected using Western blot analysis. The differentially expressed genes in the culture solutions in EGF groups and control group were analyzed with RNA-Seq technique.

RESULTSCells in EGF group had significantly stronger migration ability than in control group (P = 0.0361), inhibitor AG1478 + EGF group (P = 0.0113), inhibitor LY294002 + EGF group (P = 0.0169), and inhibitor U0126 + EGF group (P = 0.0293). EGF increased the phosphorylation levels of EGFR, AKT and ERK, and increased the expression of MMP-2. However, the increased expressions of P-AKT and P-ERK could be suppressed by AG1478 and LY294002. As shown by GO functional enrichment analysis and KEGG pathway analysis, EGF increased the transcription of genes, which were mainly involved in transcriptional regulation, protein modification, and apoptosis inhibition. Genes that were involved in the MARK pathway included DUSP5, IL1B, DUSP6, NGF, and HSPA2.

CONCLUSIONEGF-induced migration of hAMSCs may be mediated by the signaling pathways of PI3K and ERK, which needs MMP-2 expression and the co-expression of genes involved in transcriptional regulation, protein modification, and apoptosis inhibition.

Amnion ; cytology ; Cell Movement ; drug effects ; Cells, Cultured ; Epidermal Growth Factor ; pharmacology ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mesenchymal Stromal Cells ; metabolism ; physiology ; Proto-Oncogene Proteins c-akt ; metabolism

Amniotic epithelial cells (AECs) have been expected to be a good cell source for stem cell-based cardiac repair. Activin A signaling is required for cardiac differentiation in human embryonic stem cells (ESCs), and bone morphogenetic protein-4 (BMP-4) is an important regulator that controls stem cell fates. Previous study has established an efficient protocol to generate cardiomyocytes from human ESCs via induction with Activin A and BMP-4. The aim of present study was to test the hypothesis that Activin A and BMP-4 could also induce AECs to differentiate into cardiomyocytes in vitro. Human AECs (hAECs) were isolated from human term placenta by trypsin digestion according to the previous reports. Freshly isolated hAECs were examined to detect the expression of cytokeratin 19 by immunocytochemistry. High-density undifferentiated hAECs at passage 1 were sequential treated with 100 ng/ ml human recombinant Actvin A and 10 ng/ml BMP-4. The expression of cardiac-specific genes was examined before and after in vitro induction of cellular differentiation. Freshly isolated hAEC could express cytokeratin 19, the specific marker of epithelial cells. The data showed that hAECs treated with Activin A and BMP-4 were able to express cardiac-specific genes, including Nkx2.5 and alpha-actinin. Our results demonstrated that Activin A and BMP-4 could induce cardiomyocyte differentiation of hAECs, which might be a novel approach to induce differentiation of AECs into cardiomyocytes-like cells.
Activins ; pharmacology ; Amnion ; cytology ; Bone Morphogenetic Protein 4 ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cells, Cultured ; Epithelial Cells ; cytology ; Humans ; Myocytes, Cardiac ; cytology ; Recombinant Proteins ; pharmacology

BACKGROUNDHuman amniotic epithelial cells (HAECs) are able to secrete biologically active neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3, both of which exhibit trophic activities on dopamine neurons. Previous study showed that when human amniotic epithelial cells were transplanted into the striatum of 6-hydroxydopamine (6-OHDA)-induced Parkinson disease rats, the cells could survive and exert functional effects. The purpose of this study was to investigate the survival and the differentiation of human amniotic epithelial cells after being transplanted into the lateral ventricle of Parkinson's disease (PD) rats, and to investigate the effects of grafts on healing PD in models.

METHODSThe Parkinson's model was made with stereotactic microinjection of 6-hydroxydopamine (6-OHDA) into the striatum of a rat. The PD models were divided into two groups: the HAECs group and the normal saline (NS) group. Some untreated rats were taken as the control. The rotational asymmetry induced by apomorphine of the HAECs group and the NS group were measured post cell transplantation. The expression of nestin and vimentin in grafts were determined by immunohistology. Ten weeks after transplantation the density of tyrosine hydroxylase positive cells in the substantia nigra of the HAECs group, NS group and the untreated group was determined. The differentiation of grafts was determined by TH immunohistology. High performance liquid chromatography (HPLC) was used to determine monoamine neurotransmitter levels in the striatum.

RESULTSThe rotational asymmetry induced by apomorphine of the HAECs group was ameliorated significantly compared to the NS group two weeks after transplantation (P < 0.01). The grafts expressed nestin and vimentin five weeks after transplantation. TH immunohistochemistry indicated that the TH positive cells in the substantia nigra of the HAECs group increased significantly compared to the NS group (P < 0.01). Tyrosine hydroxylase (TH) positive cells in the substantia nigra of the HAEC group and the NS group were decreased compared to the untreated group (P < 0.01). Dopamine and DOPAC levels in the striatum of the HAECs group increased significantly compared to the NS group (P < 0.05). Homovanillic acid (HVA) levels in the striatum of the HAECs group increased significantly compared to the NS group (P < 0.01). In addition dopamine, DOPAC, and HVA levels in the striatum and dopamine levels in the cerebrospinal fluid of the HAECs group and the NS group were decreased compared to the untreated group (P < 0.05).

CONCLUSIONSHuman amniotic epithelial cells could be used to ameliorate the rotational asymmetry induced by apomorphine of the PD models. This could have been due to the increased content of dopamine and its metabolic products, DOPAC and HVA, in the striatum in the PD models.

Amnion ; cytology ; Animals ; Apomorphine ; pharmacology ; Chromatography, High Pressure Liquid ; Epithelial Cells ; cytology ; drug effects ; transplantation ; Female ; Homovanillic Acid ; metabolism ; Humans ; Immunohistochemistry ; Oxidopamine ; toxicity ; Parkinsonian Disorders ; chemically induced ; metabolism ; therapy ; Rats ; Rats, Sprague-Dawley