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

OBJECTIVE: To investigate the role of phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) inducing epithelial mesenchymal transition in human peritoneal mesothelial cells (HPMC). METHODS: Primary HPMC was harvested from human omental tissue and maintained under defined in vitro conditions. The expression of p-GSK-3beta and total GSK-3beta in HMPC was detected by Western blot after incubation with different concentrations (0, 5, 10, 20, and 40 mmol/L)of LiCl at different time points (0, 1, 3, 6, and 12 h). The protein expression of E-cadherin and alpha-SMA was also examined after treatment with 20 mmol/L LiCl according to different time courses. The intracellular distribution and expression of alpha-SMA were determined by indirect immunofluorescence. RESULTS: LiCl stimulated phosphorylation of GSK-3beta and the effect was time-dependent and concentration-dependent to limited extent (P<0.05). The expression of alpha-SMA increased (P<0.05) and the expression of E-cadherin decreased significantly (P<0.05) after 24 h stimulation by 20 mmol/L LiCl. The indirect immunoflurescence showed that the expression of alpha-SMA in HPMC increased significantly after 24 h incubation with 20 mmol/L LiCl. CONCLUSION The phosphorylation of GSK-3beta leads HMPC to epithelial mesenchymal transition and provides new clue for the treatment of peritoneal fibrosis.
Actins ; metabolism ; Cadherins ; metabolism ; Epithelial Cells ; cytology ; drug effects ; Epithelial-Mesenchymal Transition ; drug effects ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Lithium Chloride ; pharmacology ; Mesoderm ; cytology ; drug effects ; Peritoneum ; cytology ; Phosphorylation

Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.
Actin Cytoskeleton/*drug effects ; Actins/metabolism ; Animals ; Cell Differentiation/*drug effects ; Cells, Cultured ; Chickens ; Chondrogenesis/*drug effects ; Cytochalasin D/*pharmacology ; Mesoderm/cytology/drug effects ; Myosin Light Chains/metabolism ; Nucleic Acid Synthesis Inhibitors/*pharmacology ; Phosphorylation ; Staurosporine/*pharmacology ; Stress Fibers/drug effects

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.
Biological Markers/metabolism ; Cell Differentiation/*drug effects ; Cell Line ; Cell Lineage/*drug effects ; Cells, Cultured ; Down-Regulation/drug effects ; Embryo, Mammalian/cytology/drug effects/metabolism ; Embryonic Stem Cells/*cytology/*drug effects/enzymology ; Endoderm/*cytology/drug effects ; Enzyme Activation/drug effects ; Free Radical Scavengers/pharmacology ; Humans ; Mesoderm/*cytology/drug effects ; Mitogen-Activated Protein Kinases/metabolism ; Pluripotent Stem Cells/cytology/metabolism ; Reactive Oxygen Species/metabolism/*pharmacology ; Up-Regulation/drug effects

OBJECTIVETo investigate the effects of FGF-8 on cranial neural crest cell (CNCC) differentiating into ectomesenchymal cell of the first branchial arch, and determine the appropriate dose and stage of CNCC exposure to FGF-8.

METHODSCranial neural crest explants were cultured in free-serum medium containing modified DMEM/F12 and different doses of FGF-8. The differentiation type of CNCC were determined by in situ hybridization for Hoxa2 and immunocytochemistry for vimentin.

RESULTSPre-emigrating CNCC demonstrated the negative Hoxa2 stain and positive vimentin stain after treated by 100 ug/FGF-8. Both post-emigrating CNCC group and control group were positive for Hoxa2 and vimentin stain.

CONCLUSIONSOn the early stage of CNCC emigration, the first branchial arch phenotype of CNCC could be induced by FGF-8. This experiment could provide in vitro model for study on the mechanism of tooth-jaw regeneration.

Animals ; Branchial Region ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Cranial Nerves ; cytology ; Female ; Fibroblast Growth Factor 8 ; pharmacology ; Male ; Mesoderm ; cytology ; Mice ; Mice, Inbred Strains ; Neural Crest ; cytology

The present study investigated the effects of transforming growth factor (TGF)-beta on retinal pigment epithelial (RPE) transformation in a simplified model and also whether or not TGF-beta exhibits similar proliferation effects on transformed RPE cells that it has on primary RPE cells. Furthermore, we examined the cell proliferation effects of RPE-conditioned medium (CM). A vertical wound measuring 2 mm in diameter was made on primary RPE monolayers. The expression of alpha- smooth muscle actin (SMA) by the cells located at the wound edges was observed using a confocal microscope under immunofluorescent staining. Cell proliferation was measured by incorporating 3H-thymidine into DNA. The presence of alpha- SMA was observed in the cells within the wound after treatment with TGF-beta2, while negative expression was observed in control cells. TGF-betas inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, but the spindle-shaped late-passaged RPE cells were not inhibited by these growth factors. The medium conditioned by RPE cells stimulated the proliferation of subconjunctival fibroblasts and inhibited the proliferation of primary RPE cells, in a manner similar to TGF-beta. These findings demonstrate that TGF-beta-stimulated RPE cells may evoke proliferative vitreoretinopathy through mesenchymal transformation and cell proliferation.
Actins/analysis ; Animal ; Cell Division/drug effects ; Cells, Cultured ; Culture Media, Conditioned ; DNA/biosynthesis ; Mesoderm/*cytology ; Pigment Epithelium of Eye/*cytology ; Rabbits ; Swine ; Transforming Growth Factor beta/*physiology ; Vitreoretinopathy, Proliferative/etiology

To study the effect of mesenchymal stem cell (MSC) on immune function, MSCs were isolated and cultured from human bone marrow cells. The purity of MSCs were identified with the spindle-fibroblastic morphology characterization by microphotograph and the phenotypes were tested by flow cytometry. MSCs were plated in 96-well plates (2,000/well and 1,000/well), and cocultured for 3 days with T cells isolated from cord blood. Cord blood T cells non-cocultured with MSC acted as control group. After cord blood T cells stimulated by PHA for 60 hours, [(3)H]-thymidine was added to each well and T cell proliferation was assessed by [(3)H] thymidine incorporation. The results showed that cord blood T cell proliferation was suppressed when 2,000 MSCs were plated each well and cord blood T cell proliferation was activated when 1,000 MSCs were plated. Our results suggested that the immunomodulatory function of MSC seemed dependent on cell dose. High concentration of MSC most often resulted in inhibition, while low concentration resulted in stimulation.
Antigens, CD ; analysis ; Bone Marrow Cells ; cytology ; Cell Division ; immunology ; Cells, Cultured ; Coculture Techniques ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; Mesoderm ; cytology ; Phytohemagglutinins ; pharmacology ; Stem Cells ; cytology ; T-Lymphocytes ; cytology ; drug effects ; metabolism ; Thymidine ; metabolism ; Tritium

OBJECTIVETo study the isolation of human bone marrow mesenchymal stem cells (MSCs) and in vitro differentiation into chondrocytes as potential seed cell for condyle cartilage tissue engineering.

METHODSHuman MSCs were isolated by percoll solution from normal human bone marrow sample and cultured in flasks. Specific cell surface markers were identified by flow-cytometry. After the cells were treated with inductive medium containing insulin, transferrin, pyruvate, dexathemesone and TGF-beta for 7 - 14 days, microscopic, histological and immuno-histo-chemical studies were performed for chondrogenic phenotype identification.

RESULTSPrimary cultures of human MSCs express CD29 and CD44 positively and meanly, but CD34, CD45 and HLA-DR negatively. After 14 days of induction, the cells were positively stained by safranin O. Immunohistochemical analysis proved strong type II collagen expression.

CONCLUSIONSPercoll helps to generate a better isolation of MSCs from human bone marrow aspirates with a purity more above 95%. The isolated MSCs can be expanded and induced in vitro to differentiate into chondrocytes by inductive medium.

Bone Marrow Cells ; cytology ; Cartilage, Articular ; chemistry ; cytology ; Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chondrocytes ; chemistry ; cytology ; Collagen Type II ; analysis ; Dexamethasone ; pharmacology ; Humans ; Immunohistochemistry ; Insulin ; pharmacology ; Mesoderm ; cytology ; Pyruvates ; pharmacology ; Stem Cells ; cytology ; Tissue Engineering ; methods ; Transferrin ; pharmacology ; Transforming Growth Factor beta ; pharmacology

OBJECTIVETo investigate the role of connective tissue growth factor (CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.

METHODSHK-2 cells were randomly divided into two groups: (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 microg/L. The cells were collected at 72 h time points. Direct immunofluorescence staining and immunohistochemistry were used to evaluate the E-cadherin, Vimentin, alpha-SMA and ERK2 in cells. Western-blotting was used to detect the E-cadherin, Vimentin and ERK2 protein expression. Boyden Chamber was used to detect the migration of tubular endothelium at 1 d, 3 d and 5 d.

RESULTSThere were less E-cadherin but more Vimentin expressed in cells of the experimental group. The presence of alpha-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group. On the first day, the cellular migration in the two groups showed no difference. However, after 3 days, the transformed cells migrated surpassed the control group with peak at the 5th day [(45.0+/-1.1):(14.0+/-1.2), P<0.05)].

CONCLUSIONConnective tissue growth factor induces mesenchymal transformation of HK-2 cells, in which the ERK2 signaling pathway may play an important role.

Actins ; metabolism ; Cadherins ; metabolism ; Cell Line ; Cell Movement ; drug effects ; Connective Tissue Growth Factor ; pharmacology ; Epithelial Cells ; cytology ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; Humans ; Kidney Tubules, Proximal ; cytology ; Mesoderm ; cytology ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Random Allocation ; Signal Transduction ; Vimentin ; metabolism

PURPOSE: Down-regulation of E-cadherin is a hallmark of the epithelial-to-mesenchymal transition (EMT). EMT progression in cancer cells is associated with the loss of certain epithelial markers and the acquisition of a mesenchymal phenotype, as well as migratory activities. Cyclooxygenase-2 (COX-2) expression is associated with tumor invasion and metastasis in colon cancer. This study investigated the relationship between E-cadherin and COX-2 in colon cancer cells and human colon tumors. MATERIALS AND METHODS: Colon cancer cell lines and immunohistochemistry were used. RESULTS: E-cadherin expression was inversely related to the expressions of COX-2 and Snail in colon cancer cells. Ectopic expression of COX-2 or Snail reduced E-cadherin and induced a scattered, flattened phenotype with few intercellular contacts in colon cancer cells. Treatment of cancer cells with phorbol 12-myristate 13-acetate increased the expressions of COX-2 and Snail, decreased 15-hydroxyprostaglandin dehydrogenase expression, and increased the cells' motility. In addition, exposure to prostaglandin E2 increased Snail expression and cell motility, and decreased E-cadherin expression. Membranous E-cadherin expression was lower in adenomas and cancers than in the adjacent, non-neoplastic epithelium. In contrast, the expressions of Snail and COX-2 were higher in cancers than in normal tissues and adenomas. The expressions of COX-2 and Snail increased in areas with abnormal E-cadherin expression. Moreover, COX-2 expression was related to higher tumor stages and was significantly higher in nodal metastatic lesions than primary cancers. CONCLUSION: This study suggests that COX-2 may have a role in tumor metastasis via EMT.
Adult ; Aged ; Aged, 80 and over ; Blotting, Western ; Cadherins/genetics/metabolism ; Cell Differentiation/genetics/physiology ; Cell Line, Tumor ; Cell Movement/drug effects/genetics ; Colonic Neoplasms/*metabolism/*pathology ; Cyclooxygenase 2/genetics/metabolism/*physiology ; Dinoprostone/pharmacology ; Epithelial Cells/*cytology/metabolism ; Epithelium/*metabolism ; Female ; HT29 Cells ; Homeodomain Proteins/genetics/metabolism ; Humans ; Immunohistochemistry ; Male ; Mesoderm/*cytology/*metabolism ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; Tetradecanoylphorbol Acetate/pharmacology ; Transcription Factors/genetics/metabolism