OBJECTIVETo investigate the effects of fresh and preserved amniotic membrane on polymorphonuclear neutrophils (PMNs) so as to understand the anti-inflammatory mechanism of amniotic membrane transplantation.

METHODSConditioned medium was collected 48 hours after fresh or preserved amnions were cultured in DMEM and 5% CO(2) at 37 degrees C. Then, polymorphonuclear cells were cultured in conditioned culture or DMEM. Fluorescent microscopy with 4',6-diamidino-2-phenylindole (DAPI) staining and cytometry were performed 6, 9, 12, and 15 hours later.

RESULTSApoptotic neutrophils were found in each group at different time points. The percentage of apoptotic cells at 6, 9, 12, and 15 hours after culture in the fresh and preserved amnion groups and the control group was 17.3%, 24.4%, 29.8%, 37.1%, and 16.2%, 20.1%, 23.7%, 27.7%, and 10.2%, 13.7%, 21.1%, 26.4%, respectively (t test, P(1) < 0.01, P(2) < 0.01 and P(3) < 0.01).

CONCLUSIONAmniotic membrane can accelerate apoptosis of polymorphonuclear neutrophils, reduce inflammation, and prevent ocular surface collagen from resolution, indicating that fresh amnion might have a stronger effect than preserved amnion.

Amnion ; physiology ; Apoptosis ; Cells, Cultured ; Neutrophils ; cytology ; immunology

The aim of this study was to isolate, cultivate and phenotypically characterize two types of human amnio-tic membrane (HAM)-derived cells, and to analyze their differentiation potential in vitro. Human amnion epithelial cells (hAEC) were derived from the embryonic ectoderm, while human amnion mesenchymal cells (hAMC) were derived from the embryonic mesoderm. The cells were characterized by flow cytometry and immunofluorescence, then immunofluorescence also was performed for the analysis of multipotentiality in differentiation. The results indicated that immunophenotypic characterization of both cell types demonstrated positive for HLA-A, B, C and mesenchymal stem cell markers (CD29, CD73, CD44, CD59, CD90, CD105, CD166), but did not express the hematopoietic markers (CD31, CD34, CD45, HLA-DR) and showed the weak expression of costimulatory molecules (CD40, CD40L, CD80, CD86). Phenotypes of both cell populations were maintained from passages 3 to 7. The immunofluorescence indicated that hAEC expressed cytokeratin 19, but did not express vimentin. On the contrary, hAMC expressed vimentin but did not express cytokeratin 19. The assessment of multilineage potential demonstrated that hAMC showed greater cardiomyocytes potential, while hAEC showed greater neural potential. It is concluded that hAEC and hAMC can be successfully isolated from the HAM. Both cell populations possess similar immunophenotype. However, they differ in cell yield and multipotential for differentiation into the major lineages, hAEC possess a much greater ectodermal differentiation capacity, while hAMC possess a much greater mesodermal differentiation capacity. This conclusion will be important for use of these cells in cell therapy.
Amnion ; cytology ; Cell Differentiation ; physiology ; Cell Lineage ; Epithelial Cells ; cytology ; Humans ; Immunophenotyping ; Stromal Cells ; cytology

Adult stem cells are drawing more and more attention due to the potential application in degenerative medicine without posing any moral problem. There is growing evidence showing that the human amnion contains various types of adult stem cell. Since amniotic tissue is readily available, it has the potential to be an important source of regenerative medicine material. In this study we tried to find multipotent adult stem cells in human amnion. We isolated stem cells from amniotic mesenchymal cells by limiting dilution assay. Similar to bone marrow derived mesenchymal stem cells, these cells displayed a fibroblast like appearance. They were positive for CD105, CD29, CD44, negative for haematopoietic (GlyA, CD31, CD34, CD45) and epithelial cell (pan-CK) markers. These stem cells had the potential to differentiate not only into osteogenic, adipogenic and endothelial lineages, but also hepatocyte-like cells and neural cells at the single-cell level depending on the culture conditions. They had the capacity for self-renewal and multilineage differentiation even after being expanded for more than 30 population doublings in vitro. So they may be an ideal stem cell source for inherited or degenerative diseases treatment.
Adult Stem Cells ; cytology ; Amnion ; cytology ; Cell Differentiation ; physiology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology

OBJECTIVETo establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs).

METHODShAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry.

RESULTSThe cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45.

CONCLUSIONSThis study establishes a potential method for isolation of hAMSCs from human amnion,in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.

Amnion ; cytology ; Cell Culture Techniques ; Flow Cytometry ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; cytology

Human leukocyte antigen G (HLA-G), a kind of non-classical major histocompatibility complex class I antigens, can inhibit inflammatory reaction, assist tumor cells to escape from immune surveillance and promote the immunologic tolerance of the graft. HLA-G, expressed and secreted by human amniotic mesenchymal cells (HAMC), suppresses the functions of NK cells, T cells and B cells and modulates the activity of dendritic cells (DC). These findings provide a theoretical basis for illustrating the mechanism of immunosuppression on HAMC. In this article, the recent advances on not only the gene and the molecular structure of HLA-G, but also the possible mechanisms of HLA-G in immunoregulatory function of HAMC, as well as the relation of HLA-G with HAMC, NK, DC, T and B cells are reviewed.
Amnion ; cytology ; HLA-G Antigens ; immunology ; Humans ; Immune Tolerance ; Mesenchymal Stromal Cells ; cytology ; immunology

OBJECTIVETo investigate the feasibility of inducing differentiation of the human amniotic mesenchymal cells (hAMCs) into osteoblasts in vitro, so as to provide the seed cells for bone tissue engineering.

METHODSThe hAMCs were isolated from abandoned human amnion and cultured in osteogenic media to induce the osteogenic differentiation in vitro. After hAMCs were induced by osteogenic media for 15 days, morphological observation, immunocytochemistry and western blot were used to study the cellular morphology and expression of alkaline phosphatase (ALP), type I collagen, osteopontin and osteocalcin.

RESULTSThe primary cultured hAMCs had long spindle shape or irregular shape, which were distributed evenly. The cells were usually suheultured in 5 or 7 days. After subculture, the cells became larger. After cultured by osteogenic media for 15 days, the hAMCs were detected to express ALP, osteocalcin and osteopontin, and secrete type I collagen.

CONCLUSIONSThe hAMCs are isolated, cultured and amplified easily in vitro. The induced differentiated cells by osteogenic media have typical osteoblast morphological and functional characteristics, which can be used as seed cells for bone tissue engineering.

Amnion ; cytology ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteoblasts ; cytology ; Osteogenesis ; Tissue Engineering ; methods

OBJECTIVETo isolate human amniotic mesenchymal cells (hAMCs) and investigate their transdifferentiation ability into islet-like cells in vitro.

METHODSHuman amnion was treated with the trypsin/EDTA to remove the amniotic epithelial cells and then incubated with collagenase I and dispase at 37 degrees celsius; overnight. The cells were collected by centrifugation and identified for the expressions of vimentin and SSEA-4 using immunofluorescence assay and for CD29, CD90, CD34, and CD45 using flow cytometry. RT-PCR was performed to detect the expressions of ACTG2, ACTA2, MMP2, Cripto, Sox2, LEFTYA, nanog, and Oct-4 in the cells. The differentiation potential of the isolated cells into inslet-like cells was assessed after a 14-day induction with the inducing factors by RT-PCR and immunofluorescence assay.

RESULTSThe hAMCs were capable of in vitro proliferation and passaging for 10 passages while retaining the normal karyotype. The isolated cells were positive for staining of vimentin and SSEA-4 and negative for CD34 and CD45; the CD29 and CD90 cells accounted for (91.5∓9.93)% and (48.7∓9.47)% of the cells, respectively. The hAMCs expressed several pluripotency-related genes, including Cripto, Sox2, LEFTYA, nanog, and Oct-4. After induction, endocrine-related genes were expressed in the islet-like cells, including PDX1, ngn3, insulin and glucagon.

CONCLUSIONWe have successfully established the method for isolating hAMCs, which possess the potential of differentiation into islet-like cells in vitro.

Amnion ; cytology ; Cell Culture Techniques ; methods ; Cell Transdifferentiation ; physiology ; Cells, Cultured ; Female ; Humans ; Islets of Langerhans ; cytology ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo compare the biological characteristics and immunosuppressive activity between human amniotic mesenchymal stem cells (hAMSC) and human bone marrow mesenchymal stem cells (hBMMSC).

METHODSMSC from human amnion and bone marrow were isolated using enzymatic digestion and Ficoll-Hypaque density gradients, respectively. Their biological characteristics were compared by morphology, cell growth curves, cell cycle profile analysis, immunophenotype and immunofluorescence assay. Their immunosuppressive activities were studied on total activated T-cells with phytohemagglutinin (PHA-PBMSC). An in vitro co-culture was performed to compared the lymphocyte proliferation and the supernatant level of IFN-γ were measured by CCK-8 method and ELISA, respectively.

RESULTSBoth hAMSC and hBMMSC demonstrated fibroblast-like morphology. The hAMSC were able to be amplified for at least 15 passages, while the hBMMSC only for 6-7 passages. There was no significant difference in the proportion of G2/M phase cells of the 2 cells types (P>0.05). By FACS analysis for immunophenotype, both MSC were shown to be positive for CD105, CD90, CD73 and negative for CD34, CD45, CD11b, CD19, HLA-DR, but hAMSC were positive for Oct-3/4, which was in contrast to hBMMSC. Both of them expressed vimentin. Both the cells exhibited a inhibitory role on the lymphocyte proliferation induced by PHA in co-culture conditions, that was increased with the increase MSC proportion and both the suppressing effecs were enhanced. The supernatant IFN-γ levels of hAMSC co-cultured with lymphocyte at a ratio of 1:1 after 72 hours were measured by ELISA, and the level of IFN-γ was significantly lower than that in the same co-culture system of hBMMSC. In contrast to the IFN-γ in the PHA-stimulated group, the IFN-γ level in both co-culture groups was significantly lower.

CONCLUSIONMSC from amnion displayed a higher proliferative capacity and stem cell properties, compared with hBMMSC. Both MSC can inhibit lymphocyte proliferation and suppress IFN-γ secretion induced by PHA in vitro.

Amnion ; cytology ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunophenotyping ; Immunosuppression ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; cytology ; T-Lymphocytes ; cytology

OBJECTIVETo investigate the survival, growth and distribution of human amniotic epithelial cells (hAEC) after injected into injured rabbit vocal folds, in addition, to assess the ability of hAEC to affect the components of lamina propria extracellular matrix (ECM) and prevent vocal fold scarring.

METHODShAEC were isolated from human amnion and marked by Lenti-EGFP. Fifteen New Zealand rabbits were used for this experiment. EGFP-hAEC was injected into the left injured vocal folds in thirteen rabbits, and the contralateral thirteen vocal folds experienced an injured procedure only ("injured untreated control"), and four vocal folds were left as untreated controls. The survival, distribution, differentiation potential and secretion function of hAEC were examined by immunofluorescence method. HE staining and immunohistochemical staining were performed for the evaluation of collagen and fibronectin respectively.

RESULTShAEC showed a cobblestone-like growth. After implanted into the injured vocal folds, hAEC could survive in vocal fold lamina propria for 2 months. The immunofluorescence analysis showed the evidence of hAEC differentiation into muscle cells as well as secretion the ECM protein. Three months postoperatively, the density of collagen was higher in the injured untreated control folds than that in the injured vocal folds injected with hAEC and the untreated controls. Besides, the content of fibronectin in the injured untreated control group was significantly increased.

CONCLUSIONShAEC survived in the vocal folds lamina propria, and had the potentiality to differentiate into vocal folds tissue and secret some ECM components. The histological improvement caused by the injected cells demonstrate that hAEC had the ability to promote the repairment and regeneration of injured vocal folds.

Amnion ; cytology ; Animals ; Epithelial Cells ; transplantation ; Humans ; Rabbits ; Regeneration ; Transplantation, Heterologous ; Vocal Cords ; pathology ; surgery

This study was aimed to compare the immunoregulatory effects of mesenchymal stem cells derived from human umbilical cord amnion (AMSC) and adult bone marrow (BMMSC) in vitro, so as to provide the experimental basis for allogeneic hematopoietic stem cell transplantation in clinic. The AMSC were isolated from human umbilical cord amnion by using digestion with collagenase. They were identified by morphology, growth characteristics, immunophenotyping and differentiation ability. Furthermore, the immunoregulatory effects of AMSC and BMMSC were tested by lymphocyte transformation and mixed lymphocyte reaction. The results showed that AMSC and BMMSC possessed similar biological characteristics such as exhibition of fibroblastic morphology and strong proliferation ability in vitro. Flow cytometric analysis revealed that the AMSC highly expressed CD73, CD90, CD105, but negative for CD34, CD45, HLA-DR, and CD86 of BMMSC. Functionally, they all could differentiate into adipocyte, osteocytes and chondrocytes. Moreover, AMSC could inhibit cellular or nonspecific mitogenic stimuli-induced T cell proliferation with a dose-dependent manner. Reverse transcriptional-polymerase chain reaction also demonstrated expression of the similar immune cytokines in AMSC and BMNSC. It is concluded that the MSC derived from human umbilical cord amnion may be an excellent alternative source for experimental and clinical application.
Amnion ; cytology ; immunology ; Bone Marrow Cells ; cytology ; immunology ; Cell Differentiation ; Cells, Cultured ; Humans ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; cytology ; immunology ; Umbilical Cord ; cytology