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

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

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

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

This study was aimed to investigate the inhibitory mechanism of human amniotic mesenchymal stem cells (HAMSC) on lymphocyte proliferation and to validate the participation of the nonclassic human leukocyte antigen (HLA) class I molecule (HLA-G) in immunosuppressive action of HAMSC. HAMSC were isolated from fetal membranes of human placentas, and were cultured and expanded. The phenotypes of HAMSC were identified by flow cytometry, at same time the HLA-G levels on membrane surface and in cytoplasm were detected by flow cytometry. The soluble HLA-G (sHLA-G) level in HAMSC supernatants was determined by ELISA, MTT assay was used to examine the effect of mixed cultured HAMSC on proliferation of lymphocytes. The results showed that both surface and cytoplasm of HAMSC expressed HLA-G, the average rates of HLA-G expression on surface and in cytoplasm were (16.75 ± 3.871)% and (39.14 ± 4.274)%, respectively. The sHLA-G level in cell culture supernatant was 5.2 ng/ml. After HAMSC and culture supernatants were added in the MLR, the inhibitory rate on lymphocyte proliferation increased obviously, meanwhile the inhibitory rate on lymphocyte proliferation decreased when the HLA-G antibody was added in MLR. It is concluded that the surface and cytoplasm of HAMSC express HAL-G, at same time HAMSC secrete the HLA-G to supernatants of culture. The HLA-G is one of critical factors inhibiting immuno-function of HAMSC. This study contributes to improve the clinical therapeutic trails for using the HAMSC to prevent rejection.
Amnion ; cytology ; Cell Proliferation ; Cells, Cultured ; HLA-G Antigens ; immunology ; Humans ; Lymphocyte Activation ; Lymphocytes ; cytology ; immunology ; Mesenchymal Stromal Cells ; cytology ; immunology

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are capable of differentiating into several lineages and possess immunomodulatory properties. In this study, we investigated the soluble factor-mediated immunomodulatory effects of hAM-MSCs. Mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation was suppressed by hAM-MSCs in a dose-dependent manner as well as hAM-MSC culture supernatant. Moreover, interferon-gamma and interleukin (IL)-17 production significantly decreased from PBMC, whereas IL-10 from PBMCs and transforming growth factor beta (TGF-beta) production from hAM-MSCs significantly increased in co-cultures of hAM-MSCs and PBMCs. Production of several MSC factors, including hepatocyte growth factor (HGF), TGF-beta, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in hAM-MSCs co-cultured with PBMCs. These results indicate that the immunomodulatory effects of hAM-MSCs may be associated with soluble factors (TGF-beta, HGF, PGE2, and IDO), suggesting that hAM-MSCs may have potential clinical use in regenerative medicine.
Amnion/cytology/*immunology ; Cell Differentiation/immunology ; Coculture Techniques ; Dinoprostone/genetics/immunology ; Female ; Hepatocyte Growth Factor/genetics/immunology ; Humans ; Immunologic Factors/*immunology ; Immunophenotyping ; Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics/immunology ; Interferon-gamma/immunology ; Interleukin-10/analysis/immunology ; Interleukin-17/analysis/immunology ; Leukocytes, Mononuclear/cytology/immunology ; Mesenchymal Stem Cells/cytology/*immunology ; Pregnancy ; RNA, Messenger/chemistry/genetics ; Regenerative Medicine/methods ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta/genetics/immunology