Objectives: To evaluate the immunomodulatory properties of human amniotic mesenchymal stromal cells.

Materials and Methods: Human amniotic mesen-chymal stromal cells were isolated, characterized by flow cytometry, cultured in vitro, and evaluated in allogeneic and xenogeneic mixed lymphocyte reactions. The proliferation of T cells and the expression of interleukin 2 and interferon-γ by T cells were evaluated in the presence of human amniotic mesenchymal stromal cells.

Results: Human amniotic mesenchymal stromal cells were successfully isolated from human amniotic membranes and had well-defined human mesenchymal stem cell markers (CD90, CD73, CD105, and CD166). The human amniotic mesenchymal stromal cells inhibited the proliferation of human and rabbit T cells and the secretion of interleukin-2 and interferon-γ by human T cells.

Conclusions: Human amniotic mesenchymal stromal cells may be useful for cell therapy and tissue engineering because of availability, phenotypic plasticity, and immunomodulatory properties.

Key words : Cellular immunity, Placenta, Stem cells, Transplant

Introduction

Bone marrow is the main source of mesenchymal stem cells for experimental and clinical studies. Bone marrow stem cells are used in preclinical studies for vascular disorders, neurologic disorders, bone regeneration, and anticancer therapy.^1-3 However, bone marrow stem cells are present in low quantities and collected by an invasive procedure, and their proliferation and differentiation capacity decrease with donor age.^4-5 Therefore, alternative sources of mesenchymal stem cells may be useful for cell therapy. Human adult mesenchymal stem cells from various sources have been established including adipose tissue, placenta, blood, and cord blood.^6-8 Mesenchymal stem cells derived from placenta are especially useful because they have wide differentiation potential, common accessibility, and limited ethical issues.^9,10

The human placenta has 2 sides, fetal (amnion and chorion) and maternal (decidua). The amniotic membrane is the innermost fetal membrane and includes a single layer of epithelial cells, a basement membrane, and a stromal layer. Human amniotic epithelial cells and human amniotic mesenchymal stromal cells may be released separately by differential enzymatic digestion.^11-12 Furthermore, the placenta maintains fetomaternal tolerance during pregnancy, and placental cells may have immuno-modulatory characteristics that may decrease the risk of immunologic rejection of these cells by a transplant recipient.¹³

The 2 fetal membranes (amnion and chorion) extend from the basal surface of the placenta and encase the amniotic fluid, which suspends the fetus during pregnancy. These fetal membranes facilitate exchange of gases and wastes, provide a defense barrier, and help support pregnancy and parturition.¹⁴ When amniotic cells are xenotransplanted into immunocompetent animals that have not been treated previously with immunosuppressants, the amniotic cells may survive without overt host responses. Cells that may be transplanted across major histocompatibility complex barriers without immunosuppression may have multiple allogeneic therapeutic applications.^15-16 However, the detailed properties of mesenchymal stem cells derived from the human amniotic membrane are unknown.

This study sought to perform immunohistologic analyses on unsectioned amniotic membrane to determine the spatial distribution of cells positive for stem cell markers. In addition, we evaluated immunomodulation in mixed lymphocyte reactions in vitro by coculturing human amniotic mesenchymal stromal cells with peripheral blood mononuclear cells that were isolated from whole blood. We showed that human amniotic mesenchymal stromal cells may induce inhibitory effects on allogeneic and xenogeneic T lymphocytes.

Materials and Methods

Human tissues
Human placentas (6 placentas; gestational age, 38-40 wk) were obtained at the Department of Obstetrics and Gynecology from healthy mothers during births by cesarean section. The mothers gave informed consent. The study was approved by the Ethics Committee of Wuxi People’s Hospital, and the protocols conform with the ethical guidelines of the 1975 Helsinki Declaration.

Localization of mesenchymal stem cells in the amniotic membrane
Immunofluorescence was performed on unsectioned amniotic membranes to identify the distribution of cells that had positive markers of mesenchymal stem cells. The amnion was manually peeled from the chorion near the center, trimmed to create a square sheet (3 mm × 3 mm), and stored at -80ºC until thick cryosections were prepared (thickness, 5 μm) (Leica Microsystems Nussloch Gmbh, Heidelberger, Germany). Antibody permeability was increased by incubation with a detergent (0.1% Triton X-100, Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (30 min). Nonspecific binding of immunoglobulins was blocked by incubation with a protein blocking agent (20 min) (Millipore, Billerica, MA, USA). Sections were incubated with primary antibodies against CD90 (1:500) (BD Biosciences, Franklin Lakes, NJ, USA) and CD105 (1:500) (BD Biosciences) (16 h, 4ºC). Sections were incubated with secondary goat antirat or antimouse immuno-globulin M (1:200) conjugated with fluorescein isothiocyanate (FITC) (eBioscience, San Diego, CA, USA) and viewed on a laser scanning microscope (Olympus BX-51, Olympus, Center Valley, PA, USA) equipped with an image analysis system (Olympus DP-50, Olympus).

Isolation and culture of human amniotic mesenchymal stromal cells
Human amniotic mesenchymal stromal cells were isolated and cultured with a modification of a previously described method.^17-18 The amnion was peeled away and processed separately from the chorion and washed in phosphate-buffered saline with 1% penicillin and streptomycin (Gibco BRL, Life Technologies, Grand Island, NY, USA) to remove red blood cells. For isolation of human amniotic mesenchymal stromal cells, amnions were dissected, fragmented (2-3 mm³ pieces), and incubated (15 min, 37ºC) in Dulbecco Modified Eagle Medium with low glucose (Gibco BRL, Life Technologies) and 0.25% trypsin (Gibco BRL, Life Technologies) to release amniotic epithelial cells. After centrifugation (600g, 5 min), the supernatant was discarded and the tissue was digested with collagenase I (1 mg/mL) (Sigma-Aldrich) (30 min, 37ºC) with constant agitation. The digested tissue was filtered with nylon filters (sieving net, 100 μm) (Falcon, BD Biosciences Millipore Corporation, Billerica, MA, USA). The filtered cell suspension was centrifuged (600g, 10 min) and washed 3 times with phosphate-buffered saline. Collected cells were resuspended in cell expansion medium composed of Dulbecco Modified Eagle Medium with low glucose (Gibco BRL, Life Technologies) supplemented with 20% fetal calf serum (Gibco BRL, Life Technologies) and 1% penicillin and streptomycin (Gibco BRL, Life Technologies). Cell number and viability were assessed by manual counting with a hemacytometer and trypan blue exclusion. Cells were plated (1 × 10⁴ cells/cm²) in cell expansion medium. The medium was changed every 48 hours and the first passage of cells was performed at 14 to 21 days.

Cell surface marker analysis
Cells were treated with trypsin (Gibco BRL, Life Technologies) and added to tubes (1 × 10⁵ cells/test) for fluorescence-activated cell sorting. Cell suspensions were added to the following mouse antihuman monoclonal antibodies conjugated with FITC or phycoerythrin (PE): CD34-FITC, CD14-PE, CD90-PE, CD73-FITC, human leukocyte antigen DR-FITC (BD Bioscience), CD45-PE, CD105-FITC, CD166-PE, and CD106-FITC (Biolegend, San Diego, CA, USA). Nonspecific background was evaluated by parallel staining with isotype-matched immunoglobulin G conjugates (IgG1-FITC and IgG1-PE) (BD Bioscience). Antibodies were added to the cells in the dark to avoid bleaching. After addition of the antibody, the sample was incubated at room temperature in the dark (20 min). All samples were analyzed using fluorescence-activated cell sorting (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA). A minimum 104 gated events were acquired from each sample for analysis with software (CellQuest, Becton Dickinson).

Mesodermal differentiation assays
Differentiation was induced in cells from second to fourth passages that were seeded in 6-well plates and cultured by methods as previously reported with slight modification.^19-21 Adipogenic differentiation was induced in subconfluent cells during 15 days by cultivation in expansion medium supplemented with 1 μmol/L dexamethasone (Sigma-Aldrich), 0.5 mmol/L 1-methyl-3-isobutylxanthine (Sigma-Aldrich), 0.2 μmol/L indomethacin (Sigma-Aldrich), and 10 μg/mL insulin (Sigma-Aldrich). Adipogenesis was measured by the accumulation of neutral lipids in fat vacuoles stained with Oil Red O (Sigma-Aldrich). Cells were fixed with 4% paraformaldehyde (30 min), washed, and stained with 0.16% Oil Red O (20 min).

Osteogenic differentiation was induced by treating subconfluent cells with proliferation medium over 3 weeks supplemented with1 μM dexamethasone, 10 mM glycerol-2-phosphate, 50 μM L-ascorbic acid-2-phosphate (Sigma-Aldrich), 10% fetal bovine serum, and penicillin and streptomycin. Osteogenesis was demonstrated by accumulation of mineralized calcium phosphate assessed by von Kossa stain. Cells were stained with 1% silver nitrate (45 min) (Sigma-Aldrich) under ultraviolet light and 3% sodium thiosulfate (5 min) (Sigma-Aldrich).

Chondrogenic differentiation was induced in pellet cultures initiated from 5 × 10⁵ human amniotic mesenchymal stromal cells and cultured for 3 weeks in Dulbecco Modified Eagle Medium with high glucose (Gibco BRL, Life Technologies) supplemented with 0.1 mM dexamethasone, 1 mM sodium pyruvate (Sigma-Aldrich), 50 mM L-ascorbic acid-2-phosphate, 35 mM L-proline (Sigma-Aldrich), 10 ng/mL transforming growth factor β3 (PeproTech, Rocky Hill, NJ, USA), and 50 mg/mL insulin, human transferrin, and selenous acid (ITS Premix, BS Biosciences). Chondrogenic differentiation was evaluated with Alcian blue staining. Cultured cells were washed with phosphate-buffered saline and fixed in 10% neutral-buffered formalin (room temperature, 30 min). Cells were incubated with 3% acetic acid (room temperature, 3 min) and stained with 1% Alcian blue (room temperature, 30 min) (Sigma-Aldrich). Stained cells were washed extensively in running tap water and rinsed in double-deionized water. Experiments were performed in duplicate wells.

Mixed lymphocyte reaction
Immunomodulation was evaluated in vitro by coculturing human amniotic mesenchymal stromal cells with peripheral blood mononuclear cells in mixed lymphocyte reactions. Heparinized human whole blood samples were provided by healthy volunteers who gave informed consent. The human peripheral blood mononuclear cells were obtained from the heparinized whole blood samples or buffy coat using density gradient centrifugation (density, 1.077 kg/m³). Rabbit peripheral blood mononuclear cells were obtained with the same method but different separation medium (density, 1.0965 kg/m³). Fresh or cultured human amniotic mesenchymal stromal cells (5 × 10⁴ cells) were plated onto 96-well flat-bottom plates in Dulbecco Modified Eagle Medium (complete medium) overnight and γ-irradiated (3000 cGy). For mixed lymphocyte reactions, the responder peripheral blood mononuclear cells were mixed with an equal number of γ-irradiated (3000 cGy) allogeneic stimulator peripheral blood mononuclear cells. A mixed lymphocyte reaction without human amniotic mesenchymal stromal cells was used as a control. Experiments were performed with several ratios of human amniotic mesenchymal stromal cells to peripheral blood mononuclear cells (1:5, 1:10, 1:20, 1:50, 1:100, and 1:500). All cultures were performed in triplicate in a final volume of 200 μL Dulbecco Modified Eagle Medium (complete medium). Cell proliferation was assessed after 5 days of culture by adding 3H-thymidine (1 μCi/well) (ICN Biomedicals, Irvine, CA, USA) for 16 to 18 hours. Cells were collected (Filtermate Harvester, PerkinElmer, Waltham, MA, USA) and thymidine incorporation was measured with a microplate scintillation and luminescence counter (Top Count NXT, Perkin-Elmer).

Detection of cytokine secretion
Peripheral blood mononuclear cells from 2 humans donors were plated in triplicate on round-bottom 24-well tissue culture plates and used as reactors. There were 8 groups prepared (human amniotic mesenchymal stromal cells; mixed lymphocyte reaction; mixed lymphocyte reaction plus human amniotic mesenchymal stromal cells). Within the group of mixed lymphocyte reaction plus human amniotic mesenchymal stromal cells, experiments were performed with several ratios of human amniotic mesenchymal stromal cells to peripheral blood mononuclear cells (1:5, 1:10, 1:20, 1:50, 1:100, and 1:500). At 72 hours in culture (37ºC, 5% carbon dioxide), the culture supernatant was collected for the evaluation of interleukin 2 (IL-2) and interferon γ (IFN-γ) by an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA) or stored at -80ºC.

Statistical analyses
Average data were reported as mean ± standard error. Differences were evaluated with a t test or a 1-way analysis of variance (with or without repeated measures). Statistical significance was defined by P ≤ .05.

Results

Localization of mesenchymal stem cells in the amniotic membrane
Most cells labeled with the mesenchymal stem cell markers CD90 and CD105 were human amniotic mesenchymal stromal cells from the thick basement membrane, but there were human amniotic epithelial cells derived from the embryonic ectoderm in some membranes (Figures 1A and 1B). Therefore, immunofluorescence showed that the human amniotic membrane contained cell types having characteristics of mesenchymal stem cells.

Isolation, culture, flow cytometry, and differentiation of human amniotic mesenchymal stromal cells
The cells isolated from the amniotic membrane at passage 0 were a mixed population of epithelial and fibroblastlike cells. After passage with trypsin, the cells formed a monolayer of homogenous bipolar spindle like cells with a whirlpool-like array (Figures 2A and 2B). Evaluation of the isolated cells with flow cytometry showed that these cells were positive for CD166, CD90, CD105, and CD73, and negative for the hematopoietic surface markers CD34, CD14, CD106, and CD45. Isolated cells were negative for human leukocyte antigen DR (Figure 3).

Specific factors induced adipogenic, osteogenic, and chondrogenic differentiation of human amniotic mesenchymal stromal cells. Cultured cells showed varied morphology after 1 week. The adipocyte phenotype in induced human amniotic mesenchymal stromal cells was noted by the appearance of numerous large, round, intracytoplasmic lipid droplets that stained positive with Oil Red O (Figure 4A). After exposure to osteogenic differentiation medium for 3 weeks, human amniotic mesenchymal stromal cells differentiated and mineralized, with black spots from calcium deposition noted with von Kossa stain (Figure 4B). The chondrogenic phenotype in induced human amniotic mesenchymal stromal cells was confirmed by the accumulation of sulfated proteoglycans that stained positive with Alcian blue stain (Figure 4C).

Immunologic regulation of human amniotic mesenchymal stromal cells
The proliferation of T lymphocytes was inhibited with increased ratios of human amniotic mesenchymal stromal cells to human peripheral blood mononuclear cells. There was a significant difference for human or rabbit T-cell proliferation between the group of mixed lymphocyte reactions plus human amniotic mesenchymal stromal cells compared with mixed lymphocyte reaction alone (P ≤ .05). The inhibition effect was especially high in groups with a 1:5 ratio of human amniotic mesenchymal stromal cells to human or rabbit peripheral blood mononuclear cells (Figure 5).

After human amniotic mesenchymal stromal cells were added to the mixed lymphocyte reaction system, the levels of IL-2 and IFN-γ secreted by T cells decreased significantly. Secretion of IL-2 and IFN-γ decreased progressively as the ratio of human amniotic mesenchymal stromal cells to peripheral blood mononuclear cells was increased (P ≤ .05) (Figure 6). Therefore, human amniotic mesenchymal stromal cells inhibited the secretion of the cytokines IL-2 and IFN-γ by T cells.

Discussion

In the present study, we isolated and cultured mesenchymal stromal cells from amniotic membrane and characterized the phenotype, growth characteristics, and differentiation potential. In addition, we observed the immunologic regulation of human amniotic mesenchymal stromal cells on allogeneic and xenogeneic T cells. The human amniotic mesenchymal stromal cells can inhibited T-cell proliferation in 2-way mixed lymphocyte reactions and decreased secretion of IL-2 and IFN-γ.

Human amniotic mesenchymal stromal cells derived from amniotic membranes may be useful for cell therapy and tissue engineering because of availability, phenotypic plasticity, immuno-modulatory properties, and limited ethical concerns.^22-23 The placenta is a fetomaternal organ that maintains fetal tolerance, allows nutrient uptake and gas exchange with the mother, and contains many progenitor cells or stem cells.^24-25 The human amniotic membrane contains 2 cell types with different embryologic origins because the placenta has 2 sides (fetal [amnion and chorion] and maternal [decidua]). Human amniotic epithelial cells are derived from the epiblast, and human amniotic mesenchymal stromal cells are derived from the hypoblast.^26-28

Mesenchymal stem cells may modulate the immune response. The T lymphocyte is a major effector of the adaptive immune response, and mesenchymal stem cells may modulate the function of T cells. Mesenchymal stem cells lack expression of major histocompatibility complex class II and costimulatory molecules such as CD80, CD86, or CD40.^29-31 In addition, human amniotic mesenchymal stromal cells may modulate immune cell activities and may be transplanted across major histocompatibility complex barriers.^32-33 Amniotic cells may suppress T-cell proliferation because of cell-cell contact and secrete of cytokines. Little information is available about the effector molecules from human amniotic mesenchymal stromal cells responsible for this suppression, but these molecules may include prostaglandin E2, tumor necrosis factor γ, interleukin 10, transforming growth factor β, and soluble human leukocyte antigen G.^34-35 Studies with 1-way lymphocyte reactions have shown that human amniotic mesenchymal stromal cells do not induce human T-cell proliferation.³² In the present study, we showed that human amniotic mesenchymal stromal cells may induce inhibitory effects on allogeneic and xenogeneic T cells in 2-way mixed lymphocyte reactions.

The interest in mesenchymal stem cells for biologic and clinical application has prompted the International Society for Cellular Therapy to propose minimum criteria for defining human mesenchymal stem cells, which should (1) adhere to plastic, (2) express CD73, CD90, and CD105, (3) lack express CD45, CD34, CD14, CD11b, CD79a, CD19, and human leukocyte antigen DR and (4) differentiate to osteoblasts, adipocytes, and chondroblasts in vitro.³⁶ With immunofluorescence analysis, we identified cells positive for CD90 and CD105 dispersed in an extracellular matrix largely composed of collagen and laminin. We examined the surface marker profile of the amnion-derived cells using fluorescence-activated cell sorting. The phenotypes of these cells were positive for CD90, CD44, CD105, and CD73, and negative for CD34, CD45, and human leukocyte antigen DR. We also showed that human amniotic mesenchymal stromal cells may differentiate into bone, adipose cells, and cartilage.

In conclusion, the present study showed that human amniotic mesenchymal stromal cells may be isolated from amniotic membranes and have the capacity to differentiate into multiple cell lineages. In addition, human amniotic mesenchymal stromal cells may regulate immune responses by suppressing the number and function of effector T cells. Therefore, human amniotic mesenchymal stromal cells may be useful for clinical and tissue engineering applications because these pluripotent and abundant cells have rapid proliferation, plasticity, and immunomodulatory properties.

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