This study was aimed to investigate the effect of human umbilical vein endothelial cells (HUVEC) on dendritic cell (DC) development. First, HUVEC were isolated from human umbilical cord by collagenase digestion, and then the morphology, immunophenotypes and functions were identified. Furthermore, the HUVEC were cocultured with CD14(+) monocytes under the cytokine condition for detecting the influence of HUVEC on differentiation of CD14(+) cells to DC. The phenotype of dendritic cells derived from CD14(+) cells was analyzed by flow cytometry, the immunoregulatory function of DC was tested by mixed lymphocyte reaction (MLR). The change of IL-6 and VEGF as well as EPK and p38 signal pathway were analyzed by neutral antibody experiment and Western blot. The results showed that HUVEC isolated from human umbilical cord were characterized by spindle-shaped morphology, homogenous immunophenotypes (vWF(+)CD31(+)CD73(+)CD45(-)HLA-DR(-)CD86(-)CD34(low)), Dil-Ac-LDL incorporation ability and forming capillary-like structures. Following stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4), HUVEC cocultures could inhibit the initial differentiation of CD14(+) monocyte to DC. Interestingly, IL-6 and VEGF enhanced the suppression effect of HUVEC on generation of DC via activation of the ERK or p38 mitogen activated protein kinase pathway. It is concluded that HUVEC are involved in DC development and can suppress the differentiation of monocyte to DC.
Cell Differentiation ; Coculture Techniques ; Dendritic Cells ; cytology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Monocytes ; cytology

OBJECTIVETo explore the way of stably inducing canine bone marrow mesenchymal stem cells (BMSCs) to differentiate into fibroblasts and myofibroblasts in vitro, and provide seed cells for fabricating tissue engineering heart valves (TEHV).

METHODSAdult canine BMSCs were separated by a gradient centrifugation on Percoll (density 1.073 g/ml), then the cells were incubated in low-glucose Dulbecco Eagle's minimum essential medium (LG-DMEM) with 10% bovine calf serum. Cell phenotype were identified by immunohistochemistry staining. The second and third generation of BMSCs were committedly induced by conditioning culture medium, which were detected by immunohistochemistry staining. The induced-BMSCs were freezed, preserved and resuscitated after 7 d to observe the cell growth, proliferation and function.

RESULTSBMSCs deriving from the bone marrow mononuclear cells separated by a Percoll gradient were positive expression of alpha-smooth muscle antibody, vimentin and negative expression of CD34, laminin. About (50 +/- 3)% induced-BMSCs were positive expression of laminin. Approximately (85 +/- 3)% freezed induced-BMSCs could be resuscitated. And the growth, proliferation and function were well.

CONCLUSIONBMSCs could be committedly induced to differentiate into fibroblasts and myofibroblasts in vitro. It is suitable to be the seed cells.

Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; Dogs ; Fibroblasts ; cytology ; Mesenchymal Stromal Cells ; cytology ; Monocytes ; cytology ; Myoblasts ; cytology

Immune and hemopoiesis are one of basic project of experimental hematology. Immune function is a essential activity of white blood cells. It was puzzled for the diversity and complexity of immune response. Polarized immune response of immune cells was discovered 30 years ago, which facilitates the study on differentiation of lymphocyte. Recently recognition on multifunctional polarized immune response of lymphocyte and monocyte/macrophage would promote to elucidate the regulatory network of immune cells, diversity and complexity of immune response as well as the study on hemopoiesis. In this paper the approach of multifunctional polarized immune response of lymphocyte, monocyte/macrophage and dendritic cells were reviewed, and their role, especially in cytokine storm and tumor pro-inflammation condition were discussed.
Cell Differentiation ; Cytokines ; immunology ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; Humans ; Monocytes ; cytology ; immunology

To establish a method for quantitative measurement of phagocytosis, the phagocytic process of apoptotic granulosa cells by monocytes was imitated in vitro. Monocytes and granulosa cells were isolated from Kunming mice and cultured. Granulosa cells were induced to apoptosis by garlic, and then co-cultured with monocytes. At different time points (1 h, 2 h, 3 h, 4 h, 5 h), co-cultured cells were observed by microscope after Wright's staining. The results showed that at the beginning of morphological changes in apoptotic granulosa cells, monocytes captured the apoptotic cells. Meanwhile, the apoptosis of granulosa cells were progressing. Debris was found in phagocytic vacuole. At the point of 3 h after co-culture, the ratio of monocytes which attached to apoptotic granulosa cells to those which engulfed the apoptotic cells was close to one. Namely, half of monocytes were in the state of recognition and half were in the state of engulfment, and this time point was named as 'half phagocytic period'. Regression analysis showed that the equation of linear regression was y = -0.247x +1.644 (y represents Attachment/Engulfment ratio, x represents co-culture time), R(2)=0.912, F=31.095, P=0.011 (<0.05), T= -5.576, P=0.011 (<0.05). In conclusion, the present mode of phagocytosis in vitro can be used as a method to quantitatively assay some effective factors such as medicines which could enhance or restrain phagocytosis.
Animals ; Apoptosis ; Coculture Techniques ; Female ; Granulosa Cells ; cytology ; Mice ; Monocytes ; cytology ; Phagocytosis

OBJECTIVETo investigate the biological properties of immature dendritic cells( imDC) derived from cord blood before and after cryopreservation, so as to provide a method for preservation of imDC.

METHODSImmature dendritic cells were generated from human cord blood (CB) monocytes and cultured with rhGM-CSF and rhIL-4, and 10% DMSO was added into culture medium as cryopreservation reagent. After freezing in - 80 degrees C refrigerator, the cells were finally cryopreserved in - 196 degrees C liquid nitrogen, and then thawed with 40 'C water, and they were finally named frozen-thawed imDC. The morphology of imDC were observed with light microscope, and TBR were calculated. Cellular surface markers for DC maturation were determined with flow cytometry, and the ability of the cells to stimulate proliferation of non-sensitized T lymphocyte was determined with allogeneic mixed lymphocyte reaction.

RESULTSMonocyte (MNC) from cord blood could differentiate into DC after GM-CSF and rhIL-4 induction. Under light microscope, the cells showed irregular morphology, with branch-like prominence on the cell surface. Similar changes were also observed with scan electron microscope. The cryopreserved imDC were resistant to trypan blue staining, and TBR was (86. 8 +/- 1. 3) % . There was no obvious difference in the cell morphology between cryopreserved and fresh imDCs. The expression of cell surface markers and maturation markers in imDCs before cryopreservation were as follows: CDla(62 +/-8)% , CD14 (18 +/- 7)% , HLA-DR (67 +/- 5)% , CD80 (13+/-7)%, CD 86 (12+/- 5) % . Though the expression of CD80, CD86 and CD83 of cryopreserved imDC increased to (15 +/-5)% , (17 +/-5)% and (7.4 +/-3. 3)% , respectively( P <0.05), they still possessed the phenotype of imDC. There exhibited no obvious difference in cmp value between fresh imDC[ (463 +/- 104) min(-1) ] and cryopreserved imDC[ (512 +/-78 )min(-1) ] , ( P > 0. 05 ). The cpm in control group was (488 +/- 197 ) min'. The stimulation index in all groups was lower than 2, and both fresh imDC and cryopreserved imDC could not stimulate the proliferation of non-sensitized T lymphocyte.

CONCLUSIONThe cryopreserved imDC exhibits immature characteristic in cell phenotypes, function and good cell activity, indicating that the method of cryopreservation of imDC is feasible.

Cell Differentiation ; Cell Separation ; Cryopreservation ; methods ; Dendritic Cells ; cytology ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Monocytes ; cytology

The main aim of this study was to investigate the biological activities and immune modulation changes of chorionic villi mesenchymal stem cells (CV-MSC) after long term culture. The morphology of the CV-MSC of passage 3 and passage 9 were observed by microscopy, and their phenotypes were detected by flow cytometry. CV-MSC of passage 3 and 9 were co-cultured with PHA-stimulated PBMNC, and IFN-γ concentration in culture medium was detected by ELISA. The mRNA expression of COX-2, HGF and HLA-G in CV-MSC were detected by real-time PCR. The results showed that after long term culture, the CV-MSC kept the MSC morphology and most of the phenotypes including CD31, CD34, CD44, CD45, CD62L, CD73, CD90, CD105, CD117, CD151, CD235a, CD271 and HLA-DR, while the CD49d was significantly up-regulated. Immune modulation ability of CV-MSC was reduced and the mRNA expression of COX-2 and HGF was down regulated after long term culture, but the expression of HLA-G did not found to be obvious change. It is concluded that the long term in vitro expansion changes the expression of CD49d and reduces immune modulation of CV-MSC.
Cells, Cultured ; Chorionic Villi ; immunology ; Female ; Humans ; Integrin alpha4 ; metabolism ; Mesenchymal Stromal Cells ; cytology ; immunology ; Monocytes ; cytology ; Placenta ; cytology ; Pregnancy

OBJECTIVETo investigate the relationship between aging and the number and function of bone marrow-derived endothelial progenitor cells (EPC) in rats.

METHODSThe number and function of bone marrow-derived EPC were measured in 4, 10 and 18 months old rats (n = 10 in each group). Mononuclear cells (MNC) were isolated from rat bone marrow by Ficoll density gradient centrifugation and cultured for 7 days, EPC were identified as adherent cells double positive stained for FITC-UEA-I and DiI-acLDL under laser confocal immunofluence microscopy. Boyden chamber was used to determine the migration capacity of EPC and the number of recultured EPC was counted to test the adherent function of EPC.

RESULTThe number (42.0 +/- 5.8 vs 30.8 +/- 4.7 vs 21.5 +/- 4.7 per field) and the migration and adhesion capacities of EPC were significantly reduced with aging.

CONCLUSIONThis study shows that the number and function of EPC are decreased with aging.

Age Factors ; Animals ; Bone Marrow Cells ; cytology ; Cell Count ; Cells, Cultured ; Endothelial Cells ; cytology ; Monocytes ; physiology ; Rats ; Rats, Sprague-Dawley

Dendritic cells (DC) are highly efficient antigen-presenting cells that initiate the primary immune response. Several laboratories have developed culture systems for human DC from peripheral blood monocytes. Most of these studies have used fetal calf serum (FCS) containing culture conditions that are inappropriate for human application. GM-CSF and IL-4 were used to make immature DC. The monocyte-conditioned medium (MCM) was used to induce the final maturation of DC. Using the previously described methods, the quality of MCM has unpredictable variations. Therefore using a defined cocktail of growth factors for the generation of mature DC would be advantageous for experimental as well as clinical purposes. In this study, it is suggested that combinations of both GM-CSF/IL-4 or GM-CSF/IL-13 could be used as the first-step culture to produce immature DC, and that cytokine cocktail (GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6, PGE2) was as efficient as MCM for the second step-culture to produce fully maturated DC. Here, we have generated an easily reproducible culture system for DC that allows for the generation of large amounts of immature and mature DC, and we also now have established the method in a FCS-free system that is suitable for clinical use.
Cell Division/drug effects ; Culture Media/pharmacology ; Cytokines/pharmacology ; Cytological Techniques* ; Dendritic Cells/cytology* ; Human ; Monocytes/cytology*

OBJECTIVETo study the effects of Lipopolysaccharide (LPS) on the maturation and secretion of human peripheral dendritic cells (DCs).

METHODSDCs from healthy human peripheral monocytes (PBMCs) were induced in vitro with rhGM-CSF, rhIL-4, Flt3-L and TNFalpha. The subjects were divided into 3 groups: the long-term group stimulated with LPS 1 microg/ml at day 1, 4, 7, 9 post culture; the short-term group stimulated with LPS 1 microg/ml at day 7 and 8 post culture, and the DCs without LPS stimulation was control group. After 10 days of culture, the morphologic features of DCs were observed by light and electron microscopes, the phenotypic patterns were characterized by flow cytometry, the proliferation of T cell were evaluated with mixed leukocytes reaction (MLR) and the levels of IL-12 and IFNgamma produced by DCs were analyzed with ELISA.

RESULTSCompared with the short-term group, the expressions of HLA-DR (65.81%+/-10.96%), CD86 (48.81%+/-18.13%), CD80 (13.56%+/-5.48%), CD83 (11.52%+/-5.09%), the secretions of IFNgamma(15.60+/-5.83 pg/ml) and IL-12 (51.77+/-11.02 pg/ml) by the DCs in long-term group were decreased obviously (P is less than 0.05) and the proliferation of homogenic lymphocyte cells (1.548+/-0.365) stimulated by DCs was also impaired (P < 0.05).

CONCLUSIONLong-term LPS stimulation can suppress the maturation and secretion of DCs, which might be the reason of poor immunity in the patients with intestinal endotoxemia.

Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Humans ; Interleukin-12 ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Monocytes ; cytology ; metabolism

PURPOSE: Dendritic cell (DC) vaccination for melanoma was introduced because melanoma carries distinct tumor-associated antigens. The purpose of this study was to investigate the efficacy and safety of DC vaccination for melanoma in Korea. MATERIALS AND METHODS: Five patients with stage IV and one with stage II were enrolled. Autologous monocyte-derived DCs (MoDCs) were cultured and pulsed with tumor-lysate, keyhole limpet hemocyanin, and cytokine cocktail for mature antigen-loaded DC. DC vaccination was repeated four times at 2-week intervals and 2-4x107 DC were injected each time. RESULTS: Reduced tumor volume was observed by PET-CT in three patients after DC vaccination. Delayed type hypersensitivity responses against tumor antigen were induced in five patients. Tumor antigen-specific IFN-gamma-producing peripheral blood mononuclear cells were detected with enzyme-linked immunosorbent spot in two patients. However, the overall clinical outcome showed disease progression in all patients. CONCLUSION: In this study, DC vaccination using tumor antigen-loaded, mature MoDCs led to tumor regression in individual melanoma patients. Further standardization of DC vaccination protocol is required to determine which parameters lead to better anti-tumor responses and clinical outcomes.
Dendritic Cells/*cytology ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Immunotherapy/*methods ; Melanoma/*therapy ; Monocytes/*cytology ; Treatment Outcome