Mature dendritic cells are potent antigen-presenting cells that initiate primary immune responses, while immature dendritic cells have quite different properties from mature dendritic cells and are tolerance inducer actually. Here we describe the method of using monocyte condition medium to generate dendritic cells of different maturation phases from nonproliferating progenitors in human peripheral blood. The procedure involves two steps. The first step(or priming phase) is to work on a 6-7-day culture of plastic-adherent blood monocyte in medium supplement with GM-CSF and IL-4. The second step (or differentiation phase) requires the exposure to monocyte conditioned medium. Only the dendritic cells generated by the first step are actually immature, with strong immature dendritic cell features such as active endocytosis, the same expression of monocyte marker CD14, and much of the MHC class II still lies within intracellular compartments (MIIC). The second stage dendritic cells have all the features of mature dendritic cell, including a stellate shape, nonadherence to plastic, the expression of dendritic cells restricted marker CD83, and very strong T cell stimulatory function. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. Since progression from immature to mature dendritic cell is entirely dependent on exogenously added growth factor such as monocyte condition medium, the peripheral blood monocyte may help to harness synchronized population of mature and immature dendritic cells for studies or therapies.
Cell Culture Techniques ; Cell Differentiation ; Culture Media, Conditioned ; chemistry ; Dendritic Cells ; cytology ; Granulocyte-Macrophage Colony-Stimulating Factor ; chemistry ; Humans ; Interleukin-4 ; chemistry ; Monocytes ; cytology

We have established a bone marrow endothelial cell line. This review focuses on the elucidation and analysis of the effects of this bone marrow endothelial cell-conditioned medium (BMEC-CM) on the differentiation and proliferation of hematopoietic and endothelial progenitors as well as embryonic stem cells (ESCs). We will review that (1) BMEC-CM promotes proliferation and differentiation of hematopoietic lineage; (2) BMEC-CM promotes proliferation and differentiation of endothelial lineage; (3) BMEC-CM induces differentiation of hematopoietic stem cells/progenitors into endothelial progenitors; and (4) BMEC-CM induces differentiation of ESCs into hematopoietic cells and endothelial cells. We conclude that the soluble factors secreted by BMECs are able to support the proliferation and differentiation of hematopoietic and endothelium lineages. Moreover, these soluble factors induce hematopoietic cells to differentiate to endothelial cells, and induce ESCs to differentiate towards both endothelial cells and hematopoietic cells. Therefore, this work provides evidence that a close relationship involved in the development of hematopoietic and endothelial lineage. This disclosure will be beneficial for therapy strategy in the treatment of ischemic and tumor diseases, and improve our understanding of the relationship between hematopoietic and endothelial lineages.
Bone Marrow Cells ; chemistry ; Cell Differentiation ; Cell Proliferation ; Culture Media, Conditioned ; chemistry ; Embryonic Stem Cells ; cytology ; Endothelial Cells ; chemistry ; Hematopoietic Stem Cells ; cytology ; Humans

OBJECTIVE: To perform the proteome analysis of conditioned medium prepared from mouse embryonic fibroblast feeder layers by 2-dimensional (2D) electrophoresis and mass spectrometry and to find out the possible differentiation-inhibitory factor in conditioned medium. METHODS: Feeder layers were prepared by 60Co gamma-irradiation on mouse embryonic fibroblast. Insulin-transferrin-sodium selenite supplemented medium was used to culture the feeder layers for 24 hours. The condioned medium prepared from mouse embryonic fibroblast feeder layers were made into powder by lyophilization, the redissolved solution was applied to Sephadex G-50 gel filtration chromatography, and then cold acetone was used to precipitate the proteins in the eluted solution. The protein samples were applied to 2D electrophoresis. The 2D images were analyzed by 2D image analysis software. Selected protein spots were digested by trypsin, analyzed by mass spectrometry, and then searched against the NCBInr batabase using Mascot MS/MS Ions Search. RESULTS: The protein samples extracted from mouse embryonic fibroblast feeder layers conditioned medium could be used for 2D electrophoresis. On 2D images, there were (221+/-67) spots. Most of the proteins were located in the region of MW 20 approximately 70 kD, pI 4 approximately 8. Using mass spectrometry, we preliminarily identified 13 spots: 3 keratins, 3 transferrins, 1 trypsin precursor, 2 unknown proteins (3 spots), 1 connexin 46, 1 beta-galactoside binding protein, and 1 secreted protein, acidic and rich in cysteine. CONCLUSION Conditioned medium prepared from mouse embryonic fibroblast feeder layers contain beta-galactoside binding protein and secreted protein, acidic and rich in cysteine.
Animals ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; Cysteine ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; Embryo, Mammalian ; Fibroblasts ; cytology ; Galactosides ; chemistry ; Mass Spectrometry ; Mice ; Proteome

OBJECTIVETo study, at the cytological level, the basic concept of Chinese medicine that "the Kidney (Shen) controls the bone".

METHODSKaempferol was isolated form Rhizoma Drynariae (Gu Sui Bu, GSB) and at several concentrations was incubated with opossum kidney (OK) cells, osteoblasts (MC3T3 E1) and human fibroblasts (HF) at cell concentrations of 2×10(4)/mL. Opossum kidney cell-conditioned culture media with kaempferol at 70 nmol/L (70kaeOKM) and without kaempferol (0OKM) were used to stimulate MC3T3 E1 and HF proliferation. The bone morphological protein receptors I and II (BMPR I and II) in OK cells were identified by immune-fluorescence staining and Western blot analysis.

RESULTSKaempferol was found to increase OK cell growth (P<0.05), but alone did not promote MC3T3 E1 or HF cell proliferation. However, although OKM by itself increased MC3T3 E1 growth by 198% (P<0.01), the 70kaeOKM further increased the growth of these cells by an additional 127% (P<0.01). It indicates that the kidney cell generates a previously unknown osteoblast growth factor (OGF) and kaempferol increases kidney cell secretion of OGF. Neither of these media had any significant effect on HF growth. Kaempferol also was found to increase the level of the BMPR II in OK cells.

CONCLUSIONSThis lends strong support to the original idea that the Kidney has a significant influence over bone-formation, as suggested by some long-standing Chinese medical beliefs, kaempferol may also serve to stimulate kidney repair and indirectly stimulate bone formation.

3T3 Cells ; Animals ; Cell Line ; Culture Media, Conditioned ; Intercellular Signaling Peptides and Proteins ; secretion ; Kaempferols ; pharmacology ; Kidney Tubules ; physiology ; secretion ; Medicine, Chinese Traditional ; Mice ; Opossums ; Osteoblasts ; chemistry

OBJECTIVETo investigate the effects of endothelia progenitor cells conditioned medium (EPC-CM) on the migration, adhesion and proliferation of vascular smooth muscle cells (VSMCs).

METHODSMononuclear cells were isolated from rat bone marrow by density gradient centrifugation,plated on dishes precoated with 5% fibronectin, and then cultured with complete M199 medium (including 15% fetal calf serum, 10 microg/L VEGF and 5 microg/L bFGF). EPC-CM was collected and used to incubate VSMCs isolated from rat arteriae aorta. After 24 h, VSMCs proliferation, adhesion and migration were assayed with CCK-8, adhesion test and modified Boyden chamber assay, respectively.

RESULTSThe proliferation, adhesion and migration of VSMCs were obviously decreased when the cells were cultured with EPC-CM.

CONCLUSIONEPC-CM could inhibit VSMC functions, which would be one of the mechanisms against atherosclerosis by EPCs.

Animals ; Cell Adhesion ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; Endothelial Cells ; cytology ; Male ; Muscle, Smooth, Vascular ; cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology

OBJECTIVETo explore the differences of metabolic footprint in the conditioned culture medium of placental explants between early-onset and late-onset severe preeclampsia.

METHODSIn 13 cases of early-onset severe preeclampsia and 14 cases of late-onset severe preeclampsia, the placentas were sampled at the surface of the maternal placenta. High performance liquid chromatography-mass spectrometry (HPLC-MS) was used to determine the differences in the metabolites in the conditioned culture medium of the placental villous explants cultured in 6% atmospheric O(2) for 96 h. Standard samples were used to establish the tryptophan and kynurenine chromatography library by HPLC-MS to analyze the concentration of tryptophan and kynurenine in the conditioned culture medium.

RESULTSThirty-six metabolites showed statistically significant differences between early-onset and late-onset severe preeclampsia (P<0.05). The concentration of kynurenine was significantly higher in early-onset severe preeclampsia than in late-onset severe preeclampsia (P<0.05).

CONCLUSIONEarly-onset and late-onset severe preeclampsia may have different pathogeneses. By detecting the concentration of metabolites, metabolomic strategies provide a new means for predicting the onset time of severe preeclampsia.

Chorionic Villi ; metabolism ; Culture Media, Conditioned ; chemistry ; Female ; Humans ; In Vitro Techniques ; Kynurenine ; metabolism ; Ornithine ; metabolism ; Placenta ; metabolism ; Pre-Eclampsia ; metabolism ; Pregnancy ; Tryptophan ; metabolism

OBJECTIVETo investigate the effects of X-ray exposure on the release of soluble tumor necrosis factor receptor-p75 (sTNFR-p75) in hepatocellular carcinoma HepG2 cells in vitro.

METHODSEnzyme-linked immunosorbent assay (ELISA) was used to examine the levels of sTNFR-p75 in the supernatants of HepG2 cells before and after X-ray exposure. The cell apoptosis was analyzed by flow cytometry and transmission electron microscope(TEM), and the morphological changes of the cells were examined under optical microscope and transmission electron microscope(TEM).

RESULTSX-ray exposure of the cells resulted in a strong increase of cell apoptosis (P<0.05) and sTNFR-p75 production in the cells as compared with the those before the exposure (P<0.01). Optical microscopy revealed apoptotic changes of HepG2 cell after the exposure, shown as cell shrinkage, spherical cell morphology, cytoplasmic and nuclear condensation. Apoptotic bodies were detected by TEM.

CONCLUSIONX-ray exposure induces HepG2 cells apoptosis by inhibiting the release of sTNFR-p75 into the supernatant.

Animals ; Apoptosis ; radiation effects ; Carcinoma, Hepatocellular ; pathology ; secretion ; Cell Line, Tumor ; Culture Media, Conditioned ; chemistry ; metabolism ; radiation effects ; Humans ; Liver Neoplasms ; pathology ; secretion ; Microscopy ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; chemistry ; secretion ; Solubility ; X-Rays

OBJECTIVETo investigate the influence of the endothelial cells on the scar-derived fibroblasts in the hypertropic scar tissue.

METHODSThe endothelial cell from the human umbilical vein was cultured and its supernatant was then collected. Thereafter, it was mixed with the pre-cultured scar-derived fibroblasts from the hypertropic scar tissue. The fibroblasts were analyzed with a flow cytometer in a MTT-assay method to evaluate the cell proliferation and its division cycle.

RESULTSThe endothelial cells were significantly increasing the scar-derived fibroblast proliferation (P < 0.05), compared with the control. The proportion of the fibroblasts in the S-stage of cell cycle was also quite high.

CONCLUSIONThe endothelial cells may secrete some active products, which may enhance the proliferation of the scar-derived fibroblasts and stimulate the fibroblasts into the S-stage of cell generation cycle.

Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cell Line ; Cells, Cultured ; Cicatrix, Hypertrophic ; pathology ; Culture Media, Conditioned ; chemistry ; pharmacology ; Dose-Response Relationship, Drug ; Endothelium, Vascular ; chemistry ; cytology ; Fibroblasts ; drug effects ; pathology ; Humans

Flavonoids are a class of important active ingredients in traditional Chinese medicine, pharmacological activity and in vivo process is the focus of research in recent years. Calycosin is the main active ingredients of flavonoids in Astragali Radix, recent studies indicate that it has many kinds of pharmacological activity, but the absorption and transport characteristics in vivo is unclear. The experiment using Caco-2 cell model, with apigenin as internal standard substance, using the method for the determination of drug concentration by HPLC, were studied at different concentrations and absorption transport characteristics of respectively adding different types of protein inhibitors. Data were analyzed by Q test, the results show that low, middle, high concentration of P(app)(BL-AP)/ P(app)(AP-BL) = 1.38 < 1.5, respectively adding different types of protein inhibitors, compared with the control group of P(app)(BL-AP)/ P(app)(AP-BL), there were no significant differences. Calycosin absorption may mainly passive transport, also involved in active transport mechanism, the transport may not be affected by the P-protein, MRP2 protein, SGLT protein.
Absorption ; Biological Transport ; Caco-2 Cells ; Chromatography, High Pressure Liquid ; Culture Media, Conditioned ; chemistry ; Drug Stability ; Drugs, Chinese Herbal ; analysis ; pharmacokinetics ; Humans ; Hydrogen-Ion Concentration ; Isoflavones ; analysis ; pharmacokinetics ; Models, Biological

In this study, we aimed to investigate the influences of conditioned medium from human umbilical vein endothelial cells (HUVEC) on cancer stem cell phenotype of human hepatoma cells. HUVEC and human hepatoma cells (MHCC97H) were cultured, respectively, and then the MHCC97H cells were co-cultured with conditioned medium from HUVEC (EC-CM) with Transwell system. Anti-cancer drug sensitivity, colony-formation, migration/invasion ability, expression of cancer stem cell marker and sphere formation were performed to determine the cancer stem cell phenotype in MHCC97H cells. We found that MHCC97H cells co-cultured with EC-CM exhibited significantly higher colony-formation ability and lower sensitivity of anti-cancer drugs 5-FU and Cis. Transwell assay showed that treatment with EC-CM obviously increased migration and invasion of MHCC97H cells. Moreover, increased sphere forming capability and expression of CD133 in MHCC97H cells were observed after co-cultured with EC-CM. These results suggested that EC-CM could promote cancer stem cell phenotype of hepatoma cells.
Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Coculture Techniques ; Culture Media, Conditioned ; Fluorouracil ; pharmacology ; Human Umbilical Vein Endothelial Cells ; chemistry ; Humans ; Liver Neoplasms ; Neoplastic Stem Cells ; cytology ; Phenotype