Effects of differences in donors and passage numbers of endothelial progenitor cell (EPC)-derived vascular endothelial cells (VECs) on the phenotype determination of VECs (type-I versus type-II).
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Kumiko Saeki
PROVIDER: E-GEOD-61000 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Human VECs are categorized into two groups regarding their effects on the proliferation of vascular smooth muscle cells (VSMCs):type-I, pro-proliferative VECs and type-II anti-proliferative VECs. The effects of VECs on VSMC proliferation were quantitatively assessed according to the following method: human aortic smooth muscle cells, which were stained by PKH-26 in advance, were cultured on the layer of CFSE-stained VECs, and VSMC proliferation were evaluated after four days by flow cytometry analyses using ModFit LTM-bM-^DM-" software (Verity Software House Inc., Topsham, ME). Commercially available primary human VECs including HUVEC, HAEC and HMVEC as well as the majority of endothelial progenitor cell (EPC)-derived VECs (EPCdECs), whether EPCs were obtained from adult or fetal tissues, enhanced VSMC proliferation, showing type-I phenotype. EPCdECs of minor donors including EPC1dEC suppressed VSMC proliferation, showing type-II phenotype. However, type-II VECs turned into type-I VECs after a few rounds of subcultures. Comparative analyses on gene expression profiles between type-I VECs and type-II VECs revealed that regulator of G-protein signaling 5 (RGS5) was the only gene that showed the discriminative expression pattern: high expressions in type-I VECs and low expressions in type-II VECs. Totally six samples of type-I VECs (HUVEC, HAEC, HMVEC, EPC1dEC[P12], UCEPC1dEC, EPC2dEC[P7]) and two samples of type-II VECs (EPC1dEC[P7] and EPC1dEC[P7] purchased at a different time point) were subjected to the analyses.
2014-09-30 | E-GEOD-60999 | biostudies-arrayexpress
Project description:EPC-derived VECs (EPCdECs) are categorized into two group according to their effects on the proliferation of vascular smooth muscle cells: type-I pro-proliferative VECs and type-II anti-proliferative VECs. Type-II EPCdECs were converted to type-I VECs by repetitive subcultures. Not only subculture-dependent cellular stresses but also donor differences greatly affect the phenotype determination of VECs. By comparing the gene expression profiles of type-II EPCdEC of the first donor (EPC1dEC) at early passage and those of type-II EPC1dEC at late passage and EPCdEC of the second donor at early passage, characteristic gene expression patterns that discriminate Type-I and type-II EPCdECs will be comprehended.
2014-09-30 | GSE61000 | GEO
Project description:Human VECs are categorized into two groups regarding their effects on the proliferation of vascular smooth muscle cells (VSMCs):type-I, pro-proliferative VECs and type-II anti-proliferative VECs. The effects of VECs on VSMC proliferation were quantitatively assessed according to the following method: human aortic smooth muscle cells, which were stained by PKH-26 in advance, were cultured on the layer of CFSE-stained VECs, and VSMC proliferation were evaluated after four days by flow cytometry analyses using ModFit LT™ software (Verity Software House Inc., Topsham, ME). Commercially available primary human VECs including HUVEC, HAEC and HMVEC as well as the majority of endothelial progenitor cell (EPC)-derived VECs (EPCdECs), whether EPCs were obtained from adult or fetal tissues, enhanced VSMC proliferation, showing type-I phenotype. EPCdECs of minor donors including EPC1dEC suppressed VSMC proliferation, showing type-II phenotype. However, type-II VECs turned into type-I VECs after a few rounds of subcultures. Comparative analyses on gene expression profiles between type-I VECs and type-II VECs revealed that regulator of G-protein signaling 5 (RGS5) was the only gene that showed the discriminative expression pattern: high expressions in type-I VECs and low expressions in type-II VECs.
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Project description:Comparison of mRNA expression in human EPC vs. HUVEC vs. human monocytes. Cell-type specific gene expression under basal cell culture conditions (no stimulation). The hybridization was performed with three samples of EPC vs. three samples of HUVEC vs. 3 samples of CD14+ monocytes. â?¢ The origin of the biological sample:; Human endothelial progenitor cells (EPC): EPC were ex vivo cultivated from human peripheral blood-derived mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation from healthy human volunteers as previously described (Dimmeler et al., 2001). Pooled human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Verviers, Belgium). CD14+ monocytes were purified from PBMC by positive selection with anti-CD14-microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity assessed by FACS analysis was greater than 95%. â?¢ Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques:; EPC:; 8000000 PBMC/ml were plated on human fibronectin (Sigma, Taufkirchen, Germany) and maintained in endothelial basal medium (Cambrex) with EGM SingleQuots and 20% fetal calf serum (FCS). After 3 days, nonadherent cells were removed and adherent cells were incubated in fresh medium for 24 h before starting experiments. HUVEC:; HUVEC were cultured in endothelial basal medium (Cambrex) supplemented with hydrocortisone, bovine brain extract, gentamicin, amphotericin B, epidermal growth factor, and 10% FCS until the third passage according to the manufacturerâ??s recommendations. CD14+ monocytes: No culture. â?¢ Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol. Total RNA was extracted from EPC, HUVEC, and CD14+ monocytes using the RNeasy cleanup system (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Quantity and quality of total RNA was analyzed by the 2100 Bioanalyzer system (Agilent Technologies, Waldbronn, Germany) and agarose gel electrophoresis. â?¢ Labeling protocol(s):; The detailed protocol for the sample preparation and microarray processing is available from Affymetrix (Santa Clara, CA). Briefly, 10 µg of purified total RNA was reverse transcribed by Superscript II reverse transcriptase (Life Technologies, Grand Island, NY) using T7-(dT)24 primer containing a T7 RNA polymerase promoter. After synthesis of the second complementary DNA (cDNA) strand, this product was used in an in vitro transcription reaction to generate biotinylated complementary RNA (cRNA). â?¢ The protocol and conditions used during hybridization, blocking and washing:; Fifteen micrograms of fragmented, biotinylated cRNA were hybridized to a HG-U95Av2 microarray (Affymetrix Inc.) for 16 hours at 45° C with constant rotation at 60 rpm. This high-density oligonucleotide array targets 9,670 human genes as selected from the National Center for Biotechnology Information (NCBI) Gene Bank database with a total of 12,000 oligonucleotide sets. Each microarray was used to assay a single sample. After hybridization, the microarray was washed and stained on an Affymetrix fluidics station and scanned with an argon-ion confocal laser, with a 488 nm emission and detection at 570 nm. â?¢ GeneChip image analysis was performed using the Microarray Analysis Suite 5.0 (Affymetrix, Inc.). Expression data were analyzed utilizing the GeneSpringâ?¢ software version 4.2 (Silicon Genetics Inc., San Carlos, CA).
2008-06-11 | E-GEOD-2040 | biostudies-arrayexpress
Project description:Comparison of mRNA expression in human EPC vs. HUVEC vs. human monocytes. Cell-type specific gene expression under basal cell culture conditions (no stimulation). The hybridization was performed with three samples of EPC vs. three samples of HUVEC vs. 3 samples of CD14+ monocytes. • The origin of the biological sample: Human endothelial progenitor cells (EPC): EPC were ex vivo cultivated from human peripheral blood-derived mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation from healthy human volunteers as previously described (Dimmeler et al., 2001). Pooled human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Verviers, Belgium). CD14+ monocytes were purified from PBMC by positive selection with anti-CD14-microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity assessed by FACS analysis was greater than 95%. • Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques: EPC: 8000000 PBMC/ml were plated on human fibronectin (Sigma, Taufkirchen, Germany) and maintained in endothelial basal medium (Cambrex) with EGM SingleQuots and 20% fetal calf serum (FCS). After 3 days, nonadherent cells were removed and adherent cells were incubated in fresh medium for 24 h before starting experiments. HUVEC: HUVEC were cultured in endothelial basal medium (Cambrex) supplemented with hydrocortisone, bovine brain extract, gentamicin, amphotericin B, epidermal growth factor, and 10% FCS until the third passage according to the manufacturer’s recommendations. CD14+ monocytes: No culture. • Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol. Total RNA was extracted from EPC, HUVEC, and CD14+ monocytes using the RNeasy cleanup system (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Quantity and quality of total RNA was analyzed by the 2100 Bioanalyzer system (Agilent Technologies, Waldbronn, Germany) and agarose gel electrophoresis. • Labeling protocol(s): The detailed protocol for the sample preparation and microarray processing is available from Affymetrix (Santa Clara, CA). Briefly, 10 µg of purified total RNA was reverse transcribed by Superscript II reverse transcriptase (Life Technologies, Grand Island, NY) using T7-(dT)24 primer containing a T7 RNA polymerase promoter. After synthesis of the second complementary DNA (cDNA) strand, this product was used in an in vitro transcription reaction to generate biotinylated complementary RNA (cRNA). • The protocol and conditions used during hybridization, blocking and washing: Fifteen micrograms of fragmented, biotinylated cRNA were hybridized to a HG-U95Av2 microarray (Affymetrix Inc.) for 16 hours at 45° C with constant rotation at 60 rpm. This high-density oligonucleotide array targets 9,670 human genes as selected from the National Center for Biotechnology Information (NCBI) Gene Bank database with a total of 12,000 oligonucleotide sets. Each microarray was used to assay a single sample. After hybridization, the microarray was washed and stained on an Affymetrix fluidics station and scanned with an argon-ion confocal laser, with a 488 nm emission and detection at 570 nm. • GeneChip image analysis was performed using the Microarray Analysis Suite 5.0 (Affymetrix, Inc.). Expression data were analyzed utilizing the GeneSpring™ software version 4.2 (Silicon Genetics Inc., San Carlos, CA). Keywords: parallel sample
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Project description:Colorectal cancer (CRC) is one of the common malignancies worldwide, accounting for a significant percentage of cancer mortality. Concurrent chemoradiation (CCRT) is now a standard treatment for unresectable malignancies of anorectum. To improve quality of life, CCRT is also commonly applied in treatment of lower rectal and anal canal cancer to preserve anal sphincter function. The most commonly used chemotherapeutic drugs combined with radiation as radiosensitizers is 5-fluorouracil (5-FU). Circulating endothelial progenitor cells (EPC), which contribute to the tumor vessel formation, reflect the response to chemotherapy both in animal model and clinical trial. Thus, circulating EPC can be used as a marker for optimizing and monitoring the anti-angiogenesis therapy including angiogenesis inhibitors and chemotherapy. Whether circulating EPC can be served as a marker of CCRT efficacy or not remains undetermined. Since CCRT is now a standard treatment of locally advanced and high-risk CRC, the development of a surrogate marker for monitoring CCRT response and optimize treatment intensity is very important.
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