Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies ...Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.展开更多
In this paper, we investigate the effect and the possible mechanism of high glucose levels on the calcification of human aortic smooth muscle cells （HASMCs）. HASMCs were divided into four groups： normal glucose gro...In this paper, we investigate the effect and the possible mechanism of high glucose levels on the calcification of human aortic smooth muscle cells （HASMCs）. HASMCs were divided into four groups： normal glucose group （NG）, osmolality control group （OC）, high glucose group （HG, HASMCs culture medium containing 30 mmol/L glucose）, and high glucose plus recombinant human Noggin protein （bone morphogenetic protein-2 （BMP-2） antagonist） group （HN）. The mRNA levels and the protein expressions of BMP-2 and core binding factor alpha-1 （Cbfa-1） were measured by real-time quantitative polymerase chain reaction （PCR） and Western blot. After induced by 10 mmol/L β-glycerol phosphoric acid, cells were had＇vested for assessments of alkaline phosphatase （ALP） activities at Days 1,2, and 3, and intracellular calcium contents at Days 7 and 14, respectively. High glucose levels increased the mRNA levels and the protein expressions of BMP-2 and Cbfa-1 （P〈0.05）. The expression of Cbfa-1 was partially blocked by Noggin protein （P〈0.05）, while BMP-2 was not （P〉0.05）. After being induced by β-glycerol phosphoric acid, high glucose levels increased the ALP activity [（48.63±1.03） vs. （41.42±2.28） U/mg protein, Day 3; P〈0.05] and the intracellular calcium content [（2.76±0.09） vs. （1.75±0.07） μmol/mg protein, Day 14; P〈0.05] in a time-dependent manner when compared with the NG group, while the ALP activity could not be blocked by Noggin protein [（48.63±1.03） vs. （47.37±0.97） U/mg protein, Day 3; P〉0.05]. These results show that high glucose levels can evoke the calcification of HASMCs by inducing osteoblastic trans-differentiation and intracellular calcium deposition via the BMP-2/Cbfa-1 pathway, which can be partially blocked by Noggin protein.展开更多
文摘Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.
文摘In this paper, we investigate the effect and the possible mechanism of high glucose levels on the calcification of human aortic smooth muscle cells （HASMCs）. HASMCs were divided into four groups： normal glucose group （NG）, osmolality control group （OC）, high glucose group （HG, HASMCs culture medium containing 30 mmol/L glucose）, and high glucose plus recombinant human Noggin protein （bone morphogenetic protein-2 （BMP-2） antagonist） group （HN）. The mRNA levels and the protein expressions of BMP-2 and core binding factor alpha-1 （Cbfa-1） were measured by real-time quantitative polymerase chain reaction （PCR） and Western blot. After induced by 10 mmol/L β-glycerol phosphoric acid, cells were had＇vested for assessments of alkaline phosphatase （ALP） activities at Days 1,2, and 3, and intracellular calcium contents at Days 7 and 14, respectively. High glucose levels increased the mRNA levels and the protein expressions of BMP-2 and Cbfa-1 （P〈0.05）. The expression of Cbfa-1 was partially blocked by Noggin protein （P〈0.05）, while BMP-2 was not （P〉0.05）. After being induced by β-glycerol phosphoric acid, high glucose levels increased the ALP activity [（48.63±1.03） vs. （41.42±2.28） U/mg protein, Day 3; P〈0.05] and the intracellular calcium content [（2.76±0.09） vs. （1.75±0.07） μmol/mg protein, Day 14; P〈0.05] in a time-dependent manner when compared with the NG group, while the ALP activity could not be blocked by Noggin protein [（48.63±1.03） vs. （47.37±0.97） U/mg protein, Day 3; P〉0.05]. These results show that high glucose levels can evoke the calcification of HASMCs by inducing osteoblastic trans-differentiation and intracellular calcium deposition via the BMP-2/Cbfa-1 pathway, which can be partially blocked by Noggin protein.
