Project description:H1 hESC were exposed to BMP4 (10 ng/ml) in absence of FGF2 for 0, 3, 12, 24, 72, and 120 h under both physiological (4 % O2) and atmospheric (20 % O2) conditions to identify candidate genes and gene networks associated with specification from totipotent precursor cells to trophoblast. RNA from cells under both conditions was isolated at each time point. Approximately 32,000 probe sets (out of 40,391) provided a signal above background at one of the time points. Of these, 14,478 showed a ≥2-fold (plus or minus) change (p<0.05) under 4 % O2, and 18,580 under 20 % O2. The data indicate the potential usefulness of hESC grown in presence of BMP4 and absence of FGF2 for studying both the emergence of trophoblast from hESC precursors and for its subsequent differentiation over time, space and varied O2 concentrations. Keywords: Genes and gene networks associated with specification from totipotent precursor cells to trophoblast
Project description:Trophoblast differentiation from human ESC has been achieved by exposing the cells to BMP4 with or without supplementation of ALK4/5/7 inhibitor (A83-01) and FGF2 signaling inhibitor (PD173074) (BAP). Here the two differentiation conditions, BMP4 and BAP were applied to two sets of human PSC lines, H1 ESC and iPSC that latter was generated by DOX-inducible lentiviral (V) transductions of umbilical cord mesenchymal cells. The V-iPSC showed residual transgene expressions from the viral vectors in DOX-free culture condition. When the both ESC and iPSC lines were differentiated simultaneously, similar time dependent morphological changes were observed but BMP4 treated V-iPSC showed a minor yet consistent lag in the differentiation progression compared to BMP4 treated hESC. Although both differentiated ESC and V-iPSC showed dominant trophoblast phenotypes, the BMP4 treated V-iPSC also expressed gene markers consistent with the presence of mesoendoderm. The BAP condition provided more efficient differentiation than BMP4 alone, and the BAP-differentiated iPSC and ESC never expressed mesoendoderm markers.
Project description:Trophoblast differentiation from human ESC has been achieved by exposing the cells to BMP4 with or without supplementation of ALK4/5/7 inhibitor (A83-01) and FGF2 signaling inhibitor (PD173074) (BAP). Here the two differentiation conditions, BMP4 and BAP were applied to two sets of human PSC lines, H1 ESC and iPSC that latter was generated by DOX-inducible lentiviral (V) transductions of umbilical cord mesenchymal cells. The V-iPSC showed residual transgene expressions from the viral vectors in DOX-free culture condition. When the both ESC and iPSC lines were differentiated simultaneously, similar time dependent morphological changes were observed but BMP4 treated V-iPSC showed a minor yet consistent lag in the differentiation progression compared to BMP4 treated hESC. Although both differentiated ESC and V-iPSC showed dominant trophoblast phenotypes, the BMP4 treated V-iPSC also expressed gene markers consistent with the presence of mesoendoderm. The BAP condition provided more efficient differentiation than BMP4 alone, and the BAP-differentiated iPSC and ESC never expressed mesoendoderm markers. Five samples, one control of undifferentiated V-iPSC (FGF2) and four differentiated samples included two H1 ESC (treated with BMP4 or BMP4+A83-01) and similarly treated two V-iPSC were analyzed. Trophoblast differentiation was conducted with BMP4 (10 ng/ml) with or without supplementation of ALK4/5/7 inhibitor (A83-01; 1 μM) to H1 ESC and V-iPSC, respective reagents were added to FGF2-free MEF-CM from the second day culture of following passages for up to six additional days.
Project description:To realize the full potential of human embryonic stem cells (hESC), it is important to develop culture conditions that maintain hESC in a pluripotent, undifferentiated state. A low O2 atmosphere (~4% O2), for example, prevents spontaneous differentiation and supports self-renewal of hESC. To identify genes whose expression is sensitive to O2 conditions, microarray analysis was performed on RNA from hESC that had been maintained under either 4% or 20% O2. Of 149 genes differentially expressed, 42 were up-regulated and 107 down-regulated under 20% O2. Several of the down-regulated genes are most likely under the control of hypoxia-inducing factors and include genes encoding enzymes involved in carbohydrate catabolism and cellular redox state. Although genes associated with pluripotency, including OCT4, SOX2 and NANOG were generally unaffected, some genes controlled by these transcription factors, including LEFTY2, showed lowered expression under 20% O2, while a few genes implicated in lineage specification were up-regulated. Although the differences between O2 conditions were generally subtle, they were observed in two different hESC lines and at different passage numbers. The data are consistent with the hypothesis that 4% O2 favors the molecular mechanisms required for the maintenance of pluripotency. This report emphasizes the importance of employing physiological concentrations of O2 when culturing hESC. The transcript profiles of cultures under 20 % O2 suggest that the cells are more poised to differentiate than when they are under the lower 4 % O2 conditions and that the down-regulation of LEFTY2 under 20 % O2 may destabilize the network of genes maintaining ESC pluripotency. Finally, the association of HIF2A with undifferentiated but not differentiating cells is consistent with a particular role for that transcription factor in control of pluripotency. Keywords: different oxygen concentrations in hESC culture condition
Project description:Time course study of lung development in normal oxygen and 10% oxygen conditions in order to study the relationship between oxidant stress and gene expression changes in the developing lung around birth. Comparison to normal liver as control tissue not under oxidant stress. Keywords: time course
Project description:Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human embryonic stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (21% O2) as somatic cells. We hypothesized that O2 levels modulate gene expression and differentiation potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (1% or 5% O2) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio- and vasculogenesis including vascular endothelial growth factor and angiopoitein-like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O2 levels; after 24 hours at 5% O2, more than 50% of cells were CD34+. Transplantation of resulting endothelial-like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction. 26 samples in a time course experiment. Gene expression is measured at 5 different time points and under 3 different oxygen levels. Two control samples are provided.
Project description:We show that FCM-BMP4-treated hESC differentiate into bona fide CTB by direct comparison to primary human placental tissues and isolated CTB through gene expresson profiling. We have compared gene expression profiling of FCM-BMP4-treated hESC to a panel of fetal tissues and first trimester tissues. Moreover, we have analysed the expression profiles of cells after shRNA knock-down of p63 compared to Scramble upon BMP4 treatment. Difference in expression between CTB trimester 1 / trimester 3 and PSC were compared the difference between FCM-BMP4-treated hESC and PSC at each time point collected.