Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human megakaryocytes treated with mild hyperthermia on Mk differentiation

ABSTRACT: Physical factors can have major influences on the proliferation and differentiation fate of hematopoietic stem cells in culture. Recently, we demonstrated that cord blood CD34+ cells undergo accelerated and increased megakaryocyte differentiation when incubated at 39Â°C. In this study, we investigated in detail the impacts of mild hyperthermia on the kinetics of megakaryocyte differentiation, maturation, polyploidization and cell viability. The qualitative and quantitative effects on Mk differentiation were found to be rapidly induced, and optimization of the culture length at 39Â°C led to greater Mk yields. Mild hyperthermia did not promote endomitosis of cord blood-derived Mk, but rather led to a small reduction in the proportion of polyploid Mk. Moreover, it had little impact on viability but induced a significant shift in the proportion of cells undergoing apoptosis at 37Â°C to necrosis at 39Â°C. Finally, our results suggest that the effects on megakaryocyte differentiation could be the consequences of aberrant expression of key megakaryocyte transcription factors induced by mild hyperthermia. Experiment Overall Design: To define potential differences between megakaryocytes (Mk) derived at 37Â°C or 39Â°C, we compared their gene expression repertoire by micro-gene chip technology (Affymetrix GeneChip HG133A). Two independent experiments were carried out on Mk at 37 or 39Â°C. CD41a+ Mk issued from CB CD34+ cells were cultured for 7-days with TPO at 100 ng/ml and purified by positive magnetic selection using a CD41a-FITC monoclonal antibody (Immunotech, Marseille, France) and MACS columns according to manufacturerâs instructions (Miltenyi, Auburn, CA, USA) with a purity of >95%.

ORGANISM(S): Homo sapiens

SUBMITTER: Lars Palmqvist

PROVIDER: E-GEOD-9218 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: N-Methyl-D-aspartate receptors (NMDAr), widely located around the central nervous system, are known to be involved in behavioral disorders. Dizocilpine (commonly referred to as MK-801) is a well known non-competitive NMDAr antagonist. Methods: We treated rats with intraperitoneal injection [0.08 (low-dose) and 0.16 (high-dose) mg/kg] of MK-801. In one experiment, 40 min after NaCl (vehicle control) and MK-801 (0.08 mg/kg) injection, electrocorticogram (ECoG) signals were analyzed. In the second experiment, 40 min post-injection, the whole brain of each animal was rapidly removed and separated into amyglada, cerebral cortex, hippocampus, hypothalamus, midbrain and ventral striatum) on ice, followed by analysis using a 4x44K DNA microarray chip. Results: Spectral analysis revealed that a single subcutaneous injection of MK-801 significantly and selectively augmented the power of spontaneous gamma and higher-frequency oscillations. The results from DNA microarray analysis of 4400 genes showed the largest number (up- and down- regulations) of gene expressions in the cerebral cortex (378), midbrain (376), hippocampus (375), ventral striatum (353), amygdala (301), and hypothalamus (201) under low-dose of MK-801. Under high-dose, ventral striatum (811) showed the largest number of gene expression changes. These genes represented... Conclusions: Our results reveal that MK-801 triggered i) an increase in the power of gamma oscillations, and ii) simultaneously caused very early changes in gene expressions in the rat brain, representing a first such inventory of gene expression profiles in brain after acute MK-801 treatment. Nine male 10-weeks-old Wistar rats (300-350 g BW) were housed in acrylic cages (3/cage) at 24ÂºC and given access to tap water and laboratory chow ad libitum. The rats were divided into two groups, and each group rats received i.p. injection of 0.08 (low-dose) and 0.16 (high-dose) mg/kg of MK-801, respectively. Three rats were treated with saline as sham (vehicle control group) using the same method. After 40 min post-injection, the whole brain of each animal was rapidly removed and put on ice, and brain regions were separated according to the method of Glowinski and Iversen (1996), with minor modifications (Hirano et al., 2007). Each brain region was placed in a 2 mL Eppendorf tube, quickly immersed in liquid nitrogen before being stored in -80ÂºC prior to further analysis. Each sample was immediately weighed, flash-frozen in liquid nitrogen and stored at -80ÂºC prior to further analysis. A rat 4 x 44K whole genome oligo DNA microarray chip (G4131F, Agilent Technologies, Palo Alto, CA, USA) was used for global gene expression analysis. The effects of MK-801 were examined in the 6 brain reagion, Ventral striatum, Cerebral cortex, Midbrain, Amygdala, Hippocampus, and Hypothalamus.

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Transcription profiling of human megakaryocytes treated with mild hyperthermia on Mk differentiation

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