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1

Influence of low oxygen tensions on expression of pluripotency genes in HUCB-NSC

100%
Szablowska-Gadomska I., Zayat V., Winiarska H., Buzanska L.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
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nr 1
The Embryonic Stem Cells (ESCs) are characterized by unlimited self-renewal ability and potential to differentiate into all cell types of the body. Those cells are derived from embryos which reside in 3-5 % oxygen environment. This hypoxic condition is physiologically normal not only for ES cells but also for many other types of stem cells, for example Neural Stem Cells. These observations suggest that hypoxic condition plays a very important role in the maintenance of cell stemness. It was also demonstrated that low oxygen tensions are preferential for maintenance of a highly proliferative, pluripotent population of hES cells. Stemness is regulated by Hypoxia Inducible Factors (HIF), which depend on oxygen tensions. HIF2A (HIF-2 alpha) is an upstream regulator of Oct4, which is the main transcription factor used by Yamanaka and his group to generate the first iPSCs (induced Pluripotent Stem Cells). It has been shown that knock-down of HIF-2 alpha or HIF-3 alpha but not HIF-1 alpha, leads to a decrease in the expression of Oct4, Nanog and Sox2, which are important stem cells markers. In this study we are trying to find out the best oxygen conditions for HUCB-NSC (human umbilical cord blood neural stem cells), from which iPS cells will be generated. We investigated the difference between the level of expression of chosen genes in HUCB-NSCs cultured under atmospheric air (21% oxygen) and 5% oxygen (low oxygen tensions). The cells were cultured for two weeks in two incubators with two different oxygen concentrations. HUCB-NSCs were grown in medium containing: DMEM/F12, 1%ITS, 2%FBS, 1%AAS. For comparison of expression levels of Oct4, Sox2 and Nanog from two different oxygen environments Real-Time RTPCR was used. In summary, the cells from low oxygen conditions had higher expression of genes: Oct4, Sox2, and Nanog compared to that of cells cultivated under atmospheric air, which is in agreement with previous observations. These outcomes indicate, that the cells from 5% oxygen conditions are the better source of cells for iPS generation than those which grow in 21% of oxygen. This is due to the higher endogenous expression genes of pluripotency what suggests possible easier generation of iPS cells and more efficient responses to reprogramming program. Thus in our further investigation on reprogramming of HUCB-NSC we will apply low oxygen conditions and epigenetic modifications in order to obtain iPS cells from HUCB-NSC cell line. Sponsored by grants from Polish Ministry of Scientific Research and Higher Education: Nr 0141/B/P01/2008/35 and Nr N N302 597838.
2

Influence of low oxygen tensions on expression of pluripotency genes in stem cells

100%
Szablowska-Gadomska I., Zayat V., Buzanska L.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr 1
The stem cells are characterized by self-renewal ability and potential to differentiate into other cell types of the body. They are residuing in defined microenvironments - "stem cell niches". The embryonic stem cells (ESC) are derived from embryos which exist in 3-5 % oxygen condition. This environment is physiologically normal not only for ES cells but also for many other types of stem cells including neural stem cells (NSC). These observations suggest that low oxygen condition plays a very important role in the maintenance of cell stemness. Pluripotency is regulated by the family of hypoxia inducible factors (HIFs), which are dependent on oxygen tensions. HIF-2a is an upstream regulator of Oct4, which is one of the main transcription factors used to generate the first induced pluripotent stem cells (iPSCs). It has been shown that knock-down of HIF-2a but not HIF-1a, leads to a decrease in the expression of Oct4, Nanog and Sox2, which are important stem cells markers. The structure of hypoxia inducible factors as well as their behavior in hypoxia and normoxia was described. Therefore optimization of oxygen concentration seems to be crucial from the stem cell transplantation as well as iPS transplantation standpoint. Although many experiments with cell culture under low oxygen condition were performed, there is still much that is unknown. This short review presents some aspects on important issue of hypoxia induced regulation of stemness.
3

Changes in pattern of DNA methylation in DE promoter region of Oct3/4 gene before and after neural differentiation of HUCB-NSC

100%
Habich A., Zayat V., Buzanska L., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
|
nr 1
Umbilical cord blood is considered as a promising source of stem cells capable of self-renewal and differentiation into different cell types, including neural. Differentiation processes are governed by microenvironmental cues and by unique molecular mechanisms, where epigenetic changes of the chromatin play an important role. Emerging evidence suggests, that changes in expression of so called “stemness” gene, like Oct3/4, are associated with the specific epigenetic modifications of gene promoter. Methylation status of Oct3/4 and Nanog promoters correlates strongly with their ability to be expressed. The promoters are unmethylated in pluripotent stem cells, where those genes are expressed, and almost fully methylated in differentiated cells, where Oct3/4 and Nanog are silenced. The aim of the study was to analyze the DE (Distal Enhancer) promoter region’s methylation pattern in Oct3/4 gene in HUCB-NSC (Human Umbilical Cord Blood - Neural Stem Cells) line comparing to hESC (Human Embryonic Stem Cells) and also changes caused by neural differentiation of HUCB-NSC. Materials and Methods. HUCB-NSC were cultured in serum free, low serum (2% FBS) and in differentiating medium containing dBcAMP (300 µM) in the density of 5x104 cells per cm2 in standard conditions. After 14 DIV DNA from harvested cells was isolated. Methylation status of gene DE promoter region was analyzed by sodium bisulfite reaction. To analyze sequence of obtained PCR fragment subcloning into pGEM-T easy vector and sequencing was performed (at least 10 individual clones). DNA of hESC was received from Prof. Dvorak laboratory in Brno. Results. Methylation pattern of Oct3/4 DE promoter region was changing along differentiation process. HUCBNSC after neural differentiation revealed higher methylation status in promoter region than in undifferentiated cells. Those changes correlate with the expression of Oct3/4 gene. Supported by grant no 0141/P01/2008/35.
4

Epigenetic and molecular signature of human umbilical cord blood-derived meural stem cell (HUCB-NSC) neuronal differentiation

88%
Habich A., Szablwska-Gadomska I., Zayat V., Buzanska L., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2013
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tom 73
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nr 1
In the context of cell therapy, the epigenetic status of core stemness transcription factor (STF) genes regulating the cell proliferation/differentiation program is of primary interest. Our results confirmed that in vitro differentiation of the umbilical- cord-blood-derived-neural-stem-cells (HUCB-NSC) coincides with the progressive down-regulation of Oct3/4 and Nanog gene expression. Consistently and in parallel with the repression of gene transcription, a substantial increase in the mosaic cytosine methylation CpG dinucleotide was observed in the promoter regions of these STF genes. However none of the histone-H3 post-translational-modifications (PTM) known to be associated with transcriptionally active genes (H3Ac and H3K4me3) or repressed genes (H3K9me3 and H3K27me3) seemed to vary in relation to the progression of cell differentiation and down-regulation of STF genes. This indicates an uncoupling between STF gene expression and above mentioned histone PTMs. In contrast, the overall methylation of nuclear chromatin at repressive histone H3K9me3 was significantly higher than H3K4 trimetylation in expanding HUCB-NSC cultures and then increases through the progression of cell differentiation. These observations suggest different epigenetic programs of gene repression realized in the cell nuclei of differentiating HUCB-NSC cultures with uneven involvement of the repressive histone PTMs.
5

Generation of stable HEK293 cell lines expressing cell reprogramming factors

76%
Sypecka J., Zayat V., Szablowska-Gadomska I., Habich A., Winiarska H., Buzanska L.
Acta Neurobiologiae Experimentalis
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2011
|
tom 71
|
nr 1
Induced pluripotent stem cells (iPSCs) are the product of somatic cell reprogramming into an undifferentiated embryonic-like state. These pluripotent cells might adopt various phenotypes by means of bioengineering methods and therefore might serve for disease modeling, pharmaceutical screening and cellular replacement therapies. Transcription factors such as Oct4, Sox2, Klf4 and Myc play the crucial role in the cell converting. The aim of our study was to obtain the protein extracts for the purpose of cell reprogramming experiments. Methods The cells of HEK 293 (Human Embryonic Kidney) line (ATTC/CRL15-73) have been transfected by non-viral, HiFect method with the pCMV cDNA9R-myc plasmid, coding one of the selected factors: Oct4, Sox2 or Klf4. After transfections, cells were cultured in low density for 2-3 weeks in the presence of neomycin to select the resistant (i.e.transfected) colonies. The expression of c-myc as a marker of stable transfectants was determined by western blot analysis. The overexpressed reprogramming proteins were gently extracted with non-denaturating CellLytic buffer supplemented with protease inhibitor cocktail and stored for the future application. Results. Isolation and propagation of an individual cells from neomycin-resistant colonies allowed us to obtain about 20-30 clones for each transcription factor. The c-myc positive clones have been selected for further in vitro culturing with the purpose of continual generation of Oct4, Sox2 or Klf4 proteins. Conclusions. The presented study resulted in successful generation of stable HEK293 cell lines that could express each of the three human reprogramming factors fused with the myc tag and with polyarginine (9R) to facilitate intracellular trafficking. The extracted proteins might therefore be used in induction of cell reprogramming experiment with the aim of generating IPSCs for potential neurorestorative therapies. Supported by grant 5978/B/P01/2010/38 and No N N302 597838.
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