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1

Neutralne komorki macierzyste z ludzkiej krwi pepowinowej - charakterystyka immunocytochemiczna, fizjologiczna i molekularna

100%
Buzanska L.
Postępy Biologii Komórki
|
2006
|
tom 33
|
nr 4
667-681
2

Commitment of stem-like cells into neural fate by epigenetic and genetic stimulation in vitro

88%
Buzanska L.
Acta Neurobiologiae Experimentalis
|
2001
|
tom 61
|
nr 3
3

Bioactive domains controlling neural stem cells fate decisions

63%
Zychowicz M., Buzanska L.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr S
Studying in vitro mutual interactions of the stem cells with the components of their microenvironment mimicking in vivo conditions is crucial for the further tissue engineering and regenerative medicine applications. Bioactive domains obtained by nano/micro emerging technologies, such as microcontact printing or piezoelectric spotting, were designed to reflect the stem cell niche composition and to influence their fate decisions. Different geometry of biofunctionalized surfaces, obtained by microcontact printing of poly-L-lysine or fibronectin, was created to study the adhesion, migration, proliferation as well as the differentiation of human cord blood-derived neural stem cells (HUCB-NSCs). The bioactive domains microspotted on the cell repellant surface contained extracellular matrix protein - fibronectin enriched with small signaling molecules, (CNTF, Jagged, Wnt, Shh, Dkk) were designed to activate particular molecular pathways leading to the maintenance of the self renewal of non-differentiated cells or to promote their differentiation into different neural lineages. Our results revealed how to control neural stem cell fate decisions by manipulating with the architecture and composition of the niche bioactive domains in vitro. Moreover, such domains can be used to investigate the stem cell response to cyto- or neurotoxins (MeHgCl), thus establishing good tool for screening the effect of diverse factors influencing neural stem cell development. Sponsored by Polish Ministry of Scientific Research and Higher Education grant No: 2211/B/P01/2010/38 and European Commission Joint Research Centre NanoBioscience Action
4

Epigenetic modulation of neural stem cells: The influence on reprogramming and differentiation

63%
Buzanska L., Szablowska-Gadomska I.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr 1
The differences between pluripotent and differentiated cells include stage specific chromatin structure and transcriptional hierarchy which are both regulated by and orchestrated with the epigenetic events. Such events include alterations in DNA methylation, histone modifications, polycomb gene group and noncoding RNA expression. In this lecture the overview will be given on chromatin dynamics and epigenetic modification status during neural stem cell development. Examples of regulatory machineries responsible for gene repression at each stage of neural stem cell development will be indicated. Neural stem cells are characterized by their ability to give rise to multiple neural lineages, including neurons, astrocytes, and oligodendrocytes. Previously we have obtained neural stem cells from human cord blood (HUCB-NSC) which has been investigated by our group for their ability to be reprogrammed or differentiated using combination of small molecules as epigenetic modulators. It was demonstrated that the influence of small chemicals: histone deacetylases (Trichostatin A -TSA) and methyltransferases (RG-108) on the expression of Oct4, Sox2, Rex1 and Nanog genes depended on developmental stages of HUCB-NSC. Incubation for 5 days in reprogramming conditions followed by short time culture (3 days) in ESCM (Embryonic Stem Cell Medium) on Matrigel resulted with only partial stimulation of the investigated pluripotency markers. Nevertheless, the differences in expression pattern between tested treatment conditions were observed. Cells grown under Serum Free culture conditions treated with a combination of epigenetic inhibitors as well as recombinant proteins after longer incubation in ESCM on Matrigel were able to gain full iPS morphology and showed continuous expression of pluripotent genes. None of the mentioned above factors were alone sufficient to reprogram NSC to stable pluripotency state. Additionally the mechanism of regulation DNMTs and HDACs genes (namely DNMT 3B and HDAC1) by methyltransferases and histone deacetylases inhibitors and their role in reprogramming and differentiation process of HUCB-NSC have been tested. The present study demonstated that small molecules such as TSA and RG108 together with reprogramming proteins in lowered oxygen conditions can change epigenetic status of cells and activate and sustain pluripotent state in HUCB-NSC. In conclusion it is evident that the developmental stage of the cells and epigenetic modulation play an important role in the induction of pluripotency genes expression. Sponsored by grant from Polish Ministry of Scientific Research and Higher Education Nr 5978/B/PO1/2010/38
5

Biomimetic modulation of cord blood derived stem cells for neural fate control

63%
Buzanska L., Zychowicz M.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr 1
Bioactive surface domains were applied to investigate cellular developmental processes of human cord blood- derived stem cells and to direct their fate into desired neural lineages. Such domains should represent microenvironmental cues resembling those found in vivo. For that purpose we have created miniaturized cell growth platforms with defined arrays of cell attractive biomaterials serving as functional domains. Emerging technologies applied included a nano/micro-fabrication technique like microcontact printing and piezoelectric (noncontact) microspotting of biomolecules on plasma deposited cell repellent surface. Human Umbilical Cord Blood Neural Stem Cell (HUCB-NSC) line was plated on biodomains at different concentrations and serum conditions. HUCB-NSCs were shown to adhere and differentiate on microarray platforms in a protein type, concentration and cell density dependent manner. Receptor-mediated interactions with extracellular proteins promote neuronal differentiation, while non-specific adhesion to polyaminoacid molecules allows maintaining of stem cells immobilized to the surface in non-differentiated stage. “Smart” functional domains were created by immobilizing to the surface small signaling molecules (e.g wnt, shh, notch or jagged) together with ECM proteins. Stimulation of selected intracellular pathways by signaling molecules resulted in differentiation of HUCB-NSC to either neuronal or astroglial lineage. Miniaturization of such bioengineered active domains combined with appropriate stem cell model may allow application of such stem cell growth platforms for the multiparameter bio-tests and can provide important, additional information on the sensitivity of certain neural stem cell molecular pathways to the selected neurotoxins. Since HUCB-NSC can be cultured and harvested at different developmental stages and was shown to be a good model for developmental toxicity testing, homogenous lineage related pluripotent population is required. For that purpose iPs cells from HUCB-NSC are produced. Sponsored by grant from Polish Ministry of Scientific Research and Higher Education No 0141/B/P01/2008/35, No N N302 597838. and European Commission, JRC.
6

Cytoskeletal Rearrangements during ammonia induced apoptosis in C6 glioma cells

51%
Buzanska L., Dybel A., Domanska-Janik K., Albrecht J.
Acta Neurobiologiae Experimentalis
|
1999
|
tom 59
|
nr 3
7

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

51%
Szablowska-Gadomska I., Zayat V., Winiarska H., Buzanska L.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
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.
8

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

51%
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.
9

Differentiation of neural stem cells derived from human cord blood (HUCB-NSC) under low oxygen conditions

51%
Podobinska M., Szablowska-Gadomska I., Winiarska H., Buzanska L.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr 1
In the human body, stem cells are located in niches, which are extremely complex microenvironments (with specified oxygen conditions and cellular together with extracellular matrix components arranged as a 3D structure). The influence of signals from niches seems to play an important role in maintenance of stem cells pluripotency and in their differentiation. We have been investigating the influence of the different niche components on the proliferation and differentiation of neural stem cells into specific cell types as well as the molecular mechanisms underlying this cell responses. In this study we are investigating the influence of low oxygen tension conditions on proliferation and differentiation of Human Umbilical Cord Blood of Neural Stem Cell (HUCB-NSC). Human Neural Stem Cells (NSC) in their physiological niches are exposed to 2–8% oxygen level. For that purpose, HUCB-NSC, were cultivated in two oxygen tension conditions: 21% and 5% with or without the presence of differentiation factor dBcAMP (N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt). We compared the expression of the markers characteristic for proliferation (Ki67) as well as neuronal and astroglial lineage commitment (MAP2, GFAP, β-tubulin, NF200). The presence of tested markers was revealed on the protein (immunocytochemistry) and gene expression level (Real-Time PCR). Our data show, that the low oxygen tension promote HUCB-NSC differentation into neuronal lineage. We also observed that low concentration of oxygen increases cell proliferation.Sponsored by grant from Polish Ministry of Scientific Research and Higher Education No 5978/B/PO1/2010/38
10

Involvement of ERK/SAPK members of mitogen activated protein kinase family in PKC-inhibition triggered apoptosis in N2A cell line

51%
Bronisz-Kowalczyk A., Buzanska L., Zablocka B., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
1999
|
tom 59
|
nr 3
11
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Technologie XXI wieku i komórki macierzyste w badaniach i terapii schorzeń neurologicznych

51%
Buzanska L., Zychowicz A., Sarnowska A.
Wszechświat
|
2015
|
tom 116
|
nr 01-03
12

Correlation between activity of metalloproteinases and apoptosis in rat primary cortical cultute-the effect of CsA and FK-506

51%
Berdowska P., Ziemka-Nalecz M., Buzanska L., Zalewska T.
Acta Neurobiologiae Experimentalis
|
2001
|
tom 61
|
nr 3
13

Krew pepowinowa dla mozgu

51%
Buzanska L., Lukomska B., Domanska-Janik K.
Academia. Magazyn Polskiej Akademii Nauk
|
2005
|
nr 1
26-27
14

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

51%
Habich A., Zayat V., Buzanska L., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2011
|
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.
15

Epigenetic stimulation of pluripotency genes in cord blood derived neural stem cells

51%
Szablowska-Gadomska I., Winiarska H., Buzanska L.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr S
Epigenetic cues are potent tools for in vitro control of the stem cell fate decisions. Since human iPSc, can be derived from any tissue of the body and are characterized by unlimited self-renewal and potential to differentiation into all cell types of the body, they are considered as a good source for autologous transplantation. In this report we were studying the process of induction of pluripotency in neural stem cells derived from human cord blood using only epigenetic stimulation by small molecules and changing oxygen tension. “Pluripotency” is regulated by the set of genes including the expression of Oct4, Nanog and Sox2. Methylation status of the promoters of “pluripotency” genes as well as the chromatin histon acetylation determine self-renewal and differentiation of stem cells. However the low oxygen environmental condition and small molecules have been used only as additional factors for enhancing induction of pluripotency state. We were testing the influence of lowered (5%) oxygen conditions as well as TSA and RG-108 (histon deacetylase and DNA methyltransferase inhibitors respectively) on the expression of Oct4, Sox 2, Rex1 and Nanog genes in HUCB-NSC (human umbilical cord blood neural stem cells). Cells were cultured from 5 days until one month in serum-free medium supplemented with TSA and/or RG-108 in 5% oxygen. Our results show that low oxygen tensions can activate Oct4 and Nanog genes in HUCB-NSC. Small molecules: TSA and RG-108 enhance this process and additionally induce expression of Sox2 and Rex1. The time of cultivation of the cells in low oxygen conditions and the developmental stage of the cells are the important factors for the induction of the expression of “pluripotency” genes. Our observations confirm that the low oxygen tensions promote maintenance of undifferentiated state of the cells. Sponsored by grant from Polish Ministry of Scientific Research and Higher Education No. NN302 597838.
16

Differentation of glial cells from human umbilical cord blood-derived neural stem cell line:a potent role of growth factors and neuromorphogenes

51%
Sypecka J., Buzanska L., Winiarska H., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2005
|
tom 65
|
nr 3
17

Developmental response of human umbilical cord bIood-derived neural-like cells to selected neurotoxins

51%
Buzanska L., Sypecka J., Coecke S., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2006
|
tom 66
|
nr 4
18

Sensitivity of neural stem cells derived from human umbilical cord blood (HUCB-NSC) to developmental neurotoxin - methylmercury chloride: Dependence on non-specific and receptor mediated interactions with biomolecules

51%
Dziedzicka D., Zychowicz M., Stepien P., Buzanska L.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr 1
Human neural stem cells play an important role in in vitro developmental neurotoxicity testing. The purpose of this research was to investigate the sensitivity of neural stem cells derived from human umbilical cord blood (HUCB-NSC) to methylmercury chloride (MeHgCl), and its dependence on the type of interaction on cell membrane/biomolecule interface. MeHgCl is well known neurotoxin with documented adverse influence on human central nervous system (CNS) development. Cells were cultured in 96-well plates covered with different adhesive substrates or on Petri dishes microcontact-printed with biofunctional domains. The following biomaterials were used: poly-L-lysine, the synthetic compound, which allows to create electrostatic interactions with cells, or fibronectin and vitronectin, proteins of extracellular matrix, which create receptor mediated interactions between cells and the adhesive substrate. After the incubation with different concentrations of the neurotoxin, the cell viability, ability to proliferate, and to differentiate into neural precursors of HUCB-NSCs was measured with Alamar Blue assay and immunfluorescence stainings. High concentration of MeHgCl (1 µM) significantly decreased viability of cells and their ability to proliferate. The response of cells to the toxic effect of MeHgCl was different depending on the type of adhesive substrate. Domains covered with fibronectin or vitronectin, decreased significantly HUCB-NSC sensitivity to the neurotoxin when compared to poly-L-lysine. Our results suggest that receptor mediated interactions on cell membrane/biomolecule interface may be protective in neural stem cells’ response to certain neurotoxins. Supported by MSHE grant No 5978/B/P01/38 and NN 302663940
19

Immunomodulatory capacity of Wharton’s Jelly mesenchymal stem cells after stimulation in vitro: cytokines and growth factors transcriptional response analysis

45%
Lech W., Zychowicz M., Figiel-Dabrowska A., Domanska-Janik K., Buzanska L.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Mesenchymal Stem Cells (MSC) possess ability to release cytokines and growth factors that suppress immune responses and stimulate tissue regeneration. Wharton’s Jelly-derived MSC (WJ-MSC) in the addition to the strong adjuvant properties are characterized by low immunogenicity. AIM(S): The aim of this study is to verify immunomodulatory properties of WJ‑MSC after TNFα and IFNγ stimulation in vitro, by comparative analysis of the expression of cytokines and growth factors they produce. METHOD(S): WJ-MSC isolated from human umbilical cords were cultured in closed system that provides a constant 5% oxygen concentration. We compared immunomodulatory properties of WJ-MSC in 2D or 3D structures (scaffolds) made in our laboratory. Both of those cell populations were cultured in medium with/ without stimulate factors: TNF‑α and IFN‑γ. After stimulation, 2D and 3D cell cultures were characterized with quantitative RT-PCR for the expression of various cytokines and growth factors with non-stimulated 2D cells as an internal control. WJ-MSC grown in 3D were also characterized by live/dead cells presence which were labeled with calcein AM/ethidium homodimer. RESULTS: The obtained results indicated increased expression of mRNA in 3 D structures vs. control 2 D cells for almost all analyzed cytokines: IL‑6, TGF‑β1 , BDNF, GDNF, EGF, bFGF. Moreover, TNF‑α and IFN‑γ stimulation causes even further increase of mRNA expression of those cytokines in 3 D cultures compared to non-stimulated 2 D control. In our scaffolds models, the intercellular connections which were labeled in live cells with calcein AM have been observed already after 24h of culture and are visible also during the next days of analysis. CONCLUSIONS: Finally we can conclude, that WJ-MSC produce immunomodulatory factors, and their expression can be modulated by stimulation with chosen cytokines and 3D microenvironment. Such properties of WJ-MSC are important for the potential therapeutic application in the treatment of the diseases of inflammatory and autoimmune origin. FINANCIAL SUPPORT: The work was supported by National Centre for Research and Development grant No STRATEGMED1/234261/2/NCBR/2014.
20

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

45%
Habich A., Szablwska-Gadomska I., Zayat V., Buzanska L., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
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.
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