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1

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

100%
Buzanska L., Szablowska-Gadomska I.
Acta Neurobiologiae Experimentalis
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2013
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tom 73
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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
2

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

81%
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.
3

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

81%
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.
4

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

81%
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
5

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

81%
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.
6

Heterogeneity of the local tissue microenvironment influences differentiation of NG2 cells

71%
Sypecka J., Sarnowska A., Szablowska-Gadomska I., Lukomska D., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
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nr 1
The NG2 cells are the oligodendrocyte precursors that terminally differentiated are capable for myelination of central nervous system (CNS). They exhibit many features of neural stem cells and constitute the abundant population of dividing progenitors in the young and adult brain. A question arises if their commitment could be modulated by local tissue-specific or neuropathological signals. The aim of our study therefore was to evaluate the effect of distinct microenvironments (provided by either the spinal cord or the hippocampal slices) on the differentiation of neonatal NG2 cells. Subsequently, hippocampal slice culture subjected to an ischemic injury (the glucose-oxygen deprivation, OGD) was used in order to evaluate the cell development in microenvironment conditioned by traumatized tissue. Methods. Both the hippocampal and spinal cord slice cultures were established from the same 7-day old rats. The model of an indirect contact (i.e. exclusively by the culture media) in co-culture system was chosen to eliminate the influence of cell-cell contact. The NG2 cells were obtained from 10-day old mixed primary culture of neonatal rat hemispheres. After 7 days in co-culture, the cells were either stained with neural markers or collected for the RNA isolation and real-time PCR. Results. The medium conditioned by hippocampal slices effectively promoted neurogenesis: ~30 % of NG2 cells differentiated into TUJ 1-positive neurons. The remaining fraction mostly formed premyelinating and mature oligodendrocytes. The exposition of hippocampal slices to the OGD injury abolished the effect of pro-neuronal induction in co-cultures. In media conditioned by spinal cord slices, neurogenesis was less pronounced (20% neurons) and the oligodendrocyte differentiation was significantly slowed-down. Conclusions. The NG2 cells were shown to have intrinsic potency for neurogenesis. Heterogeneity of local microenvironment might modify the fate of endogenous or transplanted NG2 cells what should be taken into consideration in potential neurorepair strategies. Supported by grant 0345/B/P01/2010/38.
7

Generation of stable HEK293 cell lines expressing cell reprogramming factors

61%
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.
8

The modulation of HIFs, pluripotency and differentiation genes expression by oxygen conditions in neural stem cells derived from human cord blood

61%
Szablowska-Gadomska I., Drela K., Podobinska M., Ziemka-Nalecz M., Domanska-Janik K., Buzanska L.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr 1
The oxygen tension is an important factor modulating cell fate and developmental decisions. There are evidences that HIFs (Hypoxia Inducible Factors) family is implicated in the regulation of pluripotency and differentiation genes. The goal of this study is to compare the influence of close to physiological oxygen conditions (5%) to atmospheric oxygen tension on differentiation process and pluripotent activity in HUCB-NSC. The expression of Hypoxia Inducible Factors, stemness and neural differentiation markers in NSC, cultured under 5% and 21% oxygen were checked on the transcriptional and translational level. We were looking at the interaction between HIFs (HIF1 alpha, HIF 2 alpha) and activity of neural differentiations genes (MAP2, GFAP, β-tubulin) as well as expression of pluripotency genes (Oct4, Sox2, Rex1 and Nanog). In order to demonstrate the impact of increased HIF1α and/ or HIF2α level on cell differentiation we used DMOG (Sigma) which is of prolyl-4-hydroksylase inhibitor to increase HIF alpha levels. Our data show, that low oxygen conditions promote proliferation of HUCBNSC at early stage of development and can activate Oct4 and Nanog genes in HUCB-NSC. 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.Hypoxia Inducible Factors HIF 1α and HIF 2α, but not HIF3α are expressed in HUCB-NSC at all stages of development. During neuronal differentiation of HUCB-NSC by using dBcAMP, 5% oxygen level act synergistically, promoting further differentiation (enhanced MAP2 expression). Application of prolyl hydroxylase inhibitor – DMOG resulted in increased expression of HIF1α but not HIF2α and increased the expression of MAP2 (only in 21% oxygen conditions) referring to variants without DMOG. Sponsored by grant from Polish Ministry of Scientific Research and Higher Education No N N302 597838 and by NSC grant No 2011/01/B/NZ3/05401
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