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1

Searching for oligodendrocyte precursors for cell replacement therapies

100%
Sypecka J.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr 1
Inherited or acquired oligodendrocyte deficiency or abnormalities usually lead to severe neurological disorders, common in humans and animals. The wide spectrum of diseases to be treated, their frequency and lack of effective treatments result in an urgent need to elaborate alternative options such as cell replacement therapies. Elaborating the protocols for the efficient oligodendrocyte progenitor cell (OPC) generation for neurorestrative purposes still meets the essential obstacles. Searching for the most convenient, accessible and rich sources of stem-like cells is the first of them. The immunological barrier is another problem, which could however be solved by usage of either the allo- or the autografts and the pure, xeno-free compounds for the cell propagation. The third criterion is the efficiency of the progenitor derivation, proliferation and purification. The effective cellular replacement experiments carried on animal models of congenital and acquired neurodegenerative disease approached their translation into clinical practice. Notwithstanding due to urgent need for treatment of a broad spectrum of traumas and neurodegenerative disorders accompanied by hypo/demyelination, different cell sources and alternative strategies should necessarily be tested to expand the treatment options.
2

Pt point mutation in plp gene results in hyperexpression of MOG in hypomyelinated rabbit

100%
Sypecka J.
Acta Neurobiologiae Experimentalis
|
1996
|
tom 56
|
nr 1
3

In vitro model of rat perinatal hypoxia-ischemia. How temporal oxygen-glucose deprivation affects maturation of oligodendrocyte progenitor cells

63%
Janowska J., Sypecka J.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: The frequent reason of brain damage in newborns is perinatal hypoxia-ischemia. It may cause the central nervous system hypomyelination, which often results in the long-term neurobehavioral disabilities. Oligodendrocytes, responsible for myelination, differentiate from oligodendrocyte progenitor cells (OPCs) in developing brain. AIM(S): In our study we investigated how oligodendrocyte progenitor cells respond to oxygen-glucose deprivation (OGD) in the in vitro model of neonatal asphyxia. METHOD(S): OPCs were obtained from rat primary glial cultures. 24 h after seeding, the cells were exposed to short OGD procedure. Its effect on the cell proliferation was measured by immunolabeling the dividing cells for Ki67 marker and BrdU incorporation. Cell viability was evaluated with AlamarBlue testing. Impact of OGD on OPC differentiation was assessed by making microscopic examination of immunocytochemically labeled cells with antibodies against NG2 for OPCs, GalC for immature oligodendrocytes, MBP for myelinating cells. Expression of characteristic myelin proteins was additionally verified by ELISA analysis. RESULTS: The obtained data revealed that OGD stimulated proliferation of oligodendrocyte progenitor cells, which was shown in 2% higher number of Ki67‑positive cells 1 day after OGD and 17% increase in the amount of BrdU- positive cells versus control 3 days after injury. Results of AlamarBlue assay also indicated the twofold higher viability of OGD‑affected cells versus control cells 6 days after injury. Microscopic analysis, confirmed by quantitative ELISA measurement, indicated a significant inhibition of oligodendrocyte maturation after injury. CONCLUSIONS: Hypoxic‑ischemic insult, reflected in vi‑ tro by OGD procedure, alters OPCs differentiation process causing the CNS hypomyelination. In our in vitro studies we showed that this may be associated with the enhanced proliferation of oligodendrocyte progenitors as a compensative mechanism for the injury and the disrupted maturation process. FINANCIAL SUPPORT: Supported by NCN grant 2014/15/B/NZ4/01875.
4

Fate of glial progenitors is dictated by the local tissue microenvironment

63%
Sypecka J., Sarnowska A.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr 1
The oligodendrocyte progenitors (OPCs) are the abundant population of NG2-positive cells in the young and adult CNS. They are capable of myelinogenesis, but they are also among the first cells to react to CNS injuries. Over the last decade, these glia commited progenitors have been however the subject of intensive research in context of their assumed neural stem cell properties. In our studies we have addressed the question of the impact of the local tissue microenvironment on the OPC commitment and differentiation. Their susceptibility to external stimuli and assumed intrinsic neurogenic potential have been investigated in co-culture models with organotypic slices derived from two distinct CNS regions (hippocampus and spinal cord). The hippocampal slice culture exposed to oxygen glucose deprivation (OGD) was used to evaluate the cell differentiation in microenvironment conditioned by traumatized tissue. The results have shown that the local instructive clues not only trigger the neuronal commitment of oligodendrocyte progenitors, but also govern the oligodendroglial maturation. While the trophic factors secreted by hippocampal slices efficiently promoted neurogenesis, the observed effect was significantly abolished in co-cultures with the OGD-subjected tissue. The less pronounced susceptibility to adopting neuronal phenotype and the considerable slowdown of oligodendroglial differentiation was observed in the co-cultures with the spinal cord slices. Our findings indicate that OPCs actually meet some of the neural stem cell criteria. The obtained results also suggest that the specificity of the instructive clue cocktail might module the fate choice of mobilized endogenous or transplanted cells, which is important while planning neurorepair strategies. Supported by grant 0345/B/P01/2010/38.
5

The neuroprotective efFect exerted by oligodendroglial progenitors on ischemically impaired hippocampal slices

63%
Sypecka J., Sarnowska A.
Acta Neurobiologiae Experimentalis
|
2015
|
tom 75
|
nr Supl.
The role of oligodendrocytes is supposed to go beyond the process of myelinating nerve fibers in the central nervous system.  Pre-clinical studies based on oligodendrocyte progenitor cell (OPCs) therapies revealed a significant behavioral improvement in spite of failure in the remyelination process in animal models of demyelinating diseases. To address the issue of potential trophic support conferred by OPC to diseased nervous tissue, co-culture experiments with neonatal rat OPCs and organotypic hippocampal slices were designed. For this purpose, rat organotypic hippocampal slices were exposed to a brief oxygen and glucose deprivation (OGD) which allowed mimicking an ischemic injury in vitro. Soon after the OGD procedure, the hippocampal slices were co-cultured together with differentiating oligodendroglial progenitors. Cell survival and their proliferation rates in the injured slices were estimated by immunohistochemical methods. After acknowledging the beneficial influence of the neighboring oligodendrocytes on the injured tissue, a set of molecular and biochemical experiments were carried out with the aim of determining the mechanism(s) of the observed profound neuroprotective effect. The results of both molecular studies and functional assays revealed that the oligodendrocyte-derived BDNF is the major factor promoting neuronal survival in OGD-subjected hippocampal slices, while SCF and IL-10 strongly promote the cell proliferation. Among the newly-born cells, neuroblasts and microglia could most frequently be found pointing to both the neuroprotective and neuroimmunomodulatory role of differentiating oligodendrocytes. In conclusion, the presented study revealed that oligodendrocytes are able to secret BDNF and other active factors, thus providing trophic support for other neural cells. The presented study confirms the hypothesis concerning the complex functions of oligodendrocytes in the nervous tissue. Supported by MMRC statutory funds.
6

PT-rabbit mutation: genetic characteriscic and phenotypic expression

63%
Sypecka J., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
1995
|
tom 55
|
nr 5
7

Rabbit paralytic tremor phenotype - a plp1 gene mutation as a model of human Pelizaeus-Merzbacher disease

63%
Sypecka J., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2005
|
tom 65
|
nr 2
8

The crucial role of the local microenvironment in fate-decision of neonatal rat NG2 progenitors

51%
Sypecka J., Sarnowska A., Winiarska K., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2009
|
tom 69
|
nr 1
The fate choice of neural progenitors could be dictated by local cellular environment of adult CNS. The aim of our study was to investigate the effect of hippocampal tissue on the differentiation and maturation of the oligodendrocyte NG2 precursors. Methods: The hippocampal slice culture was established from the brains of 7-day old rats. The NG2 precursors, obtained from a 12-day old mixed primary culture of neonatal rat hemispheres, were labeled with CMFDA and seeded on hippocampal slices. After 7ñ14 days in co-culture, the cells were stained with neural markers. Results: The NG2 cells differentiated predominantly into oligodendrocytes, presenting various stages of maturation: progenitors NG2+, O4+ and fi nally mature Galc-positive cells. However, except for a few cells with astrocyte-specifi c S100β staining, a considerable number of these cells differentiated into TUJ+ and MAP-2+ neurons. Moreover, a certain population of these cells preserved proliferative properties of primary precursors, as revealed by the Ki67 expression. Conclusions: Neuronal microenvironment provided by the culture of hippocampal slices is potent to induce neurogenesis from oligodendrocyte NG2+/PDGFRα+/CNP+ progenitors and promotes their differentiation not only into macroglia but also into neurons. It also sustains their proliferative capacity. The results indicate a crucial role of the local cellular environment in fatedecision of primary NG2+ multipotent neural progenitors, which may affect their behavior after transplantation into CNS. Supported by MSHE grant N40101832 /0296
9

Laminin promotes oligogliogenesis and increases MMPs activity in human neural stem cells of HUCB-NSC line

51%
Sypecka J., Dragun-Szymczak P., Zalewska T., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2009
|
tom 69
|
nr 1
Oligodendrocytes, the cells responsible for myelin formation and maintenance in CNS, are depleted in many acute and chronic conditions. The stem/progenitor cells stimulation or transplantation might be seriously considered as a long hopedfor therapeutic perspective. Better understanding of the mechanism(s) regulating the activation of the cell lineage from the endogenous progenitor reservoir might be helpful. Therefore an efficient source of donor cells for transplantation in humans is being craved for. In this study we show that the application of extracellular matrix component-laminin promotes oligogliogenesis from neural stem-like cells of human cord blood cells (HUCB-NSC). Although oligodendrocytes constitute a minor subpopulation of spontaneously differentiated HUCB-NSC, the manipulation of active compounds regulating the process of cell commitment results in a several fold increase in their number. Thus cells of the HUCB-NSC line could be considered as a potential source of glial cells, fulfilling the suitable candidate criteria for oligodendrocyte replacement therapy.
10

Fibronectin-the most potent factor in neural stem cells of HUCB-NSC line development is associated with MMPs activity

51%
Dragun-Szymczuk P., Sypecka J., Zalewska T.
Acta Neurobiologiae Experimentalis
|
2009
|
tom 69
|
nr 1
Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are considered the main regulators of the cell microenvironment which governs NSC development. The aim of our study was to investigate whether the selected ECM components (laminin, fi bronectin, collagen) infl uence the neural stem cells proliferation and differentiation. In addition we checked if MMPs are engaged in the above developmental processes. Methods: The cells of HUCB-NSC line were seeded on the ECM components-coated plates. The serum-free medium was applied 48 h before experiments. On the 4th, 8th and 14th day in culture, cell proliferation assay and in situ zymography were performed, followed by the immunocytochemistry with the specifi c neural markers. Results: Our results showed that fi bronectin stimulated cell proliferation as well as MMPs activity most intensively (~20% increase in the 2-week cultured cells). It was also the most potent factor in promoting the cell differentiation, mostly toward neurons. We also checked the involvement of the MMPs in cell development. For this purpose we used MMPs inhibitors: GM6001 and TIMPs as well as inhibitors for serine and furin proteases ñ Pefabloc and DecRVKR-CMK. We found that only inhibitors of MMPs infl uence stem cells developmental processes. GM6001 down-regulated cell proliferation (~30%) and differentiation into neurons (~20%). The decrease of cell proliferation was also observed in the presence of TIMP2. In contrast, TIMP1 accelerated cell divisions. Conclusion: Our results demonstrate that fi bronectin is the potent factor in promoting the cell proliferation, differentiation, and support the participation of MMPs in the mechanism(s) responsible for neural stem cells maturation. Supported by MSRHE grants: 1266/P01/2006/31 and N40101832 /0296
11

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
12

New method of longitudinal spinal cord slice culture

51%
Koniusz S., Kawalec M., Sypecka J., Sarnowska S.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr 1
Organotypic slice cultures were established as a model that own properties of both cell culture and animal model. The most often used slice culture is derived from hippocampus but depending on the part of brain affected with pathology, researchers established cultures from cerebellum, midbrain or striatum. Above mentioned models allowed the investigation of disorders resulting from e.g. ischemia, trauma or toxic injury. Besides the brain injury, numerous studies were focused on spinal cord pathology connected with demyelination, inflammation or injury. Here, we describe the development of an in vitro model of longitudinal spinal cord slice culture. Compared to cell (neuron-oligodendrocyte) co-cultures, organotypic slices retain tissue organization as well as cell-cell contacts and therefore more closely mimic the environment in vivo. We demonstrate the applicability of this approach for xenograft transplantation of oligodendrocyte precursor cells derived from rat brain and mesenchymal stem cells derived from human umbilical cord. Stem cells fate after transplantation was observed in two paradigms: after cell transplantation on the top of spinal cord slice cultures (SCC) or cocultivation of cell culture with SCC space separated for 24 h. We observed the different morphology and protein expression of stem cells derived from different sources. Moreover, the same stem cells co-cultured with slices derived from different part of brain (hippocampus or spinal cord) expressed other markers. The method of longitudinal spinal cord slices enables observation of long fibers trajectory, new connections and neurorepair mechanisms. Moreover, it provides a time-efficient and costeffective adjunct to cell lines or in vivo transplantation models for study spinal cord pathology or experimental therapies. Furthermore, the approach can be readily used to assess the effect of pharmacological manipulations on myelin, providing a tool to better understand myelination and develop effective therapeutic strategies to treat myelin-related diseases. Supported by National Science Centre grant: 05728/B/NZ4/2011/01
13

Hippocampal microenvironment instructs NG2 precursors to become neurons

51%
Sypecka J., Sarnowska A., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2007
|
tom 67
|
nr 3
14

Effect of matrix metalloproteinases inhibition on the proliferation and differentiation of HUCB-NSCS cultured in the presence of adhesive substrates

51%
Szymczak P., Sypecka J., Wojcik-Stanaszek L., Zalewska T.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr S
Cell adhesion to extracellular matrix (ECM) generates intracellular signals that modulate cell survival, proliferation, migration of neural precursor cells and differentiation in vitro. The function of ECM in developmental processes may be associated with particular patterns of proteolysis of ECM. Among the proteases the matrix metalloproteinases (MMPs) represent family of enzymes responsible for the modification of ECM components and by this may influence cell development. The aim of our study was to determine the potential of native ECM proteins: fibronectin, laminin and collagen, on the proliferation and differentiation of HUCB-NSCs cultured in serum free condition. In an effort to elucidate the engagement of MMPs we have checked the effect of inhibitors - SB-3CT, GM6001 and doxycycline on the above development-associated processes. Among the tested substrates the highest proliferation rate (evaluated by using anti-Ki67) was noted in the presence of fibronectin. Fibronectin also occurred most permissive substrate in HUCB-NSCs differentiation toward neuronal fate. The endogenous activity of MMPs (assayed by in situ zymography) corresponds to the rate of cell proliferation. Addition of MMPs inhibitors resulted in the significant suppression of proliferation potential and inhibition of neuronal cell generation (TUJ1- and MAP2-positive) with simultaneous promotion of oligo- and astroglial cells (expressing GALC and S100β, respectively). In conclusion, our results suggest that MMPs might be an important component in neurogenesis-associated processes. Supported by MSRHE grants: 0154/B/ P01/2010/38 and 0345/B/P01/2010/38.
15

Fibronectin stimulates the MMPs activity during neural stem cells development in2vitro

51%
Dragun P., Sypecka J., Zalewska T.
Acta Neurobiologiae Experimentalis
|
2007
|
tom 67
|
nr 3
16

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
17

Relationship between extracellular matrix components and MMPs activity during development of neural stem cells from umbilical cord blood (HUCB-NSC)

51%
Szymczak P., Sypecka J., Zalewska T.
Acta Neurobiologiae Experimentalis
|
2009
|
tom 69
|
nr 3
Accumulating evidence indicates that extracellular matrix components and metalloproteinases play a pivotal role in the development of stem cells. The aim of our study was to check if the presence of laminin, fi bronectin, and collagen affect MMPs activity (MMP-9 and MMP-2) which in turn might enhance cell proliferation and differentiation. The cells of HUCB-NSC were cultured without serum for two weeks on extracellular matrix components-coated plates. On 4th, 8th, and 14th day we performed proliferation assay, and determination of MMPs activity (in situ zymography) followed by immunocytochemistry with specifi c neural markers. Our results show that among all of the investigated ECM components fi bronectin stimulated most intensively cell proliferation and differentiation, especially toward neurons. We also observed the increase of MMPs activity (~20% increase in the 2-weeks culture) in the presence of fi bronectin. To confi rm the infl uence of MMPs on developmental processes of neural stem cells we used inhibitors of MMPs – GM6001, and doxycycline. We found that the inhibition of MMPs by GM6001 decreased cell proliferation (~30%) and differentiation into neurons (~20%). As it was shown in the present study the fi bronectin occured to be the potent factor in promoting cell proliferation and differentiation and support the idea that MMPs may contribute to the mechanism involved in the development of neural stem cells. Supported by MSRHE grants: 1266/P01/2006/31 and N40101832 /0296.
18

Effect of matrix metalloproteinases inhibition on the proliferation and differentiation of HUCB-NSCs cultured in the presence of adhesive substrates

45%
Szymczak P., Wojcik-Stanaszek L., Sypecka J., Sokolowska A., Zalewska T.
Acta Neurobiologiae Experimentalis
|
2010
|
tom 70
|
nr 4
Cell adhesion to extracellular matrix (ECM) generates intracellular signals that modulate cell survival, proliferation, migration and differentiation. Because of its heterogeneous nature, ECM has the potential to induce unique responses that are composition-dependent. One approach to study the effect of ECM signals on cell development, independently on signals from other extracellular sources, has been to deprive cells of serum and to analyze the influence of specific ligands. In the current work we determine the potential of different ECM proteins (fibronectin, laminin, collagen) on the proliferation and differentiation of human umbilical cord blood-derived neural stem cells (HUCB-NSCs) cultured in serum-free conditions. The effect of tested ECM components on the above processes might be associated with the particular pattern of their proteolysis. In this context enzymes that are responsible for the modification of ECM proteins are of particular pertinence. Matrix metalloproteinases (MMPs) represent a family of enzymes known to play role in the modification of ECM and by this can change the cell-ECM substrate interaction, required for cell development. In an effort to elucidate the participation of MMPs in the proliferation and differentiation HUCB-NSCs were cultured in the presence or absence of MMPs inhibitors – GM6001 and doxycycline. Our results show that addition of the above inhibitors interfered with both the proliferation and differentiation of progenitor cells toward the neuronal lineage. This effect depends on the adhesive ECM substrate and is most pronounced in the presence of fibronectin and laminin. In conclusion, our results suggest that MMPs modulate interaction between HUCB-NSCs and their environment and therefore might be an important component in neurogenesisassociated processes.
19

Heterogeneity of the local tissue microenvironment influences differentiation of NG2 cells

45%
Sypecka J., Sarnowska A., Szablowska-Gadomska I., Lukomska D., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
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.
20

The impact of local tissue microenvironment on the oligodendrocyte progenitor differentiation

45%
Sypecka J., Sarnowska A., Winiarska H., Domanska-Janik K., Lukomska B.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr S
The oligodendrocyte precursors 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 and development could be modulated by either local tissue-specific or neuropathological signals. The aim of our study was to evaluate the effect of distinct microenvironments (provided by either the spinal cord or the hippocampal slices) on the differentiation of rat neonatal NG2 cells. The hippocampal slice cultures subjected to an ischemic injury (OGD) were used to mimic the traumatized tissue microenvironment. 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. 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 cocultures. In media conditioned by spinal cord slices, neurogenesis was less pronounced (20% neurons) and the oligodendrocyte differentiation was significantly slowed-down. The NG2 precursors have the intrinsic potency for neurogenesis. Heterogeneity of local microenviroment 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.
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