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Influence of oxygen tension on dopaminergic differentiation of human fetal stem cells of midbrain and forebrain origin.

Krabbe C, Bak ST, Jensen P, von Linstow C, Martínez Serrano A, Hansen C, Meyer M - PLoS ONE (2014)

Bottom Line: Proliferative Ki67-ir cells were found in both types of cultures, but the relative proportion of these cells was significantly higher for forebrain NSCs cultured at low, as compared to high, oxygen tension.Up-regulation of β-tubulin III was most pronounced for midbrain cells, whereas GFAP expression was higher in forebrain as compared to midbrain cells.Following mictrotransplantation into mouse striatal slice cultures predifferentiated midbrain NSCs were found to proliferate and differentiate into substantial numbers of TH-ir neurons with mature neuronal morphologies, particularly at low oxygen.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark.

ABSTRACT
Neural stem cells (NSCs) constitute a promising source of cells for transplantation in Parkinson's disease (PD), but protocols for controlled dopaminergic differentiation are not yet available. Here we investigated the influence of oxygen on dopaminergic differentiation of human fetal NSCs derived from the midbrain and forebrain. Cells were differentiated for 10 days in vitro at low, physiological (3%) versus high, atmospheric (20%) oxygen tension. Low oxygen resulted in upregulation of vascular endothelial growth factor and increased the proportion of tyrosine hydroxylase-immunoreactive (TH-ir) cells in both types of cultures (midbrain: 9.1 ± 0.5 and 17.1 ± 0.4 (P<0.001); forebrain: 1.9 ± 0.4 and 3.9 ± 0.6 (P<0.01) percent of total cells). Regardless of oxygen levels, the content of TH-ir cells with mature neuronal morphologies was higher for midbrain as compared to forebrain cultures. Proliferative Ki67-ir cells were found in both types of cultures, but the relative proportion of these cells was significantly higher for forebrain NSCs cultured at low, as compared to high, oxygen tension. No such difference was detected for midbrain-derived cells. Western blot analysis revealed that low oxygen enhanced β-tubulin III and GFAP expression in both cultures. Up-regulation of β-tubulin III was most pronounced for midbrain cells, whereas GFAP expression was higher in forebrain as compared to midbrain cells. NSCs from both brain regions displayed less cell death when cultured at low oxygen tension. Following mictrotransplantation into mouse striatal slice cultures predifferentiated midbrain NSCs were found to proliferate and differentiate into substantial numbers of TH-ir neurons with mature neuronal morphologies, particularly at low oxygen. In contrast, predifferentiated forebrain NSCs microtransplanted using identical conditions displayed little proliferation and contained few TH-ir cells, all of which had an immature appearance. Our data may reflect differences in dopaminergic differentiation capacity and region-specific requirements of NSCs, with the dopamine-depleted striatum cultured at low oxygen offering an attractive micro-environment for midbrain NSCs.

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Characterization of differentiated cells.Double immunofluorescence staining was performed for histological characterization of differentiated cultures (A). A small number of cells in both midbrain and forebrain-derived cultures co-expressed the marker of proliferating cells, Ki67, and the early neuronal marker β-tubulin III (β-tub III). The density of β-tub III-ir cells co-expressing the mature neuronal marker microtubule associated protein 2ab (MAP2) was very high for midbrain cultures, particularly when differentiated at low oxygen tension. In forebrain-derived cultures, only few β-tub III-ir/MAP2-ir cells were detected at low oxygen and even fewer at high oxygen. The density of cells co-expressing tyrosine hydroxylase (TH) and β-tub III or MAP2 was highest for midbrain cultures grown at low oxygen, and lowest for forebrain cultures at high oxygen. Scale bar  =  50 µm. Western blotting for TH, β-tub III, and the astroglial marker glial fibrillary acidic protein (GFAP) revealed marked differences depending on both cell origin and oxygen tension (B). The expression of all these markers was higher in cultures differentiated at low oxygen. The level of β-tub III and TH protein was highest for midbrain-derived cells, whereas the highest level of GFAP was detected for forebrain-derived cells.
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pone-0096465-g004: Characterization of differentiated cells.Double immunofluorescence staining was performed for histological characterization of differentiated cultures (A). A small number of cells in both midbrain and forebrain-derived cultures co-expressed the marker of proliferating cells, Ki67, and the early neuronal marker β-tubulin III (β-tub III). The density of β-tub III-ir cells co-expressing the mature neuronal marker microtubule associated protein 2ab (MAP2) was very high for midbrain cultures, particularly when differentiated at low oxygen tension. In forebrain-derived cultures, only few β-tub III-ir/MAP2-ir cells were detected at low oxygen and even fewer at high oxygen. The density of cells co-expressing tyrosine hydroxylase (TH) and β-tub III or MAP2 was highest for midbrain cultures grown at low oxygen, and lowest for forebrain cultures at high oxygen. Scale bar  =  50 µm. Western blotting for TH, β-tub III, and the astroglial marker glial fibrillary acidic protein (GFAP) revealed marked differences depending on both cell origin and oxygen tension (B). The expression of all these markers was higher in cultures differentiated at low oxygen. The level of β-tub III and TH protein was highest for midbrain-derived cells, whereas the highest level of GFAP was detected for forebrain-derived cells.

Mentions: Double immunofluorescence-stained NSCs differentiated for 10 DIV were used for general characterization of the cellular content in the cultures. Co-expression of Ki67 and β-tub III was seen for a small population of cells in midbrain and forebrain cultures at both oxygen tensions, suggesting that some of the β-tub III-ir cells were still mitotic (Fig. 4A). Many midbrain-derived β-tub III-ir neurons were found to co-express the mature neuronal marker microtubule-associated protein 2ab (MAP2), particularly for cells cultured at low oxygen tension. Quantification of the relative contents of MAP2-ir neurons in the cultures revealed a significantly higher percentage for midbrain-derived cells cultured at low oxygen tension as compared to high oxygen tension (P<0.001) (high: 5.4±0.5%; low: 15.8±1.0% MAP2-ir neurons, mean±SEM, n = 6). Less than 1.0% MAP2-ir neurons were found in forebrain cultures. Some co-expression of β-tub III and MAP2 was seen in forebrain cultures grown at low oxygen tension, whereas only very few β-tub III/MAP2-ir cells were seen at high oxygen tension. Co-expression of TH and β-tub III was found in all groups, although the density of cells co-expressing these to markers was higher in midbrain cultures. To investigate whether the different oxygen levels had affected maturation of the TH-ir cells, cultures were stained for co-expression of TH and MAP2. At both oxygen tensions the density of TH/MAP2-ir cells were much higher in the midbrain as compared to the forebrain cultures. For both cell lines, the density of TH/MAP2-ir neurons increased when cultures were differentiated at low as compared to high oxygen tension, indicating that low oxygen has important effects on the maturation of TH-ir cells. For midbrain-derived cultures, the density of TH/MAP2-ir cells was high at both oxygen levels (high/low), whereas only very few cells co-expressed these two markers in forebrain-derived cultures (Fig. 4A). No TH and GABA co-expression was seen in differentiated midbrain cultures, whereas some differentiated forebrain cells were found to co-express these markers (data not shown).


Influence of oxygen tension on dopaminergic differentiation of human fetal stem cells of midbrain and forebrain origin.

Krabbe C, Bak ST, Jensen P, von Linstow C, Martínez Serrano A, Hansen C, Meyer M - PLoS ONE (2014)

Characterization of differentiated cells.Double immunofluorescence staining was performed for histological characterization of differentiated cultures (A). A small number of cells in both midbrain and forebrain-derived cultures co-expressed the marker of proliferating cells, Ki67, and the early neuronal marker β-tubulin III (β-tub III). The density of β-tub III-ir cells co-expressing the mature neuronal marker microtubule associated protein 2ab (MAP2) was very high for midbrain cultures, particularly when differentiated at low oxygen tension. In forebrain-derived cultures, only few β-tub III-ir/MAP2-ir cells were detected at low oxygen and even fewer at high oxygen. The density of cells co-expressing tyrosine hydroxylase (TH) and β-tub III or MAP2 was highest for midbrain cultures grown at low oxygen, and lowest for forebrain cultures at high oxygen. Scale bar  =  50 µm. Western blotting for TH, β-tub III, and the astroglial marker glial fibrillary acidic protein (GFAP) revealed marked differences depending on both cell origin and oxygen tension (B). The expression of all these markers was higher in cultures differentiated at low oxygen. The level of β-tub III and TH protein was highest for midbrain-derived cells, whereas the highest level of GFAP was detected for forebrain-derived cells.
© Copyright Policy
Related In: Results  -  Collection

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pone-0096465-g004: Characterization of differentiated cells.Double immunofluorescence staining was performed for histological characterization of differentiated cultures (A). A small number of cells in both midbrain and forebrain-derived cultures co-expressed the marker of proliferating cells, Ki67, and the early neuronal marker β-tubulin III (β-tub III). The density of β-tub III-ir cells co-expressing the mature neuronal marker microtubule associated protein 2ab (MAP2) was very high for midbrain cultures, particularly when differentiated at low oxygen tension. In forebrain-derived cultures, only few β-tub III-ir/MAP2-ir cells were detected at low oxygen and even fewer at high oxygen. The density of cells co-expressing tyrosine hydroxylase (TH) and β-tub III or MAP2 was highest for midbrain cultures grown at low oxygen, and lowest for forebrain cultures at high oxygen. Scale bar  =  50 µm. Western blotting for TH, β-tub III, and the astroglial marker glial fibrillary acidic protein (GFAP) revealed marked differences depending on both cell origin and oxygen tension (B). The expression of all these markers was higher in cultures differentiated at low oxygen. The level of β-tub III and TH protein was highest for midbrain-derived cells, whereas the highest level of GFAP was detected for forebrain-derived cells.
Mentions: Double immunofluorescence-stained NSCs differentiated for 10 DIV were used for general characterization of the cellular content in the cultures. Co-expression of Ki67 and β-tub III was seen for a small population of cells in midbrain and forebrain cultures at both oxygen tensions, suggesting that some of the β-tub III-ir cells were still mitotic (Fig. 4A). Many midbrain-derived β-tub III-ir neurons were found to co-express the mature neuronal marker microtubule-associated protein 2ab (MAP2), particularly for cells cultured at low oxygen tension. Quantification of the relative contents of MAP2-ir neurons in the cultures revealed a significantly higher percentage for midbrain-derived cells cultured at low oxygen tension as compared to high oxygen tension (P<0.001) (high: 5.4±0.5%; low: 15.8±1.0% MAP2-ir neurons, mean±SEM, n = 6). Less than 1.0% MAP2-ir neurons were found in forebrain cultures. Some co-expression of β-tub III and MAP2 was seen in forebrain cultures grown at low oxygen tension, whereas only very few β-tub III/MAP2-ir cells were seen at high oxygen tension. Co-expression of TH and β-tub III was found in all groups, although the density of cells co-expressing these to markers was higher in midbrain cultures. To investigate whether the different oxygen levels had affected maturation of the TH-ir cells, cultures were stained for co-expression of TH and MAP2. At both oxygen tensions the density of TH/MAP2-ir cells were much higher in the midbrain as compared to the forebrain cultures. For both cell lines, the density of TH/MAP2-ir neurons increased when cultures were differentiated at low as compared to high oxygen tension, indicating that low oxygen has important effects on the maturation of TH-ir cells. For midbrain-derived cultures, the density of TH/MAP2-ir cells was high at both oxygen levels (high/low), whereas only very few cells co-expressed these two markers in forebrain-derived cultures (Fig. 4A). No TH and GABA co-expression was seen in differentiated midbrain cultures, whereas some differentiated forebrain cells were found to co-express these markers (data not shown).

Bottom Line: Proliferative Ki67-ir cells were found in both types of cultures, but the relative proportion of these cells was significantly higher for forebrain NSCs cultured at low, as compared to high, oxygen tension.Up-regulation of β-tubulin III was most pronounced for midbrain cells, whereas GFAP expression was higher in forebrain as compared to midbrain cells.Following mictrotransplantation into mouse striatal slice cultures predifferentiated midbrain NSCs were found to proliferate and differentiate into substantial numbers of TH-ir neurons with mature neuronal morphologies, particularly at low oxygen.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark.

ABSTRACT
Neural stem cells (NSCs) constitute a promising source of cells for transplantation in Parkinson's disease (PD), but protocols for controlled dopaminergic differentiation are not yet available. Here we investigated the influence of oxygen on dopaminergic differentiation of human fetal NSCs derived from the midbrain and forebrain. Cells were differentiated for 10 days in vitro at low, physiological (3%) versus high, atmospheric (20%) oxygen tension. Low oxygen resulted in upregulation of vascular endothelial growth factor and increased the proportion of tyrosine hydroxylase-immunoreactive (TH-ir) cells in both types of cultures (midbrain: 9.1 ± 0.5 and 17.1 ± 0.4 (P<0.001); forebrain: 1.9 ± 0.4 and 3.9 ± 0.6 (P<0.01) percent of total cells). Regardless of oxygen levels, the content of TH-ir cells with mature neuronal morphologies was higher for midbrain as compared to forebrain cultures. Proliferative Ki67-ir cells were found in both types of cultures, but the relative proportion of these cells was significantly higher for forebrain NSCs cultured at low, as compared to high, oxygen tension. No such difference was detected for midbrain-derived cells. Western blot analysis revealed that low oxygen enhanced β-tubulin III and GFAP expression in both cultures. Up-regulation of β-tubulin III was most pronounced for midbrain cells, whereas GFAP expression was higher in forebrain as compared to midbrain cells. NSCs from both brain regions displayed less cell death when cultured at low oxygen tension. Following mictrotransplantation into mouse striatal slice cultures predifferentiated midbrain NSCs were found to proliferate and differentiate into substantial numbers of TH-ir neurons with mature neuronal morphologies, particularly at low oxygen. In contrast, predifferentiated forebrain NSCs microtransplanted using identical conditions displayed little proliferation and contained few TH-ir cells, all of which had an immature appearance. Our data may reflect differences in dopaminergic differentiation capacity and region-specific requirements of NSCs, with the dopamine-depleted striatum cultured at low oxygen offering an attractive micro-environment for midbrain NSCs.

Show MeSH
Related in: MedlinePlus