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Cholera toxin regulates a signaling pathway critical for the expansion of neural stem cell cultures from the fetal and adult rodent brains.

Androutsellis-Theotokis A, Walbridge S, Park DM, Lonser RR, McKay RD - PLoS ONE (2010)

Bottom Line: Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture.Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3.This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA. AndreasTheotokis@gmail.com

ABSTRACT

Background: New mechanisms that regulate neural stem cell (NSC) expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers.

Methodology/principal findings: Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen.

Conclusions/significance: Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer.

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Related in: MedlinePlus

Cholera toxin promotes NSC proliferation in vitro.(a, b) ChAB during an 8-day FGF2 withdrawal period maintains cell division (assessed by incorporation of EdU added on days 7–8); low- and high power confocal projections are shown. (c) ChAB-treated fetal NSC cultures generate neurons, astrocytes, and oligodendrocytes (ChAB was added during FGF2 treatment, 4-days and for another 2-days following FGF2 withdrawal; cells were then switched to B27 medium with added neurotrophins for 7-days). [Size bars: 100 µm].
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pone-0010841-g005: Cholera toxin promotes NSC proliferation in vitro.(a, b) ChAB during an 8-day FGF2 withdrawal period maintains cell division (assessed by incorporation of EdU added on days 7–8); low- and high power confocal projections are shown. (c) ChAB-treated fetal NSC cultures generate neurons, astrocytes, and oligodendrocytes (ChAB was added during FGF2 treatment, 4-days and for another 2-days following FGF2 withdrawal; cells were then switched to B27 medium with added neurotrophins for 7-days). [Size bars: 100 µm].

Mentions: When mouse fetal NSC cultures were treated with CholAB in the presence of FGF2 (for 4 days), a small but significant increase in total cell number and a large increase in DCX+ cell number was measured (Figure 4a,c). A much greater effect (relative to controls) was obtained when CholAB was added immediately after FGF2 withdrawal for 2 days, as control cells under those conditions exit the cell cycle [34], [35] whereas CholAB – treated cultures increased in cell density (Figure 4b,c). Adherent fetal NSC cultures are highly homogenous, but they also contain small percentages (>1%) of DCX+ neuronal precursors. In the presence of FGF2, CholAB did not induce the acquisition of differentiation markers, but it expanded the DCX+ populations (Figure 4d,e; Figure S4a,b). This is concluded by the increase in DCX+ colony size but not number (data not shown). These results suggest that CholAB – treated cells in the absence of mitogen are still dividing. To address this possibility, fetal NSC cultures were plated in FGF2 for initial expansion, after which FGF2 was removed for 8 days, and during the last two days the thymidine analog EdU was added to the medium to detect any proliferating cells. Under those conditions, no cells in the control groups incorporated EdU, whereas 25%±5 of total cells and 15%±3 of DCX+ cells in the CholAB – treated groups still divided (Figure 5a,b). These data show that CholAB promotes the proliferation of cells in a minimal culture medium (without serum) in the absence of a mitogen. When the CholAB – treated cultures were forced to differentiate (by removal of FGF2, CholAB, and the addition of B27 cell culture supplement and neurotrophins for 7 days), neurons, astrocytes, and oligodendrocytes were generated (Figure 5c), showing that the potential of the culture en masse to generate all three major lineages of neural tissue was not lost.


Cholera toxin regulates a signaling pathway critical for the expansion of neural stem cell cultures from the fetal and adult rodent brains.

Androutsellis-Theotokis A, Walbridge S, Park DM, Lonser RR, McKay RD - PLoS ONE (2010)

Cholera toxin promotes NSC proliferation in vitro.(a, b) ChAB during an 8-day FGF2 withdrawal period maintains cell division (assessed by incorporation of EdU added on days 7–8); low- and high power confocal projections are shown. (c) ChAB-treated fetal NSC cultures generate neurons, astrocytes, and oligodendrocytes (ChAB was added during FGF2 treatment, 4-days and for another 2-days following FGF2 withdrawal; cells were then switched to B27 medium with added neurotrophins for 7-days). [Size bars: 100 µm].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2877108&req=5

pone-0010841-g005: Cholera toxin promotes NSC proliferation in vitro.(a, b) ChAB during an 8-day FGF2 withdrawal period maintains cell division (assessed by incorporation of EdU added on days 7–8); low- and high power confocal projections are shown. (c) ChAB-treated fetal NSC cultures generate neurons, astrocytes, and oligodendrocytes (ChAB was added during FGF2 treatment, 4-days and for another 2-days following FGF2 withdrawal; cells were then switched to B27 medium with added neurotrophins for 7-days). [Size bars: 100 µm].
Mentions: When mouse fetal NSC cultures were treated with CholAB in the presence of FGF2 (for 4 days), a small but significant increase in total cell number and a large increase in DCX+ cell number was measured (Figure 4a,c). A much greater effect (relative to controls) was obtained when CholAB was added immediately after FGF2 withdrawal for 2 days, as control cells under those conditions exit the cell cycle [34], [35] whereas CholAB – treated cultures increased in cell density (Figure 4b,c). Adherent fetal NSC cultures are highly homogenous, but they also contain small percentages (>1%) of DCX+ neuronal precursors. In the presence of FGF2, CholAB did not induce the acquisition of differentiation markers, but it expanded the DCX+ populations (Figure 4d,e; Figure S4a,b). This is concluded by the increase in DCX+ colony size but not number (data not shown). These results suggest that CholAB – treated cells in the absence of mitogen are still dividing. To address this possibility, fetal NSC cultures were plated in FGF2 for initial expansion, after which FGF2 was removed for 8 days, and during the last two days the thymidine analog EdU was added to the medium to detect any proliferating cells. Under those conditions, no cells in the control groups incorporated EdU, whereas 25%±5 of total cells and 15%±3 of DCX+ cells in the CholAB – treated groups still divided (Figure 5a,b). These data show that CholAB promotes the proliferation of cells in a minimal culture medium (without serum) in the absence of a mitogen. When the CholAB – treated cultures were forced to differentiate (by removal of FGF2, CholAB, and the addition of B27 cell culture supplement and neurotrophins for 7 days), neurons, astrocytes, and oligodendrocytes were generated (Figure 5c), showing that the potential of the culture en masse to generate all three major lineages of neural tissue was not lost.

Bottom Line: Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture.Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3.This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA. AndreasTheotokis@gmail.com

ABSTRACT

Background: New mechanisms that regulate neural stem cell (NSC) expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers.

Methodology/principal findings: Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen.

Conclusions/significance: Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer.

Show MeSH
Related in: MedlinePlus