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Tripotential differentiation of adherently expandable neural stem (NS) cells.

Glaser T, Pollard SM, Smith A, Brüstle O - PLoS ONE (2007)

Bottom Line: Here we provide evidence that NS cells are indeed tripotent.Mature oligodendroglial differentiation was confirmed by transplantation data showing that NS cell-derived oligodendrocytes ensheath host axons in the brain of myelin-deficient rats.In addition to delineating NS cells as a potential donor source for myelin repair, our data strongly support the view that these adherently expandable cells represent bona fide tripotential neural stem cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn and Hertie Foundation, Bonn, Germany.

ABSTRACT

Background: A recent study has shown that pure neural stem cells can be derived from embryonic stem (ES) cells and primary brain tissue. In the presence of fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF), this population can be continuously expanded in adherent conditions. In analogy to continuously self-renewing ES cells, these cells were termed 'NS' cells (Conti et al., PLoS Biol 3: e283, 2005). While NS cells have been shown to readily generate neurons and astrocytes, their differentiation into oligodendrocytes has remained enigmatic, raising concerns as to whether they truly represent tripotential neural stem cells.

Methodology/principal findings: Here we provide evidence that NS cells are indeed tripotent. Upon proliferation with FGF2, platelet-derived growth factor (PDGF) and forskolin, followed by differentiation in the presence of thyroid hormone (T3) and ascorbic acid NS cells efficiently generate oligodendrocytes ( approximately 20%) alongside astrocytes ( approximately 40%) and neurons ( approximately 10%). Mature oligodendroglial differentiation was confirmed by transplantation data showing that NS cell-derived oligodendrocytes ensheath host axons in the brain of myelin-deficient rats.

Conclusions/significance: In addition to delineating NS cells as a potential donor source for myelin repair, our data strongly support the view that these adherently expandable cells represent bona fide tripotential neural stem cells.

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Tripotential differentiation of NS cells in vitro and generation of myelinating oligodendrocytes in vivo.(A–E) Quantitative marker expression and representative immunofluorescence images. The specific culture conditions used to differentiate NS cells resulted in the generation of oligodendrocytes (∼20%) positive for O4 (B), Rip (D) and PLP (E), GFAP-expressing astrocytes (∼40%; C–D) and neurons positive for ß-III tubulin/TUJ1 (∼10%; C). (F–H) NS cells cultured in N2 medium and proliferated for 4 days in the presence of FGF2, PDGF and forskolin were transplanted into the brain of 2- to 3-day-old myelin-deficient rats. Two weeks after transplantation, the engrafted cells had formed PLP-positive myelin internodes. Shown are representative pictures from septum (F) and corpus callosum (G–H). Scale bars B–D, 100 µm; F–H, 20 µm.
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pone-0000298-g002: Tripotential differentiation of NS cells in vitro and generation of myelinating oligodendrocytes in vivo.(A–E) Quantitative marker expression and representative immunofluorescence images. The specific culture conditions used to differentiate NS cells resulted in the generation of oligodendrocytes (∼20%) positive for O4 (B), Rip (D) and PLP (E), GFAP-expressing astrocytes (∼40%; C–D) and neurons positive for ß-III tubulin/TUJ1 (∼10%; C). (F–H) NS cells cultured in N2 medium and proliferated for 4 days in the presence of FGF2, PDGF and forskolin were transplanted into the brain of 2- to 3-day-old myelin-deficient rats. Two weeks after transplantation, the engrafted cells had formed PLP-positive myelin internodes. Shown are representative pictures from septum (F) and corpus callosum (G–H). Scale bars B–D, 100 µm; F–H, 20 µm.

Mentions: The clonally derived NS cell line NS-5 generated from mouse ES cells was propagated and passaged according to Conti et al. [35] (Fig. 1A). To promote oligodendroglial differentiation cells were cultured on polyornithine/laminin coated dishes in medium containing N2 supplement plus FGF2, platelet-derived growth factor (PDGF) and forskolin, a growth factor combination known to enhance oligodendrocyte progenitor proliferation. After 4 days this condition resulted in a population of small cells with condensed cell bodies and short processes (Fig. 1B). Terminal differentiation was initiated by a 4-day-growth factor withdrawal in the presence of the thyroid hormone tri-iodothyronine (T3) and ascorbic acid [38]. Under these conditions, NS cells efficiently differentiated into oligodendrocytes, astrocytes and neurons (Fig. 1C,D). Quantitative results of the antigenic marker expression are summarized in Fig. 2A. Differentiated cultures contained 21±4% O4-positive oligodendrocytes (Fig. 2B), 45±9% of the cells expressed the astrocytic marker GFAP (Fig. 2C,D) and a fraction of 11±3% was found to be positive for the neuronal antigen ß-III tubulin/TUJ1 (Fig. 2C). Oligodendroglial differentiation could also be demonstrated by labelling with the RIP antibody, which recognizes 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) in oligodendrocytes [39] (Fig. 2D). Furthermore, cells with ramified processes and characteristic oligodendroglial morphology expressed myelin proteolipid protein (PLP) (Fig. 2E). Similar data have been obtained with NS cells derived from foetal mouse brain (Sandra G. Lopez & Steven M. Pollard, data not shown).


Tripotential differentiation of adherently expandable neural stem (NS) cells.

Glaser T, Pollard SM, Smith A, Brüstle O - PLoS ONE (2007)

Tripotential differentiation of NS cells in vitro and generation of myelinating oligodendrocytes in vivo.(A–E) Quantitative marker expression and representative immunofluorescence images. The specific culture conditions used to differentiate NS cells resulted in the generation of oligodendrocytes (∼20%) positive for O4 (B), Rip (D) and PLP (E), GFAP-expressing astrocytes (∼40%; C–D) and neurons positive for ß-III tubulin/TUJ1 (∼10%; C). (F–H) NS cells cultured in N2 medium and proliferated for 4 days in the presence of FGF2, PDGF and forskolin were transplanted into the brain of 2- to 3-day-old myelin-deficient rats. Two weeks after transplantation, the engrafted cells had formed PLP-positive myelin internodes. Shown are representative pictures from septum (F) and corpus callosum (G–H). Scale bars B–D, 100 µm; F–H, 20 µm.
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Related In: Results  -  Collection

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pone-0000298-g002: Tripotential differentiation of NS cells in vitro and generation of myelinating oligodendrocytes in vivo.(A–E) Quantitative marker expression and representative immunofluorescence images. The specific culture conditions used to differentiate NS cells resulted in the generation of oligodendrocytes (∼20%) positive for O4 (B), Rip (D) and PLP (E), GFAP-expressing astrocytes (∼40%; C–D) and neurons positive for ß-III tubulin/TUJ1 (∼10%; C). (F–H) NS cells cultured in N2 medium and proliferated for 4 days in the presence of FGF2, PDGF and forskolin were transplanted into the brain of 2- to 3-day-old myelin-deficient rats. Two weeks after transplantation, the engrafted cells had formed PLP-positive myelin internodes. Shown are representative pictures from septum (F) and corpus callosum (G–H). Scale bars B–D, 100 µm; F–H, 20 µm.
Mentions: The clonally derived NS cell line NS-5 generated from mouse ES cells was propagated and passaged according to Conti et al. [35] (Fig. 1A). To promote oligodendroglial differentiation cells were cultured on polyornithine/laminin coated dishes in medium containing N2 supplement plus FGF2, platelet-derived growth factor (PDGF) and forskolin, a growth factor combination known to enhance oligodendrocyte progenitor proliferation. After 4 days this condition resulted in a population of small cells with condensed cell bodies and short processes (Fig. 1B). Terminal differentiation was initiated by a 4-day-growth factor withdrawal in the presence of the thyroid hormone tri-iodothyronine (T3) and ascorbic acid [38]. Under these conditions, NS cells efficiently differentiated into oligodendrocytes, astrocytes and neurons (Fig. 1C,D). Quantitative results of the antigenic marker expression are summarized in Fig. 2A. Differentiated cultures contained 21±4% O4-positive oligodendrocytes (Fig. 2B), 45±9% of the cells expressed the astrocytic marker GFAP (Fig. 2C,D) and a fraction of 11±3% was found to be positive for the neuronal antigen ß-III tubulin/TUJ1 (Fig. 2C). Oligodendroglial differentiation could also be demonstrated by labelling with the RIP antibody, which recognizes 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) in oligodendrocytes [39] (Fig. 2D). Furthermore, cells with ramified processes and characteristic oligodendroglial morphology expressed myelin proteolipid protein (PLP) (Fig. 2E). Similar data have been obtained with NS cells derived from foetal mouse brain (Sandra G. Lopez & Steven M. Pollard, data not shown).

Bottom Line: Here we provide evidence that NS cells are indeed tripotent.Mature oligodendroglial differentiation was confirmed by transplantation data showing that NS cell-derived oligodendrocytes ensheath host axons in the brain of myelin-deficient rats.In addition to delineating NS cells as a potential donor source for myelin repair, our data strongly support the view that these adherently expandable cells represent bona fide tripotential neural stem cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn and Hertie Foundation, Bonn, Germany.

ABSTRACT

Background: A recent study has shown that pure neural stem cells can be derived from embryonic stem (ES) cells and primary brain tissue. In the presence of fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF), this population can be continuously expanded in adherent conditions. In analogy to continuously self-renewing ES cells, these cells were termed 'NS' cells (Conti et al., PLoS Biol 3: e283, 2005). While NS cells have been shown to readily generate neurons and astrocytes, their differentiation into oligodendrocytes has remained enigmatic, raising concerns as to whether they truly represent tripotential neural stem cells.

Methodology/principal findings: Here we provide evidence that NS cells are indeed tripotent. Upon proliferation with FGF2, platelet-derived growth factor (PDGF) and forskolin, followed by differentiation in the presence of thyroid hormone (T3) and ascorbic acid NS cells efficiently generate oligodendrocytes ( approximately 20%) alongside astrocytes ( approximately 40%) and neurons ( approximately 10%). Mature oligodendroglial differentiation was confirmed by transplantation data showing that NS cell-derived oligodendrocytes ensheath host axons in the brain of myelin-deficient rats.

Conclusions/significance: In addition to delineating NS cells as a potential donor source for myelin repair, our data strongly support the view that these adherently expandable cells represent bona fide tripotential neural stem cells.

Show MeSH
Related in: MedlinePlus