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Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons.

Belachew S, Chittajallu R, Aguirre AA, Yuan X, Kirby M, Anderson S, Gallo V - J. Cell Biol. (2003)

Bottom Line: The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming.We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs.These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroscience Research, Children's Research Institute, Children's National Medical Center, Washington, DC 20010-2970, USA.

ABSTRACT
Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan-positive progenitor cells that express the 2',3'-cyclic nucleotide 3'-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

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FACS® purification of early postnatal GFP+ cells from CNP-GFP transgenic brains reveals an NG2+/nestin+ phenotype. FACS® analysis of acutely dissociated cell suspensions of P2 whole brain from CNP-GFP transgenic mice (tg) and wild-type (wt) littermates displayed identical light forward and side scatter distributions (A). To eliminate erythrocytes and cell debris, forward scatter gating parameters were chosen for further GFP fluorescence analysis (B and C, left panels). Cells from CNP-GFP suspensions that were considered GFP+ and FACS®-selected, were in a range of fluorescence (C, bar in right panel) far above (ratio >5×) maximal background intensity yielded by the wild-type cell suspension (B, bar in right panel). Using such criteria, all FACS®-sorted cells were consistently found to be GFP+, as determined by fluorescence microscopy analysis of cell suspensions plated on coverslips immediately after sorting, and fixed 1 h later (two independent experiments, total cells counted = 1,069, 100% were GFP+; E and F). (D) Immunocytochemical characterization of pre-FACS® and post-FACS® cell suspensions (total GFP+ counted cells = 1069, two independent experiments). N.P., not present. We demonstrated here that pre-FACS® versus post-FACS® GFP+ cells were antigenically identical and mostly expressed an NG2+/nestin+ phenotype. This validated our fluorescence sorting criteria, which did not preferentially select a subset of CNP-GFP+ cells. Phase-contrast (E) and fluorescence (F) views of FACS®-selected cells that were all GFP+.
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fig1: FACS® purification of early postnatal GFP+ cells from CNP-GFP transgenic brains reveals an NG2+/nestin+ phenotype. FACS® analysis of acutely dissociated cell suspensions of P2 whole brain from CNP-GFP transgenic mice (tg) and wild-type (wt) littermates displayed identical light forward and side scatter distributions (A). To eliminate erythrocytes and cell debris, forward scatter gating parameters were chosen for further GFP fluorescence analysis (B and C, left panels). Cells from CNP-GFP suspensions that were considered GFP+ and FACS®-selected, were in a range of fluorescence (C, bar in right panel) far above (ratio >5×) maximal background intensity yielded by the wild-type cell suspension (B, bar in right panel). Using such criteria, all FACS®-sorted cells were consistently found to be GFP+, as determined by fluorescence microscopy analysis of cell suspensions plated on coverslips immediately after sorting, and fixed 1 h later (two independent experiments, total cells counted = 1,069, 100% were GFP+; E and F). (D) Immunocytochemical characterization of pre-FACS® and post-FACS® cell suspensions (total GFP+ counted cells = 1069, two independent experiments). N.P., not present. We demonstrated here that pre-FACS® versus post-FACS® GFP+ cells were antigenically identical and mostly expressed an NG2+/nestin+ phenotype. This validated our fluorescence sorting criteria, which did not preferentially select a subset of CNP-GFP+ cells. Phase-contrast (E) and fluorescence (F) views of FACS®-selected cells that were all GFP+.

Mentions: All the in vitro data were obtained from a single transgenic line (Belachew et al., 2001; Yuan et al., 2002), but were replicated in another CNP-GFP line (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200210110/DC1, and unpublished data). FACS® was used to generate pure GFP+ cell preparations from early postnatal brain of CNP-GFP mice (Fig. 1 , A–F). We characterized the nestin immunophenotype of FACS®-purified GFP+ cells because nestin is an intermediate filament protein expressed in neuroepithelium-derived NSCs during development (Lendahl et al., 1990). We observed that 99.0 ± 0.6% of P2 FACS®-sorted GFP+ cells were nestin+. Furthermore, 81.1 ± 1.6% of FACS®-sorted GFP+ cells were NG2+, whereas 17.9 ± 1.6% were NG2− (Fig. 1 D; mean ± SEM, total GFP+ counted cells = 1,069, two independent experiments). We also observed that only 6.24 ± 1.02% (mean ± SEM, total GFP+ counted cells = 678) of acutely FACS®-purified GFP+ cells expressed the CNP protein, a marker of more mature oligodendrocytes. No freshly sorted GFP+ cells expressed neuronal (neuronal nuclei protein, NeuN) or astrocytic (GFAP) markers (Fig. 1 D).


Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons.

Belachew S, Chittajallu R, Aguirre AA, Yuan X, Kirby M, Anderson S, Gallo V - J. Cell Biol. (2003)

FACS® purification of early postnatal GFP+ cells from CNP-GFP transgenic brains reveals an NG2+/nestin+ phenotype. FACS® analysis of acutely dissociated cell suspensions of P2 whole brain from CNP-GFP transgenic mice (tg) and wild-type (wt) littermates displayed identical light forward and side scatter distributions (A). To eliminate erythrocytes and cell debris, forward scatter gating parameters were chosen for further GFP fluorescence analysis (B and C, left panels). Cells from CNP-GFP suspensions that were considered GFP+ and FACS®-selected, were in a range of fluorescence (C, bar in right panel) far above (ratio >5×) maximal background intensity yielded by the wild-type cell suspension (B, bar in right panel). Using such criteria, all FACS®-sorted cells were consistently found to be GFP+, as determined by fluorescence microscopy analysis of cell suspensions plated on coverslips immediately after sorting, and fixed 1 h later (two independent experiments, total cells counted = 1,069, 100% were GFP+; E and F). (D) Immunocytochemical characterization of pre-FACS® and post-FACS® cell suspensions (total GFP+ counted cells = 1069, two independent experiments). N.P., not present. We demonstrated here that pre-FACS® versus post-FACS® GFP+ cells were antigenically identical and mostly expressed an NG2+/nestin+ phenotype. This validated our fluorescence sorting criteria, which did not preferentially select a subset of CNP-GFP+ cells. Phase-contrast (E) and fluorescence (F) views of FACS®-selected cells that were all GFP+.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172886&req=5

fig1: FACS® purification of early postnatal GFP+ cells from CNP-GFP transgenic brains reveals an NG2+/nestin+ phenotype. FACS® analysis of acutely dissociated cell suspensions of P2 whole brain from CNP-GFP transgenic mice (tg) and wild-type (wt) littermates displayed identical light forward and side scatter distributions (A). To eliminate erythrocytes and cell debris, forward scatter gating parameters were chosen for further GFP fluorescence analysis (B and C, left panels). Cells from CNP-GFP suspensions that were considered GFP+ and FACS®-selected, were in a range of fluorescence (C, bar in right panel) far above (ratio >5×) maximal background intensity yielded by the wild-type cell suspension (B, bar in right panel). Using such criteria, all FACS®-sorted cells were consistently found to be GFP+, as determined by fluorescence microscopy analysis of cell suspensions plated on coverslips immediately after sorting, and fixed 1 h later (two independent experiments, total cells counted = 1,069, 100% were GFP+; E and F). (D) Immunocytochemical characterization of pre-FACS® and post-FACS® cell suspensions (total GFP+ counted cells = 1069, two independent experiments). N.P., not present. We demonstrated here that pre-FACS® versus post-FACS® GFP+ cells were antigenically identical and mostly expressed an NG2+/nestin+ phenotype. This validated our fluorescence sorting criteria, which did not preferentially select a subset of CNP-GFP+ cells. Phase-contrast (E) and fluorescence (F) views of FACS®-selected cells that were all GFP+.
Mentions: All the in vitro data were obtained from a single transgenic line (Belachew et al., 2001; Yuan et al., 2002), but were replicated in another CNP-GFP line (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200210110/DC1, and unpublished data). FACS® was used to generate pure GFP+ cell preparations from early postnatal brain of CNP-GFP mice (Fig. 1 , A–F). We characterized the nestin immunophenotype of FACS®-purified GFP+ cells because nestin is an intermediate filament protein expressed in neuroepithelium-derived NSCs during development (Lendahl et al., 1990). We observed that 99.0 ± 0.6% of P2 FACS®-sorted GFP+ cells were nestin+. Furthermore, 81.1 ± 1.6% of FACS®-sorted GFP+ cells were NG2+, whereas 17.9 ± 1.6% were NG2− (Fig. 1 D; mean ± SEM, total GFP+ counted cells = 1,069, two independent experiments). We also observed that only 6.24 ± 1.02% (mean ± SEM, total GFP+ counted cells = 678) of acutely FACS®-purified GFP+ cells expressed the CNP protein, a marker of more mature oligodendrocytes. No freshly sorted GFP+ cells expressed neuronal (neuronal nuclei protein, NeuN) or astrocytic (GFAP) markers (Fig. 1 D).

Bottom Line: The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming.We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs.These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroscience Research, Children's Research Institute, Children's National Medical Center, Washington, DC 20010-2970, USA.

ABSTRACT
Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan-positive progenitor cells that express the 2',3'-cyclic nucleotide 3'-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

Show MeSH
Related in: MedlinePlus