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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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Clonal analysis of NG2+/CNP-GFP+ cells in vitro. (A) FACS® dot plots of acutely dissociated cells from wild-type (wt, top) and CNP-GFP transgenic brains (tg, bottom), in forward and side scatter with a polygon indicating the gate selecting the viable cells. (B–D) Sorting profiles of acutely isolated cell suspensions from P2 brains of wild-type (B) and CNP-GFP transgenic mice (C and D) dot plotted according to fluorescence intensity for GFP (x axis, logarithmic scale) and Cy-5 (fluorescence associated to secondary antibody recognizing NG2 immunoreactivity, y axis, logarithmic scale). (B) Control wild-type cells that were incubated only with the Cy-5–conjugated secondary antibody without anti-NG2 primary antibody. Crossed black lines in B–D represent thresholds of fluorescence. It was observed that <0.01% (limit of detection) of the control cells from B fell over this threshold. Thus, these lines determined the level of fluorescence above which cells from CNP-GFP brains (C) were selected as GFP+ (lower right quadrant). When CNP-GFP cell suspensions were immunostained for NG2 (D), NG2+/CNP-GFP+ cells were detected in upper right quadrant. To ensure accurate purification of NG2+/CNP-GFP+ cells, the sort gate for these cells (D, polygon) was defined by taking an additional margin (0.2–0.3 log units) with respect to background fluorescence levels. (E) FACS®-purified early postnatal (P2) NG2+/CNP-GFP+ cells were cultured at clonal density for 1 wk in SCM and the phenotype of resulting cell clones was then determined. (F) Relative proportion of the different subpopulations found in the multipotent clones, i.e., containing CNP-GFP+ cells, as well as neurons (NeuN+) and astrocytes (GFAP+). (G–J) GFP fluorescence (G, green), O4 (H, red), GFAP (I, peroxidase reaction), and NeuN (J, blue) stainings of the same microscopic field showing a representative multipotent clone derived from the growth of a single NG2+/CNP-GFP+ cell after one week in SCM. NeuN+ cells (arrowheads) are still retaining CNP-GFP fluorescence at this stage, whereas in GFAP+ astrocytes GFP expression has been lost (arrows). Bar, 50 μm for G–J.
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fig3: Clonal analysis of NG2+/CNP-GFP+ cells in vitro. (A) FACS® dot plots of acutely dissociated cells from wild-type (wt, top) and CNP-GFP transgenic brains (tg, bottom), in forward and side scatter with a polygon indicating the gate selecting the viable cells. (B–D) Sorting profiles of acutely isolated cell suspensions from P2 brains of wild-type (B) and CNP-GFP transgenic mice (C and D) dot plotted according to fluorescence intensity for GFP (x axis, logarithmic scale) and Cy-5 (fluorescence associated to secondary antibody recognizing NG2 immunoreactivity, y axis, logarithmic scale). (B) Control wild-type cells that were incubated only with the Cy-5–conjugated secondary antibody without anti-NG2 primary antibody. Crossed black lines in B–D represent thresholds of fluorescence. It was observed that <0.01% (limit of detection) of the control cells from B fell over this threshold. Thus, these lines determined the level of fluorescence above which cells from CNP-GFP brains (C) were selected as GFP+ (lower right quadrant). When CNP-GFP cell suspensions were immunostained for NG2 (D), NG2+/CNP-GFP+ cells were detected in upper right quadrant. To ensure accurate purification of NG2+/CNP-GFP+ cells, the sort gate for these cells (D, polygon) was defined by taking an additional margin (0.2–0.3 log units) with respect to background fluorescence levels. (E) FACS®-purified early postnatal (P2) NG2+/CNP-GFP+ cells were cultured at clonal density for 1 wk in SCM and the phenotype of resulting cell clones was then determined. (F) Relative proportion of the different subpopulations found in the multipotent clones, i.e., containing CNP-GFP+ cells, as well as neurons (NeuN+) and astrocytes (GFAP+). (G–J) GFP fluorescence (G, green), O4 (H, red), GFAP (I, peroxidase reaction), and NeuN (J, blue) stainings of the same microscopic field showing a representative multipotent clone derived from the growth of a single NG2+/CNP-GFP+ cell after one week in SCM. NeuN+ cells (arrowheads) are still retaining CNP-GFP fluorescence at this stage, whereas in GFAP+ astrocytes GFP expression has been lost (arrows). Bar, 50 μm for G–J.

Mentions: To enable accurate determination of cell numbers and percentages, we restricted our quantitative analysis to adherent cultures (Fig. 2 M). We assessed cellular immunophenotypes after 48 h in SCM to prevent misinterpretation of the results due to a possible amplification of an initially undetectable contamination of our FACS®-sorted samples by GFP− cells (Fig. 1 D). The fate of total FACS®-sorted CNP-GFP+ cells arising from P2 whole brain was compared with the fate of selected subpopulations of CNP-GFP+ cells double-sorted according to their NG2+ (Fig. 3 A) or O4+ phenotype (Yuan et al., 2002).


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)

Clonal analysis of NG2+/CNP-GFP+ cells in vitro. (A) FACS® dot plots of acutely dissociated cells from wild-type (wt, top) and CNP-GFP transgenic brains (tg, bottom), in forward and side scatter with a polygon indicating the gate selecting the viable cells. (B–D) Sorting profiles of acutely isolated cell suspensions from P2 brains of wild-type (B) and CNP-GFP transgenic mice (C and D) dot plotted according to fluorescence intensity for GFP (x axis, logarithmic scale) and Cy-5 (fluorescence associated to secondary antibody recognizing NG2 immunoreactivity, y axis, logarithmic scale). (B) Control wild-type cells that were incubated only with the Cy-5–conjugated secondary antibody without anti-NG2 primary antibody. Crossed black lines in B–D represent thresholds of fluorescence. It was observed that <0.01% (limit of detection) of the control cells from B fell over this threshold. Thus, these lines determined the level of fluorescence above which cells from CNP-GFP brains (C) were selected as GFP+ (lower right quadrant). When CNP-GFP cell suspensions were immunostained for NG2 (D), NG2+/CNP-GFP+ cells were detected in upper right quadrant. To ensure accurate purification of NG2+/CNP-GFP+ cells, the sort gate for these cells (D, polygon) was defined by taking an additional margin (0.2–0.3 log units) with respect to background fluorescence levels. (E) FACS®-purified early postnatal (P2) NG2+/CNP-GFP+ cells were cultured at clonal density for 1 wk in SCM and the phenotype of resulting cell clones was then determined. (F) Relative proportion of the different subpopulations found in the multipotent clones, i.e., containing CNP-GFP+ cells, as well as neurons (NeuN+) and astrocytes (GFAP+). (G–J) GFP fluorescence (G, green), O4 (H, red), GFAP (I, peroxidase reaction), and NeuN (J, blue) stainings of the same microscopic field showing a representative multipotent clone derived from the growth of a single NG2+/CNP-GFP+ cell after one week in SCM. NeuN+ cells (arrowheads) are still retaining CNP-GFP fluorescence at this stage, whereas in GFAP+ astrocytes GFP expression has been lost (arrows). Bar, 50 μm for G–J.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172886&req=5

fig3: Clonal analysis of NG2+/CNP-GFP+ cells in vitro. (A) FACS® dot plots of acutely dissociated cells from wild-type (wt, top) and CNP-GFP transgenic brains (tg, bottom), in forward and side scatter with a polygon indicating the gate selecting the viable cells. (B–D) Sorting profiles of acutely isolated cell suspensions from P2 brains of wild-type (B) and CNP-GFP transgenic mice (C and D) dot plotted according to fluorescence intensity for GFP (x axis, logarithmic scale) and Cy-5 (fluorescence associated to secondary antibody recognizing NG2 immunoreactivity, y axis, logarithmic scale). (B) Control wild-type cells that were incubated only with the Cy-5–conjugated secondary antibody without anti-NG2 primary antibody. Crossed black lines in B–D represent thresholds of fluorescence. It was observed that <0.01% (limit of detection) of the control cells from B fell over this threshold. Thus, these lines determined the level of fluorescence above which cells from CNP-GFP brains (C) were selected as GFP+ (lower right quadrant). When CNP-GFP cell suspensions were immunostained for NG2 (D), NG2+/CNP-GFP+ cells were detected in upper right quadrant. To ensure accurate purification of NG2+/CNP-GFP+ cells, the sort gate for these cells (D, polygon) was defined by taking an additional margin (0.2–0.3 log units) with respect to background fluorescence levels. (E) FACS®-purified early postnatal (P2) NG2+/CNP-GFP+ cells were cultured at clonal density for 1 wk in SCM and the phenotype of resulting cell clones was then determined. (F) Relative proportion of the different subpopulations found in the multipotent clones, i.e., containing CNP-GFP+ cells, as well as neurons (NeuN+) and astrocytes (GFAP+). (G–J) GFP fluorescence (G, green), O4 (H, red), GFAP (I, peroxidase reaction), and NeuN (J, blue) stainings of the same microscopic field showing a representative multipotent clone derived from the growth of a single NG2+/CNP-GFP+ cell after one week in SCM. NeuN+ cells (arrowheads) are still retaining CNP-GFP fluorescence at this stage, whereas in GFAP+ astrocytes GFP expression has been lost (arrows). Bar, 50 μm for G–J.
Mentions: To enable accurate determination of cell numbers and percentages, we restricted our quantitative analysis to adherent cultures (Fig. 2 M). We assessed cellular immunophenotypes after 48 h in SCM to prevent misinterpretation of the results due to a possible amplification of an initially undetectable contamination of our FACS®-sorted samples by GFP− cells (Fig. 1 D). The fate of total FACS®-sorted CNP-GFP+ cells arising from P2 whole brain was compared with the fate of selected subpopulations of CNP-GFP+ cells double-sorted according to their NG2+ (Fig. 3 A) or O4+ phenotype (Yuan et al., 2002).

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