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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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CNP-GFP+ cells gradually lose GFP expression as they differentiate into mature, excitable neurons in culture. Phase-contrast view (A), GFP green fluorescence (B), NeuN staining (C, red), and overlay (D) of the same microscopic field showing NeuN+/CNP-GFP− neuronal progeny derived from P2 FACS®-purified CNP-GFP+ cells after 48 h in SCM. Cells expressing low levels of GFP, but displaying a neuronal phenotype could also be found (E–H). These cells expressed the neuronal markers NeuN (same microscopic field with GFP green fluorescence in E, red NeuN staining in F, and overlay in G) and MAP2a,b (H, red staining). Arrowheads indicate NeuN+/CNP-GFP+ (E–G) and MAP2a,b+/CNP-GFP+ cells (H). Arrow in H points to a MAP2a,b-expressing neuron that has completely lost GFP expression. (I–K) Electrophysiological whole-cell patch-clamp experiments in current-clamp mode were performed, in order to study excitability of cell progeny arising from SCM-cultured FACS®-sorted CNP-GFP+ cells. After 2 d in SCM, cultures were switched to EGF- and FGF2-free medium supplemented with a combination of 30 ng/ml brain-derived neurotrophic factor and 30 ng/ml neurotrophin-3 for one week. GFP+ and GFP− cells were analyzed and filled with biocytin during electrophysiological recording for identification and further immunocytochemical characterization. We recorded only GFP− cells that did not display a typical astrocytic morphology. Depolarization of GFP− cells elicited single (7 out of 9 cells; I, inset), or repetitive (2 out of 9 cells; J, inset) action potentials. In five of these cells, we investigated biocytin (red) and NeuN (blue) immunoreactivities, and in all cases colocalization (purple) was observed (I–J). In contrast, all GFP+ cells tested (12 cells) did not elicit action potentials (K, inset). Out of the GFP+ cells that were immunostained after recording (6 cells), all were NeuN− (K; biocytin in red, NeuN in blue). (I–K) GFP expression could not be visualized because of dialysis associated with whole-cell recording. Bars: 25 μm (A–D), (E–G), (H), and (I–K).
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fig4: CNP-GFP+ cells gradually lose GFP expression as they differentiate into mature, excitable neurons in culture. Phase-contrast view (A), GFP green fluorescence (B), NeuN staining (C, red), and overlay (D) of the same microscopic field showing NeuN+/CNP-GFP− neuronal progeny derived from P2 FACS®-purified CNP-GFP+ cells after 48 h in SCM. Cells expressing low levels of GFP, but displaying a neuronal phenotype could also be found (E–H). These cells expressed the neuronal markers NeuN (same microscopic field with GFP green fluorescence in E, red NeuN staining in F, and overlay in G) and MAP2a,b (H, red staining). Arrowheads indicate NeuN+/CNP-GFP+ (E–G) and MAP2a,b+/CNP-GFP+ cells (H). Arrow in H points to a MAP2a,b-expressing neuron that has completely lost GFP expression. (I–K) Electrophysiological whole-cell patch-clamp experiments in current-clamp mode were performed, in order to study excitability of cell progeny arising from SCM-cultured FACS®-sorted CNP-GFP+ cells. After 2 d in SCM, cultures were switched to EGF- and FGF2-free medium supplemented with a combination of 30 ng/ml brain-derived neurotrophic factor and 30 ng/ml neurotrophin-3 for one week. GFP+ and GFP− cells were analyzed and filled with biocytin during electrophysiological recording for identification and further immunocytochemical characterization. We recorded only GFP− cells that did not display a typical astrocytic morphology. Depolarization of GFP− cells elicited single (7 out of 9 cells; I, inset), or repetitive (2 out of 9 cells; J, inset) action potentials. In five of these cells, we investigated biocytin (red) and NeuN (blue) immunoreactivities, and in all cases colocalization (purple) was observed (I–J). In contrast, all GFP+ cells tested (12 cells) did not elicit action potentials (K, inset). Out of the GFP+ cells that were immunostained after recording (6 cells), all were NeuN− (K; biocytin in red, NeuN in blue). (I–K) GFP expression could not be visualized because of dialysis associated with whole-cell recording. Bars: 25 μm (A–D), (E–G), (H), and (I–K).

Mentions: A gradual loss of GFP expression was observed during astroglial maturation of CNP-GFP+ cells (Fig. S1). Mature astrocytes derived from CNP-GFP+ cells were GFP− and displayed typical stellate shapes with intense GFAP expression in cell bodies and processes (Fig. S1, A–D). In the same cultures, we also identified GFAP+ cells expressing low levels of GFP. In all these GFAP+/CNP-GFP+ cells, GFAP expression was restricted to their cell body (Fig. S1, E–H, arrowheads), suggesting an intermediate stage of astroglial maturation. Similarly, among NeuN+ and type 2a,b microtubule-associated protein-positive (MAP2a,b) neurons generated from CNP-GFP+ cells, we identified either cells that were totally GFP− (Fig. 4 , A–D; Fig. 4 H, arrow) or cells that expressed very low levels of GFP fluorescence (Fig. 4, E–H, arrowheads).


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)

CNP-GFP+ cells gradually lose GFP expression as they differentiate into mature, excitable neurons in culture. Phase-contrast view (A), GFP green fluorescence (B), NeuN staining (C, red), and overlay (D) of the same microscopic field showing NeuN+/CNP-GFP− neuronal progeny derived from P2 FACS®-purified CNP-GFP+ cells after 48 h in SCM. Cells expressing low levels of GFP, but displaying a neuronal phenotype could also be found (E–H). These cells expressed the neuronal markers NeuN (same microscopic field with GFP green fluorescence in E, red NeuN staining in F, and overlay in G) and MAP2a,b (H, red staining). Arrowheads indicate NeuN+/CNP-GFP+ (E–G) and MAP2a,b+/CNP-GFP+ cells (H). Arrow in H points to a MAP2a,b-expressing neuron that has completely lost GFP expression. (I–K) Electrophysiological whole-cell patch-clamp experiments in current-clamp mode were performed, in order to study excitability of cell progeny arising from SCM-cultured FACS®-sorted CNP-GFP+ cells. After 2 d in SCM, cultures were switched to EGF- and FGF2-free medium supplemented with a combination of 30 ng/ml brain-derived neurotrophic factor and 30 ng/ml neurotrophin-3 for one week. GFP+ and GFP− cells were analyzed and filled with biocytin during electrophysiological recording for identification and further immunocytochemical characterization. We recorded only GFP− cells that did not display a typical astrocytic morphology. Depolarization of GFP− cells elicited single (7 out of 9 cells; I, inset), or repetitive (2 out of 9 cells; J, inset) action potentials. In five of these cells, we investigated biocytin (red) and NeuN (blue) immunoreactivities, and in all cases colocalization (purple) was observed (I–J). In contrast, all GFP+ cells tested (12 cells) did not elicit action potentials (K, inset). Out of the GFP+ cells that were immunostained after recording (6 cells), all were NeuN− (K; biocytin in red, NeuN in blue). (I–K) GFP expression could not be visualized because of dialysis associated with whole-cell recording. Bars: 25 μm (A–D), (E–G), (H), and (I–K).
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fig4: CNP-GFP+ cells gradually lose GFP expression as they differentiate into mature, excitable neurons in culture. Phase-contrast view (A), GFP green fluorescence (B), NeuN staining (C, red), and overlay (D) of the same microscopic field showing NeuN+/CNP-GFP− neuronal progeny derived from P2 FACS®-purified CNP-GFP+ cells after 48 h in SCM. Cells expressing low levels of GFP, but displaying a neuronal phenotype could also be found (E–H). These cells expressed the neuronal markers NeuN (same microscopic field with GFP green fluorescence in E, red NeuN staining in F, and overlay in G) and MAP2a,b (H, red staining). Arrowheads indicate NeuN+/CNP-GFP+ (E–G) and MAP2a,b+/CNP-GFP+ cells (H). Arrow in H points to a MAP2a,b-expressing neuron that has completely lost GFP expression. (I–K) Electrophysiological whole-cell patch-clamp experiments in current-clamp mode were performed, in order to study excitability of cell progeny arising from SCM-cultured FACS®-sorted CNP-GFP+ cells. After 2 d in SCM, cultures were switched to EGF- and FGF2-free medium supplemented with a combination of 30 ng/ml brain-derived neurotrophic factor and 30 ng/ml neurotrophin-3 for one week. GFP+ and GFP− cells were analyzed and filled with biocytin during electrophysiological recording for identification and further immunocytochemical characterization. We recorded only GFP− cells that did not display a typical astrocytic morphology. Depolarization of GFP− cells elicited single (7 out of 9 cells; I, inset), or repetitive (2 out of 9 cells; J, inset) action potentials. In five of these cells, we investigated biocytin (red) and NeuN (blue) immunoreactivities, and in all cases colocalization (purple) was observed (I–J). In contrast, all GFP+ cells tested (12 cells) did not elicit action potentials (K, inset). Out of the GFP+ cells that were immunostained after recording (6 cells), all were NeuN− (K; biocytin in red, NeuN in blue). (I–K) GFP expression could not be visualized because of dialysis associated with whole-cell recording. Bars: 25 μm (A–D), (E–G), (H), and (I–K).
Mentions: A gradual loss of GFP expression was observed during astroglial maturation of CNP-GFP+ cells (Fig. S1). Mature astrocytes derived from CNP-GFP+ cells were GFP− and displayed typical stellate shapes with intense GFAP expression in cell bodies and processes (Fig. S1, A–D). In the same cultures, we also identified GFAP+ cells expressing low levels of GFP. In all these GFAP+/CNP-GFP+ cells, GFAP expression was restricted to their cell body (Fig. S1, E–H, arrowheads), suggesting an intermediate stage of astroglial maturation. Similarly, among NeuN+ and type 2a,b microtubule-associated protein-positive (MAP2a,b) neurons generated from CNP-GFP+ cells, we identified either cells that were totally GFP− (Fig. 4 , A–D; Fig. 4 H, arrow) or cells that expressed very low levels of GFP fluorescence (Fig. 4, E–H, arrowheads).

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