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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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Early postnatal CNP-GFP+ cells generate multipotent neurospheres and give rise to neurons, astrocytes, and oligodendrocytes in vitro. (A) Culture conditions. Phase-contrast (B) and fluorescence (C) views of GFP+ neurospheres grown in suspension (5 d in vitro) on uncoated substrate in EGF- and FGF2-containing medium (SCM). (D) A clonally expanded GFP+ neurosphere expressed high levels of nestin immunostaining (red). Clonal spheres gave rise to GFAP+/GFP− astrocytes (E, red) and NeuN+/GFP− neurons (F, red) within 2 d post-plating on polyornithine-coated coverslips. G shows GFAP+ (blue) astrocytes, NeuN+ (red) neurons, and GFP+ oligodendroglial cells derived from a single clonal GFP+ sphere. When cultured in adherent conditions, i.e., on polyornithine-coated surface directly after FACS® sorting, NG2+(H, red)/nestin+(I, red) GFP+ cells also expressed a multipotent fate within 2 d in SCM, and generated mature O1+(J, red)/GFP+ oligodendrocytes, GFAP+(K, red)/GFP− astrocytes and NeuN+(L, red)/GFP− neurons. Bar: 50 μm (B–D and E–F), 25 μm (G, K, and L), and 20 μm (H–J). M shows quantitative analysis of the multipotent properties of CNP-GFP+ cells. Histograms represent immunocytochemical characterization (% of total cells in y axis, immunophenotypes in x axis) of the progeny of early postnatal (P2) FACS®-purified CNP-GFP+ cells cultured directly under adherent conditions for 48 h in SCM. Comparison was made between the fate of total CNP-GFP+ cells (blue) versus selected subsets of CNP-GFP+ cells that were FACS®-purified according to their NG2+/CNP-GFP+ (red) or O4+/CNP-GFP+ (yellow) phenotype. Nestin, NG2, O4, O1, NeuN, and GFAP phenotypes were analyzed. Values (mean ± SEM) represent averages of 2–3 independent experiments. Counting was performed separately for each staining, and the number of total cells counted (from at least 15 separate microscopic fields) ranged between 403 and 714. Significant differences reported in the Results section were all with a P value <0.001 (t test).
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fig2: Early postnatal CNP-GFP+ cells generate multipotent neurospheres and give rise to neurons, astrocytes, and oligodendrocytes in vitro. (A) Culture conditions. Phase-contrast (B) and fluorescence (C) views of GFP+ neurospheres grown in suspension (5 d in vitro) on uncoated substrate in EGF- and FGF2-containing medium (SCM). (D) A clonally expanded GFP+ neurosphere expressed high levels of nestin immunostaining (red). Clonal spheres gave rise to GFAP+/GFP− astrocytes (E, red) and NeuN+/GFP− neurons (F, red) within 2 d post-plating on polyornithine-coated coverslips. G shows GFAP+ (blue) astrocytes, NeuN+ (red) neurons, and GFP+ oligodendroglial cells derived from a single clonal GFP+ sphere. When cultured in adherent conditions, i.e., on polyornithine-coated surface directly after FACS® sorting, NG2+(H, red)/nestin+(I, red) GFP+ cells also expressed a multipotent fate within 2 d in SCM, and generated mature O1+(J, red)/GFP+ oligodendrocytes, GFAP+(K, red)/GFP− astrocytes and NeuN+(L, red)/GFP− neurons. Bar: 50 μm (B–D and E–F), 25 μm (G, K, and L), and 20 μm (H–J). M shows quantitative analysis of the multipotent properties of CNP-GFP+ cells. Histograms represent immunocytochemical characterization (% of total cells in y axis, immunophenotypes in x axis) of the progeny of early postnatal (P2) FACS®-purified CNP-GFP+ cells cultured directly under adherent conditions for 48 h in SCM. Comparison was made between the fate of total CNP-GFP+ cells (blue) versus selected subsets of CNP-GFP+ cells that were FACS®-purified according to their NG2+/CNP-GFP+ (red) or O4+/CNP-GFP+ (yellow) phenotype. Nestin, NG2, O4, O1, NeuN, and GFAP phenotypes were analyzed. Values (mean ± SEM) represent averages of 2–3 independent experiments. Counting was performed separately for each staining, and the number of total cells counted (from at least 15 separate microscopic fields) ranged between 403 and 714. Significant differences reported in the Results section were all with a P value <0.001 (t test).

Mentions: To study the developmental fate of early postnatal CNP-GFP+ cells, pure FACS®-sorted GFP+ cells from P2 brains were cultured in stem cell medium (SCM) under two different conditions (Fig. 2 A): (1) “suspension” cultures; cells seeded on uncoated plastic to form floating clonal spheres; and (2) “adherent” monolayer cultures; cells directly plated on polyornithine-coated plastic. In suspension cultures, CNP-GFP+ cells rapidly gave rise to GFP+ neurospheres (Fig. 2, B and C) that displayed high levels of nestin expression (Fig. 2 D). Single clonally expanded GFP+ spheres (8–10 d of suspension culture in SCM) plated on coated plastic generated both NeuN+ neurons and GFAP+ astrocytes in SCM (Fig. 2, E–G). Interestingly, these neurons and astrocytes derived from CNP-GFP+ cells were GFP−, illustrating that CNP gene expression is down-regulated when GFP+ cells undergo nonoligodendroglial differentiation. Similarly, in adherent cultures, FACS®-purified CNP-GFP+ cells that were nestin+ and mostly NG2+ (Fig. 1 D and Fig. 2, H–I) rapidly generated NeuN+/GFP− neurons, GFAP+/GFP− astrocytes, and mature O1+/GFP+ oligodendrocytes within 48 h (Fig. 2, J–L).


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)

Early postnatal CNP-GFP+ cells generate multipotent neurospheres and give rise to neurons, astrocytes, and oligodendrocytes in vitro. (A) Culture conditions. Phase-contrast (B) and fluorescence (C) views of GFP+ neurospheres grown in suspension (5 d in vitro) on uncoated substrate in EGF- and FGF2-containing medium (SCM). (D) A clonally expanded GFP+ neurosphere expressed high levels of nestin immunostaining (red). Clonal spheres gave rise to GFAP+/GFP− astrocytes (E, red) and NeuN+/GFP− neurons (F, red) within 2 d post-plating on polyornithine-coated coverslips. G shows GFAP+ (blue) astrocytes, NeuN+ (red) neurons, and GFP+ oligodendroglial cells derived from a single clonal GFP+ sphere. When cultured in adherent conditions, i.e., on polyornithine-coated surface directly after FACS® sorting, NG2+(H, red)/nestin+(I, red) GFP+ cells also expressed a multipotent fate within 2 d in SCM, and generated mature O1+(J, red)/GFP+ oligodendrocytes, GFAP+(K, red)/GFP− astrocytes and NeuN+(L, red)/GFP− neurons. Bar: 50 μm (B–D and E–F), 25 μm (G, K, and L), and 20 μm (H–J). M shows quantitative analysis of the multipotent properties of CNP-GFP+ cells. Histograms represent immunocytochemical characterization (% of total cells in y axis, immunophenotypes in x axis) of the progeny of early postnatal (P2) FACS®-purified CNP-GFP+ cells cultured directly under adherent conditions for 48 h in SCM. Comparison was made between the fate of total CNP-GFP+ cells (blue) versus selected subsets of CNP-GFP+ cells that were FACS®-purified according to their NG2+/CNP-GFP+ (red) or O4+/CNP-GFP+ (yellow) phenotype. Nestin, NG2, O4, O1, NeuN, and GFAP phenotypes were analyzed. Values (mean ± SEM) represent averages of 2–3 independent experiments. Counting was performed separately for each staining, and the number of total cells counted (from at least 15 separate microscopic fields) ranged between 403 and 714. Significant differences reported in the Results section were all with a P value <0.001 (t test).
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Related In: Results  -  Collection

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fig2: Early postnatal CNP-GFP+ cells generate multipotent neurospheres and give rise to neurons, astrocytes, and oligodendrocytes in vitro. (A) Culture conditions. Phase-contrast (B) and fluorescence (C) views of GFP+ neurospheres grown in suspension (5 d in vitro) on uncoated substrate in EGF- and FGF2-containing medium (SCM). (D) A clonally expanded GFP+ neurosphere expressed high levels of nestin immunostaining (red). Clonal spheres gave rise to GFAP+/GFP− astrocytes (E, red) and NeuN+/GFP− neurons (F, red) within 2 d post-plating on polyornithine-coated coverslips. G shows GFAP+ (blue) astrocytes, NeuN+ (red) neurons, and GFP+ oligodendroglial cells derived from a single clonal GFP+ sphere. When cultured in adherent conditions, i.e., on polyornithine-coated surface directly after FACS® sorting, NG2+(H, red)/nestin+(I, red) GFP+ cells also expressed a multipotent fate within 2 d in SCM, and generated mature O1+(J, red)/GFP+ oligodendrocytes, GFAP+(K, red)/GFP− astrocytes and NeuN+(L, red)/GFP− neurons. Bar: 50 μm (B–D and E–F), 25 μm (G, K, and L), and 20 μm (H–J). M shows quantitative analysis of the multipotent properties of CNP-GFP+ cells. Histograms represent immunocytochemical characterization (% of total cells in y axis, immunophenotypes in x axis) of the progeny of early postnatal (P2) FACS®-purified CNP-GFP+ cells cultured directly under adherent conditions for 48 h in SCM. Comparison was made between the fate of total CNP-GFP+ cells (blue) versus selected subsets of CNP-GFP+ cells that were FACS®-purified according to their NG2+/CNP-GFP+ (red) or O4+/CNP-GFP+ (yellow) phenotype. Nestin, NG2, O4, O1, NeuN, and GFAP phenotypes were analyzed. Values (mean ± SEM) represent averages of 2–3 independent experiments. Counting was performed separately for each staining, and the number of total cells counted (from at least 15 separate microscopic fields) ranged between 403 and 714. Significant differences reported in the Results section were all with a P value <0.001 (t test).
Mentions: To study the developmental fate of early postnatal CNP-GFP+ cells, pure FACS®-sorted GFP+ cells from P2 brains were cultured in stem cell medium (SCM) under two different conditions (Fig. 2 A): (1) “suspension” cultures; cells seeded on uncoated plastic to form floating clonal spheres; and (2) “adherent” monolayer cultures; cells directly plated on polyornithine-coated plastic. In suspension cultures, CNP-GFP+ cells rapidly gave rise to GFP+ neurospheres (Fig. 2, B and C) that displayed high levels of nestin expression (Fig. 2 D). Single clonally expanded GFP+ spheres (8–10 d of suspension culture in SCM) plated on coated plastic generated both NeuN+ neurons and GFAP+ astrocytes in SCM (Fig. 2, E–G). Interestingly, these neurons and astrocytes derived from CNP-GFP+ cells were GFP−, illustrating that CNP gene expression is down-regulated when GFP+ cells undergo nonoligodendroglial differentiation. Similarly, in adherent cultures, FACS®-purified CNP-GFP+ cells that were nestin+ and mostly NG2+ (Fig. 1 D and Fig. 2, H–I) rapidly generated NeuN+/GFP− neurons, GFAP+/GFP− astrocytes, and mature O1+/GFP+ oligodendrocytes within 48 h (Fig. 2, J–L).

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