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Novel stem/progenitor cells with neuronal differentiation potential reside in the leptomeningeal niche.

Bifari F, Decimo I, Chiamulera C, Bersan E, Malpeli G, Johansson J, Lisi V, Bonetti B, Fumagalli G, Pizzolo G, Krampera M - J. Cell. Mol. Med. (2009)

Bottom Line: Once injected into the adult brain, these cells survive and differentiate into neurons, thus showing that their neuronal differentiation potential is operational also in vivo.In conclusion, our data provide evidence that a specific population of immature cells endowed of neuronal differentiation potential is resident in the leptomeninges throughout the life.As leptomeninges cover the entire central nervous system, these findings could have relevant implications for studies on cortical development and for regenerative medicine applied to neurological disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Experimental Medicine, Stem Cell Research Laboratory, Section of Hematology, University of Verona, Verona, Italy.

ABSTRACT
Stem cells capable of generating neural differentiated cells are recognized by the expression of nestin and reside in specific regions of the brain, namely, hippocampus, subventricular zone and olfactory bulb. For other brain structures, such as leptomeninges, which contribute to the correct cortex development and functions, there is no evidence so far that they may contain stem/precursor cells. In this work, we show for the first time that nestin-positive cells are present in rat leptomeninges during development up to adulthood. The newly identified nestin-positive cells can be extracted and expanded in vitro both as neurospheres, displaying high similarity with subventricular zone-derived neural stem cells, and as homogeneous cell population with stem cell features. In vitro expanded stem cell population can differentiate with high efficiency into excitable cells with neuronal phenotype and morphology. Once injected into the adult brain, these cells survive and differentiate into neurons, thus showing that their neuronal differentiation potential is operational also in vivo. In conclusion, our data provide evidence that a specific population of immature cells endowed of neuronal differentiation potential is resident in the leptomeninges throughout the life. As leptomeninges cover the entire central nervous system, these findings could have relevant implications for studies on cortical development and for regenerative medicine applied to neurological disorders.

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In vitro expansion of the tissue extract in adherent cells culture conditions. (A) Time course experiment. Confocal images of nestin+/NG2- cells in adherent culture at 5 hrs, 1, 4, 8 and 12 days after plating. (B) CFU derived from leptomeningeal cells at different passages. Bars represent the CFU number/million of plated cells ± S.D. (B’) May–Grunwald Giemsa staining of a single colony of leptomeningeal adherent cells. (C) Transmitted light and (D) immunofluorescence, nestin (red), of adherent leptomeningeal cells. (E) FACS analysis, carried out on expanded cells obtained from adult rats, shows high expression of nestin and CD90. In vitro expanded cell population does not express neuro-glial (MAP2, GFAP and O4), leukocyte (CD45), endothelial (CD31, CD106) and haematopoietic stem cell markers (CD34). (F) Relative gene expression analysis. Leptomeningeal-derived nestin+ cells and BM-MSCs fold changes in transcription, normalized to actin mRNA, have been shown compared to SVZ-NSCs.
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fig05: In vitro expansion of the tissue extract in adherent cells culture conditions. (A) Time course experiment. Confocal images of nestin+/NG2- cells in adherent culture at 5 hrs, 1, 4, 8 and 12 days after plating. (B) CFU derived from leptomeningeal cells at different passages. Bars represent the CFU number/million of plated cells ± S.D. (B’) May–Grunwald Giemsa staining of a single colony of leptomeningeal adherent cells. (C) Transmitted light and (D) immunofluorescence, nestin (red), of adherent leptomeningeal cells. (E) FACS analysis, carried out on expanded cells obtained from adult rats, shows high expression of nestin and CD90. In vitro expanded cell population does not express neuro-glial (MAP2, GFAP and O4), leukocyte (CD45), endothelial (CD31, CD106) and haematopoietic stem cell markers (CD34). (F) Relative gene expression analysis. Leptomeningeal-derived nestin+ cells and BM-MSCs fold changes in transcription, normalized to actin mRNA, have been shown compared to SVZ-NSCs.

Mentions: Adherent stem cell growth condition. We tried to overcome the complexity [36, 43, 44] of neurosphere assay by modifying the culture conditions. The whole tissue extract was plated in flasks with growing medium (see Materials and Methods). After 48 hrs, non-adherent cells were removed and the medium completely changed. In 1–3 weeks, cell colonies could be recognized. Only a minority of cells from the whole tissue extract adhered and gave rise to CFUs. Non-adherent cells underwent apoptosis or were removed by medium change. All the adherent cells proliferating after several days derived from CFU and displayed a homogeneous immunophenotype, as shown in Fig. 5A and E. This cell population was expanded for several passages and was clonogenic, since CFU were present at each passage (Fig. 5B). Time course experiments were performed to assess whether a cell population of nestin+/GFAP-/NG2-cells (similar to what found in vivo in the leptomeninges) was present in the culture. As shown in Fig. 5A, nestin+/NG2- cells were present as early as 5 hrs after plating (approximately 90% of attached cells). Similar results were obtained with GFAP staining (data not shown). From day 8, nestin+/GFAP–/NG2– cell colonies are detectable.


Novel stem/progenitor cells with neuronal differentiation potential reside in the leptomeningeal niche.

Bifari F, Decimo I, Chiamulera C, Bersan E, Malpeli G, Johansson J, Lisi V, Bonetti B, Fumagalli G, Pizzolo G, Krampera M - J. Cell. Mol. Med. (2009)

In vitro expansion of the tissue extract in adherent cells culture conditions. (A) Time course experiment. Confocal images of nestin+/NG2- cells in adherent culture at 5 hrs, 1, 4, 8 and 12 days after plating. (B) CFU derived from leptomeningeal cells at different passages. Bars represent the CFU number/million of plated cells ± S.D. (B’) May–Grunwald Giemsa staining of a single colony of leptomeningeal adherent cells. (C) Transmitted light and (D) immunofluorescence, nestin (red), of adherent leptomeningeal cells. (E) FACS analysis, carried out on expanded cells obtained from adult rats, shows high expression of nestin and CD90. In vitro expanded cell population does not express neuro-glial (MAP2, GFAP and O4), leukocyte (CD45), endothelial (CD31, CD106) and haematopoietic stem cell markers (CD34). (F) Relative gene expression analysis. Leptomeningeal-derived nestin+ cells and BM-MSCs fold changes in transcription, normalized to actin mRNA, have been shown compared to SVZ-NSCs.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4516477&req=5

fig05: In vitro expansion of the tissue extract in adherent cells culture conditions. (A) Time course experiment. Confocal images of nestin+/NG2- cells in adherent culture at 5 hrs, 1, 4, 8 and 12 days after plating. (B) CFU derived from leptomeningeal cells at different passages. Bars represent the CFU number/million of plated cells ± S.D. (B’) May–Grunwald Giemsa staining of a single colony of leptomeningeal adherent cells. (C) Transmitted light and (D) immunofluorescence, nestin (red), of adherent leptomeningeal cells. (E) FACS analysis, carried out on expanded cells obtained from adult rats, shows high expression of nestin and CD90. In vitro expanded cell population does not express neuro-glial (MAP2, GFAP and O4), leukocyte (CD45), endothelial (CD31, CD106) and haematopoietic stem cell markers (CD34). (F) Relative gene expression analysis. Leptomeningeal-derived nestin+ cells and BM-MSCs fold changes in transcription, normalized to actin mRNA, have been shown compared to SVZ-NSCs.
Mentions: Adherent stem cell growth condition. We tried to overcome the complexity [36, 43, 44] of neurosphere assay by modifying the culture conditions. The whole tissue extract was plated in flasks with growing medium (see Materials and Methods). After 48 hrs, non-adherent cells were removed and the medium completely changed. In 1–3 weeks, cell colonies could be recognized. Only a minority of cells from the whole tissue extract adhered and gave rise to CFUs. Non-adherent cells underwent apoptosis or were removed by medium change. All the adherent cells proliferating after several days derived from CFU and displayed a homogeneous immunophenotype, as shown in Fig. 5A and E. This cell population was expanded for several passages and was clonogenic, since CFU were present at each passage (Fig. 5B). Time course experiments were performed to assess whether a cell population of nestin+/GFAP-/NG2-cells (similar to what found in vivo in the leptomeninges) was present in the culture. As shown in Fig. 5A, nestin+/NG2- cells were present as early as 5 hrs after plating (approximately 90% of attached cells). Similar results were obtained with GFAP staining (data not shown). From day 8, nestin+/GFAP–/NG2– cell colonies are detectable.

Bottom Line: Once injected into the adult brain, these cells survive and differentiate into neurons, thus showing that their neuronal differentiation potential is operational also in vivo.In conclusion, our data provide evidence that a specific population of immature cells endowed of neuronal differentiation potential is resident in the leptomeninges throughout the life.As leptomeninges cover the entire central nervous system, these findings could have relevant implications for studies on cortical development and for regenerative medicine applied to neurological disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Experimental Medicine, Stem Cell Research Laboratory, Section of Hematology, University of Verona, Verona, Italy.

ABSTRACT
Stem cells capable of generating neural differentiated cells are recognized by the expression of nestin and reside in specific regions of the brain, namely, hippocampus, subventricular zone and olfactory bulb. For other brain structures, such as leptomeninges, which contribute to the correct cortex development and functions, there is no evidence so far that they may contain stem/precursor cells. In this work, we show for the first time that nestin-positive cells are present in rat leptomeninges during development up to adulthood. The newly identified nestin-positive cells can be extracted and expanded in vitro both as neurospheres, displaying high similarity with subventricular zone-derived neural stem cells, and as homogeneous cell population with stem cell features. In vitro expanded stem cell population can differentiate with high efficiency into excitable cells with neuronal phenotype and morphology. Once injected into the adult brain, these cells survive and differentiate into neurons, thus showing that their neuronal differentiation potential is operational also in vivo. In conclusion, our data provide evidence that a specific population of immature cells endowed of neuronal differentiation potential is resident in the leptomeninges throughout the life. As leptomeninges cover the entire central nervous system, these findings could have relevant implications for studies on cortical development and for regenerative medicine applied to neurological disorders.

Show MeSH
Related in: MedlinePlus