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Convergence of cells from the progenitor fraction of adult olfactory bulb tissue to remyelinating glia in demyelinating spinal cord lesions.

Markakis EA, Sasaki M, Lankford KL, Kocsis JD - PLoS ONE (2009)

Bottom Line: OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient.The GFP(+) cells were found among primarily P0(+) myelin profiles, although some myelin basic protein (MBP) profiles were present.Immuno-electron microscopy for GFP revealed GFP(+) cell bodies adjacent to and surrounding peripheral-type myelin rings.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America. eleni.markakis@yale.edu

ABSTRACT

Background: Progenitor cells isolated from adult brain tissue are important tools for experimental studies of remyelination. Cells harvested from neurogenic regions in the adult brain such as the subependymal zone have demonstrated remyelination potential. Multipotent cells from the progenitor fraction have been isolated from the adult olfactory bulb (OB) but their potential to remyelinate has not been studied.

Methodology/principal findings: We used the buoyant density gradient centrifugation method to isolate the progenitor fraction and harvest self-renewing multipotent neural cells grown in monolayers from the adult green-fluorescent protein (GFP) transgenic rat OB. OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient. The progenitor fraction was isolated and these cells were plated in growth media with serum for 24 hrs. Cells were then propagated in N2 supplemented serum-free media containing b-FGF. Cells at passage 4 (P4) were introduced into a demyelinated spinal cord lesion. The GFP(+) cells survived and integrated into the lesion, and extensive remyelination was observed in plastic sections. Immunohistochemistry revealed GFP(+) cells in the spinal cord to be glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and neurofilament negative. The GFP(+) cells were found among primarily P0(+) myelin profiles, although some myelin basic protein (MBP) profiles were present. Immuno-electron microscopy for GFP revealed GFP(+) cell bodies adjacent to and surrounding peripheral-type myelin rings.

Conclusions/significance: We report that neural cells from the progenitor fraction of the adult rat OB grown in monolayers can be expanded for several passages in culture and that upon transplantation into a demyelinated spinal cord lesion provide extensive remyelination without ectopic neuronal differentiation.

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Related in: MedlinePlus

Photomicrographs of cells in vitro.A. Phase contrast light photomicrograph of cells isolated from the progenitor fraction of the adult rat OB at P5. Cells are of four basic morphologies: round cells with central nuclei (red crossed arrow), bipolar with long processes (green arrows), large, amorphous cells with eccentric nuclei (thick orange arrow), and ovoid nucleus with long, fine processes (blue arrows). The scale bar is: 10 µm.
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pone-0007260-g001: Photomicrographs of cells in vitro.A. Phase contrast light photomicrograph of cells isolated from the progenitor fraction of the adult rat OB at P5. Cells are of four basic morphologies: round cells with central nuclei (red crossed arrow), bipolar with long processes (green arrows), large, amorphous cells with eccentric nuclei (thick orange arrow), and ovoid nucleus with long, fine processes (blue arrows). The scale bar is: 10 µm.

Mentions: OB cells from the progenitor fraction were plated in serum-supplemented media onto polyornithine-laminin coated plates. Within 24 hrs the serum media and all non-adherent cells were suctioned off the plate. Cultures were fed with a 1∶1 mixture of DMEM/F12 supplemented with N2, conditioned media from established progenitor cultures, and b-FGF. The cells that adhered to the plate were of four basic morphologies (Fig. 1): large round cells with central round nuclei, prominent bipolar cells with thick processes and dense nuclei, large amorphous cells with eccentric nuclei with radial processes, and small bipolar cells with translucent nuclei and long fine processes. Cultures expanded rapidly in the presence of b-FGF and reached confluence in less than one week. Confluent cultures were passaged 1∶2 and continued to expand for at least eight passages; cells at P4 were chosen for grafting.


Convergence of cells from the progenitor fraction of adult olfactory bulb tissue to remyelinating glia in demyelinating spinal cord lesions.

Markakis EA, Sasaki M, Lankford KL, Kocsis JD - PLoS ONE (2009)

Photomicrographs of cells in vitro.A. Phase contrast light photomicrograph of cells isolated from the progenitor fraction of the adult rat OB at P5. Cells are of four basic morphologies: round cells with central nuclei (red crossed arrow), bipolar with long processes (green arrows), large, amorphous cells with eccentric nuclei (thick orange arrow), and ovoid nucleus with long, fine processes (blue arrows). The scale bar is: 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747269&req=5

pone-0007260-g001: Photomicrographs of cells in vitro.A. Phase contrast light photomicrograph of cells isolated from the progenitor fraction of the adult rat OB at P5. Cells are of four basic morphologies: round cells with central nuclei (red crossed arrow), bipolar with long processes (green arrows), large, amorphous cells with eccentric nuclei (thick orange arrow), and ovoid nucleus with long, fine processes (blue arrows). The scale bar is: 10 µm.
Mentions: OB cells from the progenitor fraction were plated in serum-supplemented media onto polyornithine-laminin coated plates. Within 24 hrs the serum media and all non-adherent cells were suctioned off the plate. Cultures were fed with a 1∶1 mixture of DMEM/F12 supplemented with N2, conditioned media from established progenitor cultures, and b-FGF. The cells that adhered to the plate were of four basic morphologies (Fig. 1): large round cells with central round nuclei, prominent bipolar cells with thick processes and dense nuclei, large amorphous cells with eccentric nuclei with radial processes, and small bipolar cells with translucent nuclei and long fine processes. Cultures expanded rapidly in the presence of b-FGF and reached confluence in less than one week. Confluent cultures were passaged 1∶2 and continued to expand for at least eight passages; cells at P4 were chosen for grafting.

Bottom Line: OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient.The GFP(+) cells were found among primarily P0(+) myelin profiles, although some myelin basic protein (MBP) profiles were present.Immuno-electron microscopy for GFP revealed GFP(+) cell bodies adjacent to and surrounding peripheral-type myelin rings.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America. eleni.markakis@yale.edu

ABSTRACT

Background: Progenitor cells isolated from adult brain tissue are important tools for experimental studies of remyelination. Cells harvested from neurogenic regions in the adult brain such as the subependymal zone have demonstrated remyelination potential. Multipotent cells from the progenitor fraction have been isolated from the adult olfactory bulb (OB) but their potential to remyelinate has not been studied.

Methodology/principal findings: We used the buoyant density gradient centrifugation method to isolate the progenitor fraction and harvest self-renewing multipotent neural cells grown in monolayers from the adult green-fluorescent protein (GFP) transgenic rat OB. OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient. The progenitor fraction was isolated and these cells were plated in growth media with serum for 24 hrs. Cells were then propagated in N2 supplemented serum-free media containing b-FGF. Cells at passage 4 (P4) were introduced into a demyelinated spinal cord lesion. The GFP(+) cells survived and integrated into the lesion, and extensive remyelination was observed in plastic sections. Immunohistochemistry revealed GFP(+) cells in the spinal cord to be glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and neurofilament negative. The GFP(+) cells were found among primarily P0(+) myelin profiles, although some myelin basic protein (MBP) profiles were present. Immuno-electron microscopy for GFP revealed GFP(+) cell bodies adjacent to and surrounding peripheral-type myelin rings.

Conclusions/significance: We report that neural cells from the progenitor fraction of the adult rat OB grown in monolayers can be expanded for several passages in culture and that upon transplantation into a demyelinated spinal cord lesion provide extensive remyelination without ectopic neuronal differentiation.

Show MeSH
Related in: MedlinePlus