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Optimizing culture medium composition to improve oligodendrocyte progenitor cell yields in vitro from subventricular zone-derived neural progenitor cell neurospheres.

Franco PG, Pasquini JM, Silvestroff L - PLoS ONE (2015)

Bottom Line: We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield.The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation.As a whole, we describe an optimized in vitro method for increasing OPC.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, and Instituto de Química y Fisicoquímica Biológicas "Profesor Alejandro C. Paladini" (IQUIFIB), UBA-CONICET, Ciudad Autónoma de Buenos Aires, Argentina.

ABSTRACT
Neural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC.

No MeSH data available.


Related in: MedlinePlus

Confocal and epi-fluorescence microscopy images of WT neuron and Act::EGF NS co-cultures.Axonal fibers are labelled with βTubulin III (magenta) for all images. A, B) Epi-fluorescence images of MBP+ (green) and βTubIII cells belonging to bFGF/PDGF-BB cultures treated without (A) or with Heparin (B) during proliferation. The insets in A and B are shown in a and b, respectively. C, D) Confocal images of co-cultures of neurons and bFGF/PDGF-BB (with no added Heparin) pre-treated neurospheres. C) Image of EGFP+ cells from Act::EGFP mice and β-TubIII+ neurons. D) Confocal image of an MBP+ OL (from WT-derived neurospheres) interacting with a βTubIII+ neuron projection. The insets C and D is shown enlarged in c and d, respectively. Scale bar in A = 100 μm for A and B, scale bar in a = 50 μm for a and b.
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pone.0121774.g007: Confocal and epi-fluorescence microscopy images of WT neuron and Act::EGF NS co-cultures.Axonal fibers are labelled with βTubulin III (magenta) for all images. A, B) Epi-fluorescence images of MBP+ (green) and βTubIII cells belonging to bFGF/PDGF-BB cultures treated without (A) or with Heparin (B) during proliferation. The insets in A and B are shown in a and b, respectively. C, D) Confocal images of co-cultures of neurons and bFGF/PDGF-BB (with no added Heparin) pre-treated neurospheres. C) Image of EGFP+ cells from Act::EGFP mice and β-TubIII+ neurons. D) Confocal image of an MBP+ OL (from WT-derived neurospheres) interacting with a βTubIII+ neuron projection. The insets C and D is shown enlarged in c and d, respectively. Scale bar in A = 100 μm for A and B, scale bar in a = 50 μm for a and b.

Mentions: A neuron-OPC in vitro co-culture model was used as a functional assay for OPC-enriched cultures. NS from WT mice were pre-treated with bFGF/PDGF-BB and then plated on fetal brain neurons from WT mice (Fig. 7). We made sure that no MBP+ OL were present in the neuron cultures before the plating the NS-derived OPC (data not shown). Cells were co-cultured for 6 days in DMEM/F12-B27 culture media. We detected the interaction of MBP+ cells with βTubulin III+ axonal projections, either in the absence or presence of Heparin using a conventional epi-fluorescence microscope (Fig. 7A, B). The OPC from Act::EGFP mice pretreated with bFGF/PDGF-BB were also plated onto WT-derived neurons (Fig. 7C). A 3D reconstruction of z-stack confocal images showed GFP expressing cells were able to accommodate their ramifications together with the neuronal axons (S7 Fig.).


Optimizing culture medium composition to improve oligodendrocyte progenitor cell yields in vitro from subventricular zone-derived neural progenitor cell neurospheres.

Franco PG, Pasquini JM, Silvestroff L - PLoS ONE (2015)

Confocal and epi-fluorescence microscopy images of WT neuron and Act::EGF NS co-cultures.Axonal fibers are labelled with βTubulin III (magenta) for all images. A, B) Epi-fluorescence images of MBP+ (green) and βTubIII cells belonging to bFGF/PDGF-BB cultures treated without (A) or with Heparin (B) during proliferation. The insets in A and B are shown in a and b, respectively. C, D) Confocal images of co-cultures of neurons and bFGF/PDGF-BB (with no added Heparin) pre-treated neurospheres. C) Image of EGFP+ cells from Act::EGFP mice and β-TubIII+ neurons. D) Confocal image of an MBP+ OL (from WT-derived neurospheres) interacting with a βTubIII+ neuron projection. The insets C and D is shown enlarged in c and d, respectively. Scale bar in A = 100 μm for A and B, scale bar in a = 50 μm for a and b.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383518&req=5

pone.0121774.g007: Confocal and epi-fluorescence microscopy images of WT neuron and Act::EGF NS co-cultures.Axonal fibers are labelled with βTubulin III (magenta) for all images. A, B) Epi-fluorescence images of MBP+ (green) and βTubIII cells belonging to bFGF/PDGF-BB cultures treated without (A) or with Heparin (B) during proliferation. The insets in A and B are shown in a and b, respectively. C, D) Confocal images of co-cultures of neurons and bFGF/PDGF-BB (with no added Heparin) pre-treated neurospheres. C) Image of EGFP+ cells from Act::EGFP mice and β-TubIII+ neurons. D) Confocal image of an MBP+ OL (from WT-derived neurospheres) interacting with a βTubIII+ neuron projection. The insets C and D is shown enlarged in c and d, respectively. Scale bar in A = 100 μm for A and B, scale bar in a = 50 μm for a and b.
Mentions: A neuron-OPC in vitro co-culture model was used as a functional assay for OPC-enriched cultures. NS from WT mice were pre-treated with bFGF/PDGF-BB and then plated on fetal brain neurons from WT mice (Fig. 7). We made sure that no MBP+ OL were present in the neuron cultures before the plating the NS-derived OPC (data not shown). Cells were co-cultured for 6 days in DMEM/F12-B27 culture media. We detected the interaction of MBP+ cells with βTubulin III+ axonal projections, either in the absence or presence of Heparin using a conventional epi-fluorescence microscope (Fig. 7A, B). The OPC from Act::EGFP mice pretreated with bFGF/PDGF-BB were also plated onto WT-derived neurons (Fig. 7C). A 3D reconstruction of z-stack confocal images showed GFP expressing cells were able to accommodate their ramifications together with the neuronal axons (S7 Fig.).

Bottom Line: We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield.The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation.As a whole, we describe an optimized in vitro method for increasing OPC.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, and Instituto de Química y Fisicoquímica Biológicas "Profesor Alejandro C. Paladini" (IQUIFIB), UBA-CONICET, Ciudad Autónoma de Buenos Aires, Argentina.

ABSTRACT
Neural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC.

No MeSH data available.


Related in: MedlinePlus