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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

Heparin effects during oligodendrogenesis and oligodendrocyte maturation in CNP::EGFP mice cultures.A) The number of NG2+ cells at the end of the proliferation stage are expressed as a proportion respect to the total number of nuclei analyzed per treatment. More than 300 nuclei were analyzed for each treatment in 4 independent cultures. B, C) The proportion of GFP+ and MBP+ cells after differentiation is compared in Heparin (1.25 U/L) treated cultures against culture lacking Heparin. More than 600 nuclei were analyzed for a each treatment. Data analysis in graphs was performed using Student´s t Test. D) Representative images of CNP::EGFP mice derived cutlures demonstrating EGFP+ (green) and MBP+ (magenta) cells. Scale bar in D (i) represents 100 μm for all images in D. Each pair of columns in A (with or without Heparin) was analyzed with a Student´s t test. ns = not significant.
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pone.0121774.g006: Heparin effects during oligodendrogenesis and oligodendrocyte maturation in CNP::EGFP mice cultures.A) The number of NG2+ cells at the end of the proliferation stage are expressed as a proportion respect to the total number of nuclei analyzed per treatment. More than 300 nuclei were analyzed for each treatment in 4 independent cultures. B, C) The proportion of GFP+ and MBP+ cells after differentiation is compared in Heparin (1.25 U/L) treated cultures against culture lacking Heparin. More than 600 nuclei were analyzed for a each treatment. Data analysis in graphs was performed using Student´s t Test. D) Representative images of CNP::EGFP mice derived cutlures demonstrating EGFP+ (green) and MBP+ (magenta) cells. Scale bar in D (i) represents 100 μm for all images in D. Each pair of columns in A (with or without Heparin) was analyzed with a Student´s t test. ns = not significant.

Mentions: We used the CNP::EGFP mice to analyze the OPC maturation capacity in the presence or absence of Heparin. The proportion of NG2+ cells generated by PDGF-BB treatment was not significantly different when Heparin was used in the culture during proliferation at 1.25 U/L (Fig. 6A). These findings were evaluated in parallel by analyzing the EGFP expression in the same cultures of CNP::EGFP mice and we observed that adding Heparin to the cultures during proliferation did not alter the EGFP expression in either of the treatments (S6 Fig.). The proportions of EGFP and MBP expressing cells were used as indicators of OL commitment when using NS cultures derived from CNP::EGFP mice under standard differentiating conditions. The addition of Heparin 1.25 U/L to the culture media did not hamper the generation of cells belonging to the OL lineage when evaluating the EGFP+ or MBP+ cells by ICC (Fig. 6B, C). The OL generated during differentiation did not differ morphologically among Heparin treated or non-treated cultures (Fig. 6D).


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)

Heparin effects during oligodendrogenesis and oligodendrocyte maturation in CNP::EGFP mice cultures.A) The number of NG2+ cells at the end of the proliferation stage are expressed as a proportion respect to the total number of nuclei analyzed per treatment. More than 300 nuclei were analyzed for each treatment in 4 independent cultures. B, C) The proportion of GFP+ and MBP+ cells after differentiation is compared in Heparin (1.25 U/L) treated cultures against culture lacking Heparin. More than 600 nuclei were analyzed for a each treatment. Data analysis in graphs was performed using Student´s t Test. D) Representative images of CNP::EGFP mice derived cutlures demonstrating EGFP+ (green) and MBP+ (magenta) cells. Scale bar in D (i) represents 100 μm for all images in D. Each pair of columns in A (with or without Heparin) was analyzed with a Student´s t test. ns = not significant.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383518&req=5

pone.0121774.g006: Heparin effects during oligodendrogenesis and oligodendrocyte maturation in CNP::EGFP mice cultures.A) The number of NG2+ cells at the end of the proliferation stage are expressed as a proportion respect to the total number of nuclei analyzed per treatment. More than 300 nuclei were analyzed for each treatment in 4 independent cultures. B, C) The proportion of GFP+ and MBP+ cells after differentiation is compared in Heparin (1.25 U/L) treated cultures against culture lacking Heparin. More than 600 nuclei were analyzed for a each treatment. Data analysis in graphs was performed using Student´s t Test. D) Representative images of CNP::EGFP mice derived cutlures demonstrating EGFP+ (green) and MBP+ (magenta) cells. Scale bar in D (i) represents 100 μm for all images in D. Each pair of columns in A (with or without Heparin) was analyzed with a Student´s t test. ns = not significant.
Mentions: We used the CNP::EGFP mice to analyze the OPC maturation capacity in the presence or absence of Heparin. The proportion of NG2+ cells generated by PDGF-BB treatment was not significantly different when Heparin was used in the culture during proliferation at 1.25 U/L (Fig. 6A). These findings were evaluated in parallel by analyzing the EGFP expression in the same cultures of CNP::EGFP mice and we observed that adding Heparin to the cultures during proliferation did not alter the EGFP expression in either of the treatments (S6 Fig.). The proportions of EGFP and MBP expressing cells were used as indicators of OL commitment when using NS cultures derived from CNP::EGFP mice under standard differentiating conditions. The addition of Heparin 1.25 U/L to the culture media did not hamper the generation of cells belonging to the OL lineage when evaluating the EGFP+ or MBP+ cells by ICC (Fig. 6B, C). The OL generated during differentiation did not differ morphologically among Heparin treated or non-treated cultures (Fig. 6D).

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