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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

Oligodendrocyte maturation in vitro using WT-derived NS.A) Immunocytochemistry of NS cultures pre-treated during proliferation with bFGF/EGF or bFGF/PDGF-BB, and then exposed to differentiating conditions; No growth factors (CTL), Tri-iodothyronine (T3), Insulin-like Growth Factor 1 (IGF-1) and Fetal Calf Serum (FCS). B) Comparison of the proportion of MBP+ cells detected after differentiation between bFGF/EGF and bFGF/PDGF-BB pre-treated NS. C) Quantitation of the MBP+ area/nuclei ratio after differentiation in two independent cultures. Data in B and C was analyzed with a Two-way ANOVA and Bonferroni post-test to compare the effect of NS pre-treatment during proliferation (*) or the differentiation treatment compared to its CTL (#). D) Fold change of the MBP+ area/nuclei in bFGF/PDGF-BB compared to bFGF/EGF cultures. The bars in B, C and D represent the mean + SD. The scale bar in A (i) represents 100 μm for (i) and (ii), while the scale bar in A (iii) represents 100 μm for (iii) to (x). * = p < 0.05. ** = p < 0.01 and # = p < 0.05. DIFF.; Differentiation.
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pone.0121774.g003: Oligodendrocyte maturation in vitro using WT-derived NS.A) Immunocytochemistry of NS cultures pre-treated during proliferation with bFGF/EGF or bFGF/PDGF-BB, and then exposed to differentiating conditions; No growth factors (CTL), Tri-iodothyronine (T3), Insulin-like Growth Factor 1 (IGF-1) and Fetal Calf Serum (FCS). B) Comparison of the proportion of MBP+ cells detected after differentiation between bFGF/EGF and bFGF/PDGF-BB pre-treated NS. C) Quantitation of the MBP+ area/nuclei ratio after differentiation in two independent cultures. Data in B and C was analyzed with a Two-way ANOVA and Bonferroni post-test to compare the effect of NS pre-treatment during proliferation (*) or the differentiation treatment compared to its CTL (#). D) Fold change of the MBP+ area/nuclei in bFGF/PDGF-BB compared to bFGF/EGF cultures. The bars in B, C and D represent the mean + SD. The scale bar in A (i) represents 100 μm for (i) and (ii), while the scale bar in A (iii) represents 100 μm for (iii) to (x). * = p < 0.05. ** = p < 0.01 and # = p < 0.05. DIFF.; Differentiation.

Mentions: To confirm that bFGF/PDGF-BB pre-treated cultures from Act::EGFP mice were able to generate mature OL, we exposed them to differentiating media lacking growth factors. We also induce cell differentiation by addition of T3, IGF-1 or FCS as described in the S4 Fig. The presence of mature MBP+ OL was analyzed by ICC. During proliferation, cultures grown in bFGF/EGF or bFGF/PDGF-BB contained few MBP+ OL that had a poorly developed morphology; diameter and ramifications (Fig. 3Ai, ii). After differentiating bFGF/EGF pre-treated cultures (Fig. 3Aiii, v, vii and ix), there were no evident changes in mature OL proportions between the various differentiating conditions (white bars in Fig. 3B). However, there was a significant increase in MBP+ cell proportions after differentiating bFGF/PDGF-BB pre-treated cultures in the presence of T3, IGF-1 (Fig. 3Aiv, vi, viii and x, and black bars in Fig. 3B). The OL morphology is compared in Fig. 3C and D after differentiation. Adding T3, IGF-1 or FCS during differentiation had a significant impact on MBP+ cell proportions in bFGF/PDGF-BB pre-treated cultures compared to bFGF/EGF treated cultures. As a whole, we found a significant increase in the complexity of MBP+ OL cell morphology in bFGF/PDGF-BB pre-treated cultures compared to the bFGF/EGF pre-treatment (# in Fig. 3C). Nonetheless, FCS was the only differentiation treatment to significantly increase the cell morphology when analyzing the data with the Bonferroni post-test (* in Fig. 3C).


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)

Oligodendrocyte maturation in vitro using WT-derived NS.A) Immunocytochemistry of NS cultures pre-treated during proliferation with bFGF/EGF or bFGF/PDGF-BB, and then exposed to differentiating conditions; No growth factors (CTL), Tri-iodothyronine (T3), Insulin-like Growth Factor 1 (IGF-1) and Fetal Calf Serum (FCS). B) Comparison of the proportion of MBP+ cells detected after differentiation between bFGF/EGF and bFGF/PDGF-BB pre-treated NS. C) Quantitation of the MBP+ area/nuclei ratio after differentiation in two independent cultures. Data in B and C was analyzed with a Two-way ANOVA and Bonferroni post-test to compare the effect of NS pre-treatment during proliferation (*) or the differentiation treatment compared to its CTL (#). D) Fold change of the MBP+ area/nuclei in bFGF/PDGF-BB compared to bFGF/EGF cultures. The bars in B, C and D represent the mean + SD. The scale bar in A (i) represents 100 μm for (i) and (ii), while the scale bar in A (iii) represents 100 μm for (iii) to (x). * = p < 0.05. ** = p < 0.01 and # = p < 0.05. DIFF.; Differentiation.
© Copyright Policy
Related In: Results  -  Collection

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pone.0121774.g003: Oligodendrocyte maturation in vitro using WT-derived NS.A) Immunocytochemistry of NS cultures pre-treated during proliferation with bFGF/EGF or bFGF/PDGF-BB, and then exposed to differentiating conditions; No growth factors (CTL), Tri-iodothyronine (T3), Insulin-like Growth Factor 1 (IGF-1) and Fetal Calf Serum (FCS). B) Comparison of the proportion of MBP+ cells detected after differentiation between bFGF/EGF and bFGF/PDGF-BB pre-treated NS. C) Quantitation of the MBP+ area/nuclei ratio after differentiation in two independent cultures. Data in B and C was analyzed with a Two-way ANOVA and Bonferroni post-test to compare the effect of NS pre-treatment during proliferation (*) or the differentiation treatment compared to its CTL (#). D) Fold change of the MBP+ area/nuclei in bFGF/PDGF-BB compared to bFGF/EGF cultures. The bars in B, C and D represent the mean + SD. The scale bar in A (i) represents 100 μm for (i) and (ii), while the scale bar in A (iii) represents 100 μm for (iii) to (x). * = p < 0.05. ** = p < 0.01 and # = p < 0.05. DIFF.; Differentiation.
Mentions: To confirm that bFGF/PDGF-BB pre-treated cultures from Act::EGFP mice were able to generate mature OL, we exposed them to differentiating media lacking growth factors. We also induce cell differentiation by addition of T3, IGF-1 or FCS as described in the S4 Fig. The presence of mature MBP+ OL was analyzed by ICC. During proliferation, cultures grown in bFGF/EGF or bFGF/PDGF-BB contained few MBP+ OL that had a poorly developed morphology; diameter and ramifications (Fig. 3Ai, ii). After differentiating bFGF/EGF pre-treated cultures (Fig. 3Aiii, v, vii and ix), there were no evident changes in mature OL proportions between the various differentiating conditions (white bars in Fig. 3B). However, there was a significant increase in MBP+ cell proportions after differentiating bFGF/PDGF-BB pre-treated cultures in the presence of T3, IGF-1 (Fig. 3Aiv, vi, viii and x, and black bars in Fig. 3B). The OL morphology is compared in Fig. 3C and D after differentiation. Adding T3, IGF-1 or FCS during differentiation had a significant impact on MBP+ cell proportions in bFGF/PDGF-BB pre-treated cultures compared to bFGF/EGF treated cultures. As a whole, we found a significant increase in the complexity of MBP+ OL cell morphology in bFGF/PDGF-BB pre-treated cultures compared to the bFGF/EGF pre-treatment (# in Fig. 3C). Nonetheless, FCS was the only differentiation treatment to significantly increase the cell morphology when analyzing the data with the Bonferroni post-test (* in Fig. 3C).

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