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Sorting of Sox1-GFP Mouse Embryonic Stem Cells Enhances Neuronal Identity Acquisition upon Factor-Free Monolayer Differentiation.

Incitti T, Messina A, Bozzi Y, Casarosa S - Biores Open Access (2014)

Bottom Line: In this study, we modified a monolayer differentiation protocol by selecting green fluorescent protein (GFP) positive neural precursors with fluorescence-activated cell sorting (FACS).The enhancement of neural differentiation was obtained by positively selecting for neural precursors, while specific neuronal subtypes spontaneously differentiated without additional cues; a comparable but delayed behavior was also observed in the GFP negative population, indicating that sorting settings per se eliminated nonneural and undifferentiated ESCs.This highly reproducible approach could be applied as a strategy to enhance neuronal differentiation and could be the first step toward the selection of pure populations of neurons, to be generated by the administration of specific factors in high throughput screening assays.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Biology, University of Trento , Trento, Italy .

ABSTRACT
Embryonic stem cells (ESCs) can give rise to all the differentiated cell types of the organism, including neurons. However, the efficiency and specificity of neural differentiation protocols still needs to be improved in order to plan their use in cell replacement therapies. In this study, we modified a monolayer differentiation protocol by selecting green fluorescent protein (GFP) positive neural precursors with fluorescence-activated cell sorting (FACS). The enhancement of neural differentiation was obtained by positively selecting for neural precursors, while specific neuronal subtypes spontaneously differentiated without additional cues; a comparable but delayed behavior was also observed in the GFP negative population, indicating that sorting settings per se eliminated nonneural and undifferentiated ESCs. This highly reproducible approach could be applied as a strategy to enhance neuronal differentiation and could be the first step toward the selection of pure populations of neurons, to be generated by the administration of specific factors in high throughput screening assays.

No MeSH data available.


Related in: MedlinePlus

Positional identity of sorted Sox1-GFP cells. RT-qPCR showing the expression of anteroposterior (A-P) and dorsoventral (D-V) markers during differentiation, expressed as ΔΔCt values, in GFP+ (black lines) and GFP− (dark gray lines) from day 0 (d0) to day 13 (d13), and in unsorted cells (light gray indicator) at day 13. d5p, day 5 pre-sorting. Error bars represent±SEM with n=3 independent experiments. *p<0.05, **p value<0.01.
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f3: Positional identity of sorted Sox1-GFP cells. RT-qPCR showing the expression of anteroposterior (A-P) and dorsoventral (D-V) markers during differentiation, expressed as ΔΔCt values, in GFP+ (black lines) and GFP− (dark gray lines) from day 0 (d0) to day 13 (d13), and in unsorted cells (light gray indicator) at day 13. d5p, day 5 pre-sorting. Error bars represent±SEM with n=3 independent experiments. *p<0.05, **p value<0.01.

Mentions: We further investigated the positional identity of the terminally differentiated neurons. Due to the delayed behavior of GFP− cells, we focused our attention only on GFP+ cells. Figure 3 shows the results of RT-qPCR analyses before sorting and on sorted Sox1-GFP+ cells. The expression of forebrain (FoxG1, Emx2), fore-midbrain (Otx2), midbrain (En1), hindbrain (HoxB4, Krox20), and spinal cord (HoxB9) markers along the anteroposterior (A-P) axis of the developing neural tube was assessed. All of the analyzed markers appeared to be expressed with no significant differences with respect to the unsorted population at day 13 (d13, gray bar); the only exceptions were midbrain marker En1 and hindbrain marker Krox20, which were significantly more expressed at the end of the protocol in Sox1-GFP+ cells (d13, black bars). This evidence suggests that despite the positional heterogeneity, a trend toward mid- and hindbrain can be recognized in the sorted population, consistent with recent findings showing that the absence of exogenous signals allow the generation of midbrain neurons.28 Analyzing dorsoventral (D-V) patterning, we observed that sorted cells, upon differentiation, express Math1, Pax6, Nkx2.2, and Shh transcripts.6,7 Interestingly, at the end of the differentiation protocol the expression of dorsal markers Math1 and Pax6 was comparable between the Sox1-GFP+ (black bar) and unsorted (gray bar) populations, while ventral markers Nkx2.2 and Shh were significantly upregulated in sorted cells. These data suggest that the purified population could be committed toward a more ventral fate differentiation, but this hypothesis needs to be further validated. In conclusion, these results confirmed that sorted cells can give rise to a wide range of neuronal types found in vivo along the A-P and D-V axes, including the most anterior part of the developing neural tube. These data also suggest that sorted cells are not biased toward a specific region; interestingly, they can potentially give rise also to the more rostral neurons along the rostrocaudal axis, thus making this improved simple protocol suitable for the differentiation of telencephalic neurons. In summary, sorting purification and medium modifications added to this differentiation protocol did not impair the possibility of obtaining a variety of neuronal cells widely distributed along the neural tube.


Sorting of Sox1-GFP Mouse Embryonic Stem Cells Enhances Neuronal Identity Acquisition upon Factor-Free Monolayer Differentiation.

Incitti T, Messina A, Bozzi Y, Casarosa S - Biores Open Access (2014)

Positional identity of sorted Sox1-GFP cells. RT-qPCR showing the expression of anteroposterior (A-P) and dorsoventral (D-V) markers during differentiation, expressed as ΔΔCt values, in GFP+ (black lines) and GFP− (dark gray lines) from day 0 (d0) to day 13 (d13), and in unsorted cells (light gray indicator) at day 13. d5p, day 5 pre-sorting. Error bars represent±SEM with n=3 independent experiments. *p<0.05, **p value<0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048977&req=5

f3: Positional identity of sorted Sox1-GFP cells. RT-qPCR showing the expression of anteroposterior (A-P) and dorsoventral (D-V) markers during differentiation, expressed as ΔΔCt values, in GFP+ (black lines) and GFP− (dark gray lines) from day 0 (d0) to day 13 (d13), and in unsorted cells (light gray indicator) at day 13. d5p, day 5 pre-sorting. Error bars represent±SEM with n=3 independent experiments. *p<0.05, **p value<0.01.
Mentions: We further investigated the positional identity of the terminally differentiated neurons. Due to the delayed behavior of GFP− cells, we focused our attention only on GFP+ cells. Figure 3 shows the results of RT-qPCR analyses before sorting and on sorted Sox1-GFP+ cells. The expression of forebrain (FoxG1, Emx2), fore-midbrain (Otx2), midbrain (En1), hindbrain (HoxB4, Krox20), and spinal cord (HoxB9) markers along the anteroposterior (A-P) axis of the developing neural tube was assessed. All of the analyzed markers appeared to be expressed with no significant differences with respect to the unsorted population at day 13 (d13, gray bar); the only exceptions were midbrain marker En1 and hindbrain marker Krox20, which were significantly more expressed at the end of the protocol in Sox1-GFP+ cells (d13, black bars). This evidence suggests that despite the positional heterogeneity, a trend toward mid- and hindbrain can be recognized in the sorted population, consistent with recent findings showing that the absence of exogenous signals allow the generation of midbrain neurons.28 Analyzing dorsoventral (D-V) patterning, we observed that sorted cells, upon differentiation, express Math1, Pax6, Nkx2.2, and Shh transcripts.6,7 Interestingly, at the end of the differentiation protocol the expression of dorsal markers Math1 and Pax6 was comparable between the Sox1-GFP+ (black bar) and unsorted (gray bar) populations, while ventral markers Nkx2.2 and Shh were significantly upregulated in sorted cells. These data suggest that the purified population could be committed toward a more ventral fate differentiation, but this hypothesis needs to be further validated. In conclusion, these results confirmed that sorted cells can give rise to a wide range of neuronal types found in vivo along the A-P and D-V axes, including the most anterior part of the developing neural tube. These data also suggest that sorted cells are not biased toward a specific region; interestingly, they can potentially give rise also to the more rostral neurons along the rostrocaudal axis, thus making this improved simple protocol suitable for the differentiation of telencephalic neurons. In summary, sorting purification and medium modifications added to this differentiation protocol did not impair the possibility of obtaining a variety of neuronal cells widely distributed along the neural tube.

Bottom Line: In this study, we modified a monolayer differentiation protocol by selecting green fluorescent protein (GFP) positive neural precursors with fluorescence-activated cell sorting (FACS).The enhancement of neural differentiation was obtained by positively selecting for neural precursors, while specific neuronal subtypes spontaneously differentiated without additional cues; a comparable but delayed behavior was also observed in the GFP negative population, indicating that sorting settings per se eliminated nonneural and undifferentiated ESCs.This highly reproducible approach could be applied as a strategy to enhance neuronal differentiation and could be the first step toward the selection of pure populations of neurons, to be generated by the administration of specific factors in high throughput screening assays.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Biology, University of Trento , Trento, Italy .

ABSTRACT
Embryonic stem cells (ESCs) can give rise to all the differentiated cell types of the organism, including neurons. However, the efficiency and specificity of neural differentiation protocols still needs to be improved in order to plan their use in cell replacement therapies. In this study, we modified a monolayer differentiation protocol by selecting green fluorescent protein (GFP) positive neural precursors with fluorescence-activated cell sorting (FACS). The enhancement of neural differentiation was obtained by positively selecting for neural precursors, while specific neuronal subtypes spontaneously differentiated without additional cues; a comparable but delayed behavior was also observed in the GFP negative population, indicating that sorting settings per se eliminated nonneural and undifferentiated ESCs. This highly reproducible approach could be applied as a strategy to enhance neuronal differentiation and could be the first step toward the selection of pure populations of neurons, to be generated by the administration of specific factors in high throughput screening assays.

No MeSH data available.


Related in: MedlinePlus