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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

Neural induction of sorted Sox1-GFP cells. (A) RT-qPCR analyses showing the expression of Sox1, Nestin, βIII Tubulin, NCAM, O4, and GFAP 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. Error bars represent±SEM with n=3 independent experiments. d5p, day 5 pre-sorting; ns, not significant. *p<0.05. (B) Western blot analysis showing the expression of Nestin and βIII-Tubulin during differentiation. GAPDH was used for normalization. (C) Immunocytochemistry showing expression of Nestin, βIII Tubulin, MAP2, and GFAP in Sox1-GFP+ (left), Sox1-GFP− (middle), and unsorted (right) cells at day 13 of differentiation. Hoechst 33342 (Life Technologies) was used to counterstain nuclei. Scale bars, 100 μm.
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f2: Neural induction of sorted Sox1-GFP cells. (A) RT-qPCR analyses showing the expression of Sox1, Nestin, βIII Tubulin, NCAM, O4, and GFAP 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. Error bars represent±SEM with n=3 independent experiments. d5p, day 5 pre-sorting; ns, not significant. *p<0.05. (B) Western blot analysis showing the expression of Nestin and βIII-Tubulin during differentiation. GAPDH was used for normalization. (C) Immunocytochemistry showing expression of Nestin, βIII Tubulin, MAP2, and GFAP in Sox1-GFP+ (left), Sox1-GFP− (middle), and unsorted (right) cells at day 13 of differentiation. Hoechst 33342 (Life Technologies) was used to counterstain nuclei. Scale bars, 100 μm.

Mentions: We subsequently assessed the expression of neuroectodermal precursors' markers. Figure 2A shows a RT-qPCR panel in which the extent of neural differentiation was investigated: we analyzed the neural precursors marker Sox1 and Nestin, the neuronal markers βIII Tubulin and neural cell adhesion molecule (NCAM), the glial marker GFAP and the oligodendrocyte marker O4.23 Sox1 expression perfectly paralleled GFP behavior, with a robust increase between days 3 and 4 and a peak at day 5 (d5p); sorting consistently separated Sox1-expressing cells from the Sox1-GFP negative subpopulation (d5, black line and dark gray line respectively). Despite the GFP appearance observed in culture (Fig. 1B), GFP− cells (dark gray line) only showed a very slight increase in Sox1 expression around day 9–10, suggesting that the total amount of Sox1 transcript in the GFP− population is too low with respect to the day 5 sorted GFP+ cells and cannot be properly appreciated on the histograms. However, Sox1 expression completely turned off at the end of the protocol in all three of the populations analyzed (GFP+, GFP−, and unsorted), as expected from the acquisition of a more mature neuronal fate. Nestin expression rapidly increased during the first 5 days of differentiation and was highly upregulated in GFP+ sorted cells (Fig. 2A d5, black line; Supplementary Fig. S2A), in a way comparable to Sox1 behavior. This finding is in line with what previously known about temporal expression of early neural markers24,26 and was also confirmed by Western blot and ICC analyses (Fig. 2B, C and Supplementary Fig. S2A respectively). Interestingly, Nestin expression also slightly increased in the GFP− samples (dark gray lines), further suggesting that GFP− cells are acquiring a neural fate in a delayed fashion (Supplementary Fig. S2A, d7 GFP−). At the same time, the expression of the later marker βIII Tubulin23 and NCAM increased at the end of the protocol in GFP+ cells, and their levels were significantly higher than those of the unsorted cells. ICC analyses performed at day 13 (Fig. 2C) confirmed the expression and localization of βIII Tubulin in all three populations (Sox1-GFP+, Sox1-GFP−, and unsorted). Neuronal terminal differentiation was assessed by MAP2 staining,27 confirming that mature postmitotic neurons were present in Sox1-GFP+ as well as in unsorted cells, to a lower extent. In contrast, GFP− cells did not show a robust expression of these later markers at day 13, as was also confirmed by ICC analysis (Fig. 2C). However, at day 15, only two days later, the GFP− population showed a widespread localization of Nestin and βIII Tubulin proteins, as well as MAP2 expression, thus confirming the delayed acquisition of a neural fate (Supplementary Fig. S2B). Interestingly, βIII Tubulin positive neurites in GFP negative cells show a less regular localization with respect to the GFP+ and unsorted cells (Fig. 2C), where neurites usually appear as radial outgrowths from a group of cell bodies. Glial differentiation was instead verified by GFAP expression. Even though expressed at the end of the protocol only in GFP+ cells (Fig. 2A), which show a significantly higher amount of GFAP transcript with respect to the unsorted population, this glia-specific protein was poorly expressed in culture, as assessed by ICC (Fig. 2C). This observation indicates that day 13 might be too early to assess for glial fate, in line with what is known from literature.23 Moreover, O4 expression also did not show any difference among undifferentiated and differentiating cells, or among GFP+, GFP−, and unsorted populations, suggesting that the oligodendrocyte lineage is not arising at the time points within this protocol. These data demonstrate that sorting followed by an optimized cell density for replating and a simple culture medium modification was able not only to improve the acquisition of neuroectodermal fate with respect to the unsorted population, but also to provide a more robust neuronal differentiation.


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)

Neural induction of sorted Sox1-GFP cells. (A) RT-qPCR analyses showing the expression of Sox1, Nestin, βIII Tubulin, NCAM, O4, and GFAP 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. Error bars represent±SEM with n=3 independent experiments. d5p, day 5 pre-sorting; ns, not significant. *p<0.05. (B) Western blot analysis showing the expression of Nestin and βIII-Tubulin during differentiation. GAPDH was used for normalization. (C) Immunocytochemistry showing expression of Nestin, βIII Tubulin, MAP2, and GFAP in Sox1-GFP+ (left), Sox1-GFP− (middle), and unsorted (right) cells at day 13 of differentiation. Hoechst 33342 (Life Technologies) was used to counterstain nuclei. Scale bars, 100 μm.
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Related In: Results  -  Collection

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f2: Neural induction of sorted Sox1-GFP cells. (A) RT-qPCR analyses showing the expression of Sox1, Nestin, βIII Tubulin, NCAM, O4, and GFAP 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. Error bars represent±SEM with n=3 independent experiments. d5p, day 5 pre-sorting; ns, not significant. *p<0.05. (B) Western blot analysis showing the expression of Nestin and βIII-Tubulin during differentiation. GAPDH was used for normalization. (C) Immunocytochemistry showing expression of Nestin, βIII Tubulin, MAP2, and GFAP in Sox1-GFP+ (left), Sox1-GFP− (middle), and unsorted (right) cells at day 13 of differentiation. Hoechst 33342 (Life Technologies) was used to counterstain nuclei. Scale bars, 100 μm.
Mentions: We subsequently assessed the expression of neuroectodermal precursors' markers. Figure 2A shows a RT-qPCR panel in which the extent of neural differentiation was investigated: we analyzed the neural precursors marker Sox1 and Nestin, the neuronal markers βIII Tubulin and neural cell adhesion molecule (NCAM), the glial marker GFAP and the oligodendrocyte marker O4.23 Sox1 expression perfectly paralleled GFP behavior, with a robust increase between days 3 and 4 and a peak at day 5 (d5p); sorting consistently separated Sox1-expressing cells from the Sox1-GFP negative subpopulation (d5, black line and dark gray line respectively). Despite the GFP appearance observed in culture (Fig. 1B), GFP− cells (dark gray line) only showed a very slight increase in Sox1 expression around day 9–10, suggesting that the total amount of Sox1 transcript in the GFP− population is too low with respect to the day 5 sorted GFP+ cells and cannot be properly appreciated on the histograms. However, Sox1 expression completely turned off at the end of the protocol in all three of the populations analyzed (GFP+, GFP−, and unsorted), as expected from the acquisition of a more mature neuronal fate. Nestin expression rapidly increased during the first 5 days of differentiation and was highly upregulated in GFP+ sorted cells (Fig. 2A d5, black line; Supplementary Fig. S2A), in a way comparable to Sox1 behavior. This finding is in line with what previously known about temporal expression of early neural markers24,26 and was also confirmed by Western blot and ICC analyses (Fig. 2B, C and Supplementary Fig. S2A respectively). Interestingly, Nestin expression also slightly increased in the GFP− samples (dark gray lines), further suggesting that GFP− cells are acquiring a neural fate in a delayed fashion (Supplementary Fig. S2A, d7 GFP−). At the same time, the expression of the later marker βIII Tubulin23 and NCAM increased at the end of the protocol in GFP+ cells, and their levels were significantly higher than those of the unsorted cells. ICC analyses performed at day 13 (Fig. 2C) confirmed the expression and localization of βIII Tubulin in all three populations (Sox1-GFP+, Sox1-GFP−, and unsorted). Neuronal terminal differentiation was assessed by MAP2 staining,27 confirming that mature postmitotic neurons were present in Sox1-GFP+ as well as in unsorted cells, to a lower extent. In contrast, GFP− cells did not show a robust expression of these later markers at day 13, as was also confirmed by ICC analysis (Fig. 2C). However, at day 15, only two days later, the GFP− population showed a widespread localization of Nestin and βIII Tubulin proteins, as well as MAP2 expression, thus confirming the delayed acquisition of a neural fate (Supplementary Fig. S2B). Interestingly, βIII Tubulin positive neurites in GFP negative cells show a less regular localization with respect to the GFP+ and unsorted cells (Fig. 2C), where neurites usually appear as radial outgrowths from a group of cell bodies. Glial differentiation was instead verified by GFAP expression. Even though expressed at the end of the protocol only in GFP+ cells (Fig. 2A), which show a significantly higher amount of GFAP transcript with respect to the unsorted population, this glia-specific protein was poorly expressed in culture, as assessed by ICC (Fig. 2C). This observation indicates that day 13 might be too early to assess for glial fate, in line with what is known from literature.23 Moreover, O4 expression also did not show any difference among undifferentiated and differentiating cells, or among GFP+, GFP−, and unsorted populations, suggesting that the oligodendrocyte lineage is not arising at the time points within this protocol. These data demonstrate that sorting followed by an optimized cell density for replating and a simple culture medium modification was able not only to improve the acquisition of neuroectodermal fate with respect to the unsorted population, but also to provide a more robust neuronal differentiation.

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