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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

Neuronal subtype specification of sorted cells. (A) RT-qPCR showing the expression of markers for dopaminergic (TH and DAT), glutamatergic (VGlut2), GABAergic (GAD67), serotoninergic (Tph2), and motor (HB9) neuronal subtypes, expressed as ΔΔCt values, in GFP+ (black bars) from day 0 to day 13, and in unsorted cells (gray bar) at day 13. Error bars represent±SEM with n=3 independent experiments. d5p, day 5 pre-sorting. *p<0.05, ***p<0.001. (B) Western blot analysis showing the expression of the presynaptic marker Synaptophysin 1 (SYN1) and the post-synaptic marker Post Synaptic Density (PSD) 95. GAPDH was used to normalize signals. (C) Immunocytochemistry showing expression of MAP2 and VGlut2 (upper panel) and MAP2 and SYN1 (lower panel) in Sox1-GFP+ cells at day 20. Hoechst 33342 (Life Technologies) was used to counterstain nuclei. Scale bar, 50 μm.
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f4: Neuronal subtype specification of sorted cells. (A) RT-qPCR showing the expression of markers for dopaminergic (TH and DAT), glutamatergic (VGlut2), GABAergic (GAD67), serotoninergic (Tph2), and motor (HB9) neuronal subtypes, expressed as ΔΔCt values, in GFP+ (black bars) from day 0 to day 13, and in unsorted cells (gray bar) at day 13. Error bars represent±SEM with n=3 independent experiments. d5p, day 5 pre-sorting. *p<0.05, ***p<0.001. (B) Western blot analysis showing the expression of the presynaptic marker Synaptophysin 1 (SYN1) and the post-synaptic marker Post Synaptic Density (PSD) 95. GAPDH was used to normalize signals. (C) Immunocytochemistry showing expression of MAP2 and VGlut2 (upper panel) and MAP2 and SYN1 (lower panel) in Sox1-GFP+ cells at day 20. Hoechst 33342 (Life Technologies) was used to counterstain nuclei. Scale bar, 50 μm.

Mentions: We finally checked for the presence of specific neuronal populations among the differentiated cells before sorting and in the sorted Sox1-GFP+ population. The early tyrosine hydroxylase (TH) and later dopamine transporter (DAT) dopaminergic markers,29 the VGlut2),30 the GABAergic biosynthetic enzyme GAD67,31 the serotonergic tryptophan hydroxylase 2 (Tph2)32 and the motor neuron marker HB933 were analyzed by RT-qPCR (Fig. 4A). First of all we compared their expression between the Sox1-GFP+ (black bar) and unsorted (gray bar) populations at the end of the protocol. All markers were expressed and significantly upregulated in the purified cells with respect to the unsorted population. In particular, TH, VGlut2, and Tph2 showed the most robust up-regulation with respect to undifferentiated cells and after sorting. Specifically, most dopaminergic and serotonergic neurons are born in the midbrain and hindbrain respectively,34 so that these data could be consistent with the observed trend toward mid- and hindbrain (see Fig. 3). They also suggest that the corresponding neuronal subtypes could be enriched in our cultures; moreover, the absence of upregulation of DAT might signify that cells are still in an early phase of terminal differentiation, as DAT is considered to be a late marker of mature dopaminergic neurons.35 Finally, to analyze the extent of differentiation, we checked for the presence of the synaptic proteins (Syn136 and PSD,9537 which are a pre- and post-synaptic marker respectively. Western blot analyses confirmed their expression (Fig. 4B) at the latest time points of the differentiation protocol. However, it was difficult to observe their localization by ICC at day 13, suggesting that the formation of functional synapses is undergoing but not yet terminated. For this reason, we decided to perform ICC analysis at day 20. At this later time point, we observed the localization of Syn1 in a spotted fashion along neurites (Fig. 4C, upper panel). A similar pattern of expression was also observed for the vesicular protein VGlut2 (Fig. 4C, lower panel), confirming that glutamatergic neurons were indeed present. From these observations, we can thus conclude that our modifications to the differentiation protocol can potentially allow a more efficient differentiation toward specific neuronal subtypes whose markers were otherwise very poorly expressed in the unsorted cells.


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)

Neuronal subtype specification of sorted cells. (A) RT-qPCR showing the expression of markers for dopaminergic (TH and DAT), glutamatergic (VGlut2), GABAergic (GAD67), serotoninergic (Tph2), and motor (HB9) neuronal subtypes, expressed as ΔΔCt values, in GFP+ (black bars) from day 0 to day 13, and in unsorted cells (gray bar) at day 13. Error bars represent±SEM with n=3 independent experiments. d5p, day 5 pre-sorting. *p<0.05, ***p<0.001. (B) Western blot analysis showing the expression of the presynaptic marker Synaptophysin 1 (SYN1) and the post-synaptic marker Post Synaptic Density (PSD) 95. GAPDH was used to normalize signals. (C) Immunocytochemistry showing expression of MAP2 and VGlut2 (upper panel) and MAP2 and SYN1 (lower panel) in Sox1-GFP+ cells at day 20. Hoechst 33342 (Life Technologies) was used to counterstain nuclei. Scale bar, 50 μm.
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f4: Neuronal subtype specification of sorted cells. (A) RT-qPCR showing the expression of markers for dopaminergic (TH and DAT), glutamatergic (VGlut2), GABAergic (GAD67), serotoninergic (Tph2), and motor (HB9) neuronal subtypes, expressed as ΔΔCt values, in GFP+ (black bars) from day 0 to day 13, and in unsorted cells (gray bar) at day 13. Error bars represent±SEM with n=3 independent experiments. d5p, day 5 pre-sorting. *p<0.05, ***p<0.001. (B) Western blot analysis showing the expression of the presynaptic marker Synaptophysin 1 (SYN1) and the post-synaptic marker Post Synaptic Density (PSD) 95. GAPDH was used to normalize signals. (C) Immunocytochemistry showing expression of MAP2 and VGlut2 (upper panel) and MAP2 and SYN1 (lower panel) in Sox1-GFP+ cells at day 20. Hoechst 33342 (Life Technologies) was used to counterstain nuclei. Scale bar, 50 μm.
Mentions: We finally checked for the presence of specific neuronal populations among the differentiated cells before sorting and in the sorted Sox1-GFP+ population. The early tyrosine hydroxylase (TH) and later dopamine transporter (DAT) dopaminergic markers,29 the VGlut2),30 the GABAergic biosynthetic enzyme GAD67,31 the serotonergic tryptophan hydroxylase 2 (Tph2)32 and the motor neuron marker HB933 were analyzed by RT-qPCR (Fig. 4A). First of all we compared their expression between the Sox1-GFP+ (black bar) and unsorted (gray bar) populations at the end of the protocol. All markers were expressed and significantly upregulated in the purified cells with respect to the unsorted population. In particular, TH, VGlut2, and Tph2 showed the most robust up-regulation with respect to undifferentiated cells and after sorting. Specifically, most dopaminergic and serotonergic neurons are born in the midbrain and hindbrain respectively,34 so that these data could be consistent with the observed trend toward mid- and hindbrain (see Fig. 3). They also suggest that the corresponding neuronal subtypes could be enriched in our cultures; moreover, the absence of upregulation of DAT might signify that cells are still in an early phase of terminal differentiation, as DAT is considered to be a late marker of mature dopaminergic neurons.35 Finally, to analyze the extent of differentiation, we checked for the presence of the synaptic proteins (Syn136 and PSD,9537 which are a pre- and post-synaptic marker respectively. Western blot analyses confirmed their expression (Fig. 4B) at the latest time points of the differentiation protocol. However, it was difficult to observe their localization by ICC at day 13, suggesting that the formation of functional synapses is undergoing but not yet terminated. For this reason, we decided to perform ICC analysis at day 20. At this later time point, we observed the localization of Syn1 in a spotted fashion along neurites (Fig. 4C, upper panel). A similar pattern of expression was also observed for the vesicular protein VGlut2 (Fig. 4C, lower panel), confirming that glutamatergic neurons were indeed present. From these observations, we can thus conclude that our modifications to the differentiation protocol can potentially allow a more efficient differentiation toward specific neuronal subtypes whose markers were otherwise very poorly expressed in the unsorted cells.

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