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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

Differentiation potential of sorted Sox1-green fluorescent protein (Sox1-GFP) mouse embryonic stem cells (mESCs). (A) Time course analysis showing the percentage of Sox1-GFP positive cells during neural differentiation from day 0 (d0) to day 7 (d7). Error bars represent±SEM with n=3 independent experiments. (B) Brightfield pictures of sorted Sox1-GFP+ (left panels), Sox1-GFP− cells (middle panels) and unsorted cells (right panels) at day 7 (top) and at day 13 (bottom). Scale bars, 100 μm. (C) Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) showing the expression of the pluripotency marker Oct3/4, the epithelial marker Keratin (K)18 and the mesodermal marker Brachyury, 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, ***p<0.001. Ct, cycle threshold.
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f1: Differentiation potential of sorted Sox1-green fluorescent protein (Sox1-GFP) mouse embryonic stem cells (mESCs). (A) Time course analysis showing the percentage of Sox1-GFP positive cells during neural differentiation from day 0 (d0) to day 7 (d7). Error bars represent±SEM with n=3 independent experiments. (B) Brightfield pictures of sorted Sox1-GFP+ (left panels), Sox1-GFP− cells (middle panels) and unsorted cells (right panels) at day 7 (top) and at day 13 (bottom). Scale bars, 100 μm. (C) Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) showing the expression of the pluripotency marker Oct3/4, the epithelial marker Keratin (K)18 and the mesodermal marker Brachyury, 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, ***p<0.001. Ct, cycle threshold.

Mentions: The mouse knock-in embryonic stem cell line 46C Sox1-GFP8 was cultured in monolayer on gelatin-coated dishes with minimal medium containing KSR. As previously shown,7 this medium is able to support neural differentiation of mouse ESCs (mESCs) without additional growth factors or embryoid bodies formation. Transferrin, insulin, and albumin are the only proteins contained in KSR16 and are sufficient to sustain neural survival and proliferation while giving little or no bias toward specific cell identities.17,18 In fact, the absence of other specific growth factors leads to the differentiation of a very heterogeneous population, which expresses mesodermal markers along with neuroectodermal genes.7 In this study, we improved this very simple protocol in order to select neuroectodermal precursors and obtain a purer neural population. We cultured both the Sox1-GFP cells and the E14Tg2a.4 parental ESC line, and we assessed the reproducibility of the protocol between the two cell lines (data not shown). We first performed a cytofluorimetric analysis in order to quantify the number of GFP-expressing cells at different time points (Fig. 1A). In 46C cells cultured with this protocol, the GFP, corresponding to Sox1 expression, started to be expressed around day 3. The analyses showed a highly reproducible increase in GFP positive cells from day 3, reaching a peak at day 5 and then slightly decreasing. We thus decided to perform FACS analysis at day 5. E14 cells at day 5 of differentiation were used as GFP negative control to set the proper FITC baseline (Supplementary Fig. S1A, left panel). The Sox1–GFP positive (GFP+) population was easily recognized and separated: we decided to choose the brighter subpopulation for further analysis, as well as the GFP negative (GFP−) population. Cells with an intermediate amount of GFP expression were discarded in order to avoid the presence of the earliest precursors19 and also to avoid cross-contamination between the two groups (Supplementary Fig. S1A, left panel and black box). GFP+ cells represented about 65% of the total population (Fig. 1A), a slightly lower percentage with respect to other published results, in which the differentiation media were supplemented with signaling pathway antagonists like Dkk1 and Lefty A11 or with neural-specific supplements like N2 and B278; this evidence clearly indicates that the absence of specific growth factors and supplements can impair the more efficient acquisition of a neuroectodermal fate. Finally, we checked for the purity of the sorting procedures by performing a further FACS analysis on the two sorted populations (GFP− and GFP+; Supplementary Fig. S1A, right panel). Different cell densities were tested for replating after sorting, ranging from 2×104 to 25×104 cells/cm2: due to the best output in terms of viability and/or neurogenic potential (Supplementary Fig. S1B), the 5×104 cells/cm2 density was chosen. Along with replating, we decided to make a further improvement to the original protocol by lowering KSR percentage from 15% to 5% in order to increase differentiation.20 GFP was slowly turned off starting from day 7 in both GFP+ and unsorted cells (Fig. 1B, top left and top right respectively). Cells were collected for molecular analyses at day 7, day 9, and then every other day starting from day 10. At day 13, cells showed good viability and a neuronal-like morphology (Fig. 1B, bottom left), with dense neurite outgrowth comparable with that observed in the unsorted population (Fig. 1B, bottom right). For this reason, the protocol was stopped and cells collected or fixed for molecular and immunocytochemical analyses. Interestingly, GFP− cells, which were completely GFP negative at day 7 (Fig. 1B, top middle), started expressing GFP around day 10 and fluorescence peaked at day 13 (Fig. 1B, bottom middle). Total RNA was extracted from all the samples and RT-qPCR analyses were performed in order to assess the differentiation potential of the cells. Unsorted cells collected at day 13 were analyzed in order to provide a direct comparison with the published differentiation protocol. First of all, we assessed whether pluripotent cells and nonneural ectodermal precursors such as epithelial cells were present in our cultures. As shown in Fig. 1C, the expression of Oct3/4 decreases from the third day of the culture; it is maintained at levels lower than undifferentiated cells (day 0) until the end, in both GFP+ (black line) and GFP− (dark gray line) samples, as well as in the unsorted population (light gray indicator), indicating that most of the cells were already committed to differentiate at the time of sorting, regardless of the purification procedure. GFP− cells only showed an increase of Oct3/4 expression at day 10 (d10): this could suggest the presence of undifferentiated ESCs in the GFP− population that, after recovering from the stress of the sorting procedure, are able to undergo intense proliferation, as observed between day 7 and 13 (see Fig.1B, middle panels). Early keratinocyte marker keratin (K)1821 also decreased at day 5 (d5) immediately after sorting. Interestingly, the unsorted population showed a significantly higher expression level of K18, suggesting that the purification step was able to discard nonneural ectoderm. The expression of the mesodermal marker Brachyury,22 which increased at day 5 of differentiation after a significant down-regulation at day 4, was again down-regulated after sorting in both GFP+ and GFP− populations; once again, the unsorted population showed a significant higher expression of this mesodermal marker. This finding indicates that FACS analysis efficiently discarded nonneuroectodermal precursors and also suggests that the mesodermal progenitors could have been contained in the middle population that was excluded by the sorting, since in unsorted cells we also find cells with nonneural morphology (data not shown). We also checked for endodermal markers such as Sox17, but no expression was observed throughout the protocol in both GFP+ and GFP− cells, as well as in the unsorted population, in line with what has been previously described.7 Altogether, this data indicates that sorting was able to discard the non neuroectodermal lineages from both GFP+ and GFP− subpopulations.


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)

Differentiation potential of sorted Sox1-green fluorescent protein (Sox1-GFP) mouse embryonic stem cells (mESCs). (A) Time course analysis showing the percentage of Sox1-GFP positive cells during neural differentiation from day 0 (d0) to day 7 (d7). Error bars represent±SEM with n=3 independent experiments. (B) Brightfield pictures of sorted Sox1-GFP+ (left panels), Sox1-GFP− cells (middle panels) and unsorted cells (right panels) at day 7 (top) and at day 13 (bottom). Scale bars, 100 μm. (C) Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) showing the expression of the pluripotency marker Oct3/4, the epithelial marker Keratin (K)18 and the mesodermal marker Brachyury, 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, ***p<0.001. Ct, cycle threshold.
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f1: Differentiation potential of sorted Sox1-green fluorescent protein (Sox1-GFP) mouse embryonic stem cells (mESCs). (A) Time course analysis showing the percentage of Sox1-GFP positive cells during neural differentiation from day 0 (d0) to day 7 (d7). Error bars represent±SEM with n=3 independent experiments. (B) Brightfield pictures of sorted Sox1-GFP+ (left panels), Sox1-GFP− cells (middle panels) and unsorted cells (right panels) at day 7 (top) and at day 13 (bottom). Scale bars, 100 μm. (C) Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) showing the expression of the pluripotency marker Oct3/4, the epithelial marker Keratin (K)18 and the mesodermal marker Brachyury, 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, ***p<0.001. Ct, cycle threshold.
Mentions: The mouse knock-in embryonic stem cell line 46C Sox1-GFP8 was cultured in monolayer on gelatin-coated dishes with minimal medium containing KSR. As previously shown,7 this medium is able to support neural differentiation of mouse ESCs (mESCs) without additional growth factors or embryoid bodies formation. Transferrin, insulin, and albumin are the only proteins contained in KSR16 and are sufficient to sustain neural survival and proliferation while giving little or no bias toward specific cell identities.17,18 In fact, the absence of other specific growth factors leads to the differentiation of a very heterogeneous population, which expresses mesodermal markers along with neuroectodermal genes.7 In this study, we improved this very simple protocol in order to select neuroectodermal precursors and obtain a purer neural population. We cultured both the Sox1-GFP cells and the E14Tg2a.4 parental ESC line, and we assessed the reproducibility of the protocol between the two cell lines (data not shown). We first performed a cytofluorimetric analysis in order to quantify the number of GFP-expressing cells at different time points (Fig. 1A). In 46C cells cultured with this protocol, the GFP, corresponding to Sox1 expression, started to be expressed around day 3. The analyses showed a highly reproducible increase in GFP positive cells from day 3, reaching a peak at day 5 and then slightly decreasing. We thus decided to perform FACS analysis at day 5. E14 cells at day 5 of differentiation were used as GFP negative control to set the proper FITC baseline (Supplementary Fig. S1A, left panel). The Sox1–GFP positive (GFP+) population was easily recognized and separated: we decided to choose the brighter subpopulation for further analysis, as well as the GFP negative (GFP−) population. Cells with an intermediate amount of GFP expression were discarded in order to avoid the presence of the earliest precursors19 and also to avoid cross-contamination between the two groups (Supplementary Fig. S1A, left panel and black box). GFP+ cells represented about 65% of the total population (Fig. 1A), a slightly lower percentage with respect to other published results, in which the differentiation media were supplemented with signaling pathway antagonists like Dkk1 and Lefty A11 or with neural-specific supplements like N2 and B278; this evidence clearly indicates that the absence of specific growth factors and supplements can impair the more efficient acquisition of a neuroectodermal fate. Finally, we checked for the purity of the sorting procedures by performing a further FACS analysis on the two sorted populations (GFP− and GFP+; Supplementary Fig. S1A, right panel). Different cell densities were tested for replating after sorting, ranging from 2×104 to 25×104 cells/cm2: due to the best output in terms of viability and/or neurogenic potential (Supplementary Fig. S1B), the 5×104 cells/cm2 density was chosen. Along with replating, we decided to make a further improvement to the original protocol by lowering KSR percentage from 15% to 5% in order to increase differentiation.20 GFP was slowly turned off starting from day 7 in both GFP+ and unsorted cells (Fig. 1B, top left and top right respectively). Cells were collected for molecular analyses at day 7, day 9, and then every other day starting from day 10. At day 13, cells showed good viability and a neuronal-like morphology (Fig. 1B, bottom left), with dense neurite outgrowth comparable with that observed in the unsorted population (Fig. 1B, bottom right). For this reason, the protocol was stopped and cells collected or fixed for molecular and immunocytochemical analyses. Interestingly, GFP− cells, which were completely GFP negative at day 7 (Fig. 1B, top middle), started expressing GFP around day 10 and fluorescence peaked at day 13 (Fig. 1B, bottom middle). Total RNA was extracted from all the samples and RT-qPCR analyses were performed in order to assess the differentiation potential of the cells. Unsorted cells collected at day 13 were analyzed in order to provide a direct comparison with the published differentiation protocol. First of all, we assessed whether pluripotent cells and nonneural ectodermal precursors such as epithelial cells were present in our cultures. As shown in Fig. 1C, the expression of Oct3/4 decreases from the third day of the culture; it is maintained at levels lower than undifferentiated cells (day 0) until the end, in both GFP+ (black line) and GFP− (dark gray line) samples, as well as in the unsorted population (light gray indicator), indicating that most of the cells were already committed to differentiate at the time of sorting, regardless of the purification procedure. GFP− cells only showed an increase of Oct3/4 expression at day 10 (d10): this could suggest the presence of undifferentiated ESCs in the GFP− population that, after recovering from the stress of the sorting procedure, are able to undergo intense proliferation, as observed between day 7 and 13 (see Fig.1B, middle panels). Early keratinocyte marker keratin (K)1821 also decreased at day 5 (d5) immediately after sorting. Interestingly, the unsorted population showed a significantly higher expression level of K18, suggesting that the purification step was able to discard nonneural ectoderm. The expression of the mesodermal marker Brachyury,22 which increased at day 5 of differentiation after a significant down-regulation at day 4, was again down-regulated after sorting in both GFP+ and GFP− populations; once again, the unsorted population showed a significant higher expression of this mesodermal marker. This finding indicates that FACS analysis efficiently discarded nonneuroectodermal precursors and also suggests that the mesodermal progenitors could have been contained in the middle population that was excluded by the sorting, since in unsorted cells we also find cells with nonneural morphology (data not shown). We also checked for endodermal markers such as Sox17, but no expression was observed throughout the protocol in both GFP+ and GFP− cells, as well as in the unsorted population, in line with what has been previously described.7 Altogether, this data indicates that sorting was able to discard the non neuroectodermal lineages from both GFP+ and GFP− subpopulations.

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