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Manipulation of signaling thresholds in "engineered stem cell niches" identifies design criteria for pluripotent stem cell screens.

Peerani R, Onishi K, Mahdavi A, Kumacheva E, Zandstra PW - PLoS ONE (2009)

Bottom Line: The functional consequences of this niche-size-dependent signaling control are confirmed by demonstrating that direct and indirect transcriptional targets of Stat3, including members of the Jak-Stat pathway and pluripotency-associated genes, are regulated by colony size.Modeling results and empirical observations demonstrate that colonies less than 100 microm in diameter are too small to maximize endogenous Stat3 activation and that colonies separated by more than 400 microm can be considered independent from each other.These results define parameter boundaries for the use of ESCs in screening studies, demonstrate the importance of context in stem cell responsiveness to exogenous cues, and suggest that niche size is an important parameter in stem cell fate control.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
In vivo, stem cell fate is regulated by local microenvironmental parameters. Governing parameters in this stem cell niche include soluble factors, extra-cellular matrix, and cell-cell interactions. The complexity of this in vivo niche limits analyses into how individual niche parameters regulate stem cell fate. Herein we use mouse embryonic stem cells (mESC) and micro-contact printing (microCP) to investigate how niche size controls endogenous signaling thresholds. microCP is used to restrict colony diameter, separation, and degree of clustering. We show, for the first time, spatial control over the activation of the Janus kinase/signal transducer and activator of transcription pathway (Jak-Stat). The functional consequences of this niche-size-dependent signaling control are confirmed by demonstrating that direct and indirect transcriptional targets of Stat3, including members of the Jak-Stat pathway and pluripotency-associated genes, are regulated by colony size. Modeling results and empirical observations demonstrate that colonies less than 100 microm in diameter are too small to maximize endogenous Stat3 activation and that colonies separated by more than 400 microm can be considered independent from each other. These results define parameter boundaries for the use of ESCs in screening studies, demonstrate the importance of context in stem cell responsiveness to exogenous cues, and suggest that niche size is an important parameter in stem cell fate control.

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Model validation using the activation of the Jak-Stat signaling pathway in non-patterned mouse ESCs cultures.A) Single-field Hoechst 33342 micrographs of mESCs seeded at various densities in 96-well plates after being cultured for 24 hours in serum-free media without LIF. Scale-bar is 200 µm. B) Quantification of the average single-cell nuclear pStat3 accumulation after 24 hours of culture. While differences between exogenous supplementations were statistically significant at a given seeding density (p<0.05), no significance was present between cell seeding densities (p>0.40) for a given treatment. C) Heat maps indicating the increase and distribution in local cell density which is defined as the number of neighbouring cells within a 400 µm radius. The local cell density was calculated using an already published algorithm (See Materials and Methods)[46]. D) Histogram for the localized cell density as a function of initial seeding density showing that as initial cell density increases, the mean localized cell density increases and the distribution of cell densities broaden. E) Quantification of pStat3 signaling gradients present within single wells using the Neighbours Analysis algorithm. At low seeding densities, 10,000 cell/well (E–i), pStat3 can be seen to be increasing with cell density only in the presence of LIF whereas at a higher cell density, 40,000 cells/well (E–ii), signaling gradients can be found under all three treatments. Error bars represent the SEM of 9 wells cultures in three separate trials. F) Predicted and actual measurements of single-cell pStat3 levels as function of localized cell density demonstrating that the mathematical model developed in this study can be used to predict spatial fluctuations in signal activation in the Jak-Stat in mESCs. Data for this plot is taken for a single 96-well plate seeded at 40,000/well in the no LIF condition. While the percent increase in pStat3 in this figure is approximately 40%, the range in percent change is 10–40% for n = 9 wells. Error bars represent the SEM of at least 500 cells within each bin along the x-axis.
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pone-0006438-g002: Model validation using the activation of the Jak-Stat signaling pathway in non-patterned mouse ESCs cultures.A) Single-field Hoechst 33342 micrographs of mESCs seeded at various densities in 96-well plates after being cultured for 24 hours in serum-free media without LIF. Scale-bar is 200 µm. B) Quantification of the average single-cell nuclear pStat3 accumulation after 24 hours of culture. While differences between exogenous supplementations were statistically significant at a given seeding density (p<0.05), no significance was present between cell seeding densities (p>0.40) for a given treatment. C) Heat maps indicating the increase and distribution in local cell density which is defined as the number of neighbouring cells within a 400 µm radius. The local cell density was calculated using an already published algorithm (See Materials and Methods)[46]. D) Histogram for the localized cell density as a function of initial seeding density showing that as initial cell density increases, the mean localized cell density increases and the distribution of cell densities broaden. E) Quantification of pStat3 signaling gradients present within single wells using the Neighbours Analysis algorithm. At low seeding densities, 10,000 cell/well (E–i), pStat3 can be seen to be increasing with cell density only in the presence of LIF whereas at a higher cell density, 40,000 cells/well (E–ii), signaling gradients can be found under all three treatments. Error bars represent the SEM of 9 wells cultures in three separate trials. F) Predicted and actual measurements of single-cell pStat3 levels as function of localized cell density demonstrating that the mathematical model developed in this study can be used to predict spatial fluctuations in signal activation in the Jak-Stat in mESCs. Data for this plot is taken for a single 96-well plate seeded at 40,000/well in the no LIF condition. While the percent increase in pStat3 in this figure is approximately 40%, the range in percent change is 10–40% for n = 9 wells. Error bars represent the SEM of at least 500 cells within each bin along the x-axis.

Mentions: To demonstrate the ability of the mathematical model to predict endogenous activation of Stat3, mESCs were seeded at densities ranging from 10,000–40,000 cells per well in a 96-well plate and cultured for 24 hrs. Cells were cultured in serum-free media containing LIF, no LIF, and 600 nM Jak inhibitor (JakI) to inhibit the pathway. The cells spontaneously produced colonies with an average size that depended upon the initial seeding density (Fig. 2A). The average single-cell pStat3 activation was calculated for each treatment and seeding density (Fig. 2B). Statistical significance (p<0.05) was found between treatment conditions for a given cell seeding density. However, no significance was found (p>0.4) between cell seeding densities for a given treatment. As a quantitative metric of the microenvironment, the local cell density, defined as the number of neighbours cells within a 400 µm was calculated using a previously developed algorithm, Neighbours Analysis (Supplementary Fig. S2). By using this algorithm, heat maps (Fig. 2C) and histograms (Fig. 2D) were generated to analyze the variance in local cell density in each well. Mean localized cell density increased with higher seeding densities as expected. However, the histograms also indicate that increasing seeding density leads to a broader distribution of localized cell densities across the well. Such an observation suggests that one can attempt to increase paracrine signaling by simply seeding more cells into the well, however, such an action will simultaneously increase the heterogeneity within the well by creating multiple local microenvironments consisting of a differing number of cells.


Manipulation of signaling thresholds in "engineered stem cell niches" identifies design criteria for pluripotent stem cell screens.

Peerani R, Onishi K, Mahdavi A, Kumacheva E, Zandstra PW - PLoS ONE (2009)

Model validation using the activation of the Jak-Stat signaling pathway in non-patterned mouse ESCs cultures.A) Single-field Hoechst 33342 micrographs of mESCs seeded at various densities in 96-well plates after being cultured for 24 hours in serum-free media without LIF. Scale-bar is 200 µm. B) Quantification of the average single-cell nuclear pStat3 accumulation after 24 hours of culture. While differences between exogenous supplementations were statistically significant at a given seeding density (p<0.05), no significance was present between cell seeding densities (p>0.40) for a given treatment. C) Heat maps indicating the increase and distribution in local cell density which is defined as the number of neighbouring cells within a 400 µm radius. The local cell density was calculated using an already published algorithm (See Materials and Methods)[46]. D) Histogram for the localized cell density as a function of initial seeding density showing that as initial cell density increases, the mean localized cell density increases and the distribution of cell densities broaden. E) Quantification of pStat3 signaling gradients present within single wells using the Neighbours Analysis algorithm. At low seeding densities, 10,000 cell/well (E–i), pStat3 can be seen to be increasing with cell density only in the presence of LIF whereas at a higher cell density, 40,000 cells/well (E–ii), signaling gradients can be found under all three treatments. Error bars represent the SEM of 9 wells cultures in three separate trials. F) Predicted and actual measurements of single-cell pStat3 levels as function of localized cell density demonstrating that the mathematical model developed in this study can be used to predict spatial fluctuations in signal activation in the Jak-Stat in mESCs. Data for this plot is taken for a single 96-well plate seeded at 40,000/well in the no LIF condition. While the percent increase in pStat3 in this figure is approximately 40%, the range in percent change is 10–40% for n = 9 wells. Error bars represent the SEM of at least 500 cells within each bin along the x-axis.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2713412&req=5

pone-0006438-g002: Model validation using the activation of the Jak-Stat signaling pathway in non-patterned mouse ESCs cultures.A) Single-field Hoechst 33342 micrographs of mESCs seeded at various densities in 96-well plates after being cultured for 24 hours in serum-free media without LIF. Scale-bar is 200 µm. B) Quantification of the average single-cell nuclear pStat3 accumulation after 24 hours of culture. While differences between exogenous supplementations were statistically significant at a given seeding density (p<0.05), no significance was present between cell seeding densities (p>0.40) for a given treatment. C) Heat maps indicating the increase and distribution in local cell density which is defined as the number of neighbouring cells within a 400 µm radius. The local cell density was calculated using an already published algorithm (See Materials and Methods)[46]. D) Histogram for the localized cell density as a function of initial seeding density showing that as initial cell density increases, the mean localized cell density increases and the distribution of cell densities broaden. E) Quantification of pStat3 signaling gradients present within single wells using the Neighbours Analysis algorithm. At low seeding densities, 10,000 cell/well (E–i), pStat3 can be seen to be increasing with cell density only in the presence of LIF whereas at a higher cell density, 40,000 cells/well (E–ii), signaling gradients can be found under all three treatments. Error bars represent the SEM of 9 wells cultures in three separate trials. F) Predicted and actual measurements of single-cell pStat3 levels as function of localized cell density demonstrating that the mathematical model developed in this study can be used to predict spatial fluctuations in signal activation in the Jak-Stat in mESCs. Data for this plot is taken for a single 96-well plate seeded at 40,000/well in the no LIF condition. While the percent increase in pStat3 in this figure is approximately 40%, the range in percent change is 10–40% for n = 9 wells. Error bars represent the SEM of at least 500 cells within each bin along the x-axis.
Mentions: To demonstrate the ability of the mathematical model to predict endogenous activation of Stat3, mESCs were seeded at densities ranging from 10,000–40,000 cells per well in a 96-well plate and cultured for 24 hrs. Cells were cultured in serum-free media containing LIF, no LIF, and 600 nM Jak inhibitor (JakI) to inhibit the pathway. The cells spontaneously produced colonies with an average size that depended upon the initial seeding density (Fig. 2A). The average single-cell pStat3 activation was calculated for each treatment and seeding density (Fig. 2B). Statistical significance (p<0.05) was found between treatment conditions for a given cell seeding density. However, no significance was found (p>0.4) between cell seeding densities for a given treatment. As a quantitative metric of the microenvironment, the local cell density, defined as the number of neighbours cells within a 400 µm was calculated using a previously developed algorithm, Neighbours Analysis (Supplementary Fig. S2). By using this algorithm, heat maps (Fig. 2C) and histograms (Fig. 2D) were generated to analyze the variance in local cell density in each well. Mean localized cell density increased with higher seeding densities as expected. However, the histograms also indicate that increasing seeding density leads to a broader distribution of localized cell densities across the well. Such an observation suggests that one can attempt to increase paracrine signaling by simply seeding more cells into the well, however, such an action will simultaneously increase the heterogeneity within the well by creating multiple local microenvironments consisting of a differing number of cells.

Bottom Line: The functional consequences of this niche-size-dependent signaling control are confirmed by demonstrating that direct and indirect transcriptional targets of Stat3, including members of the Jak-Stat pathway and pluripotency-associated genes, are regulated by colony size.Modeling results and empirical observations demonstrate that colonies less than 100 microm in diameter are too small to maximize endogenous Stat3 activation and that colonies separated by more than 400 microm can be considered independent from each other.These results define parameter boundaries for the use of ESCs in screening studies, demonstrate the importance of context in stem cell responsiveness to exogenous cues, and suggest that niche size is an important parameter in stem cell fate control.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
In vivo, stem cell fate is regulated by local microenvironmental parameters. Governing parameters in this stem cell niche include soluble factors, extra-cellular matrix, and cell-cell interactions. The complexity of this in vivo niche limits analyses into how individual niche parameters regulate stem cell fate. Herein we use mouse embryonic stem cells (mESC) and micro-contact printing (microCP) to investigate how niche size controls endogenous signaling thresholds. microCP is used to restrict colony diameter, separation, and degree of clustering. We show, for the first time, spatial control over the activation of the Janus kinase/signal transducer and activator of transcription pathway (Jak-Stat). The functional consequences of this niche-size-dependent signaling control are confirmed by demonstrating that direct and indirect transcriptional targets of Stat3, including members of the Jak-Stat pathway and pluripotency-associated genes, are regulated by colony size. Modeling results and empirical observations demonstrate that colonies less than 100 microm in diameter are too small to maximize endogenous Stat3 activation and that colonies separated by more than 400 microm can be considered independent from each other. These results define parameter boundaries for the use of ESCs in screening studies, demonstrate the importance of context in stem cell responsiveness to exogenous cues, and suggest that niche size is an important parameter in stem cell fate control.

Show MeSH
Related in: MedlinePlus