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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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Micro-patterning mESC cultures regulates transcription of known Stat3 targets.Quantitative real-time PCR results of mESCs cultured on D = 50 µm, P = 200 µm and D = 200 µm, P = 400 µm pattern cultured for 24 hours in serum-free media without LIF. The data is normalized to the house-keeping gene GAPDH and each gene is plotted relative to its expression found in the D = 200 µm, P = 400 µm pattern. Pluripotency-associated genes, Oct-4, Nanog, Klf4, and Sal4 decrease in expression with smaller colony size. Likewise, members of the Jak-Stat pathway, including LIF receptor (LIFR), gp130, Stat3, and Socs3 all decrease with smaller colony size demonstrating spatial control of this auto-regulatory pathway. C-myc, another target of pStat3 is also down-regulated in small colonies. Epigenetic modifiers of mESCs including HP1, Dmnt1, and Jmd1ja are also down-regulated in small colonies. Error bars represent the S.E.M for n = 3 biological replicates. Asterisks indicate statistical significance of p<0.05 by the t-test.
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pone-0006438-g004: Micro-patterning mESC cultures regulates transcription of known Stat3 targets.Quantitative real-time PCR results of mESCs cultured on D = 50 µm, P = 200 µm and D = 200 µm, P = 400 µm pattern cultured for 24 hours in serum-free media without LIF. The data is normalized to the house-keeping gene GAPDH and each gene is plotted relative to its expression found in the D = 200 µm, P = 400 µm pattern. Pluripotency-associated genes, Oct-4, Nanog, Klf4, and Sal4 decrease in expression with smaller colony size. Likewise, members of the Jak-Stat pathway, including LIF receptor (LIFR), gp130, Stat3, and Socs3 all decrease with smaller colony size demonstrating spatial control of this auto-regulatory pathway. C-myc, another target of pStat3 is also down-regulated in small colonies. Epigenetic modifiers of mESCs including HP1, Dmnt1, and Jmd1ja are also down-regulated in small colonies. Error bars represent the S.E.M for n = 3 biological replicates. Asterisks indicate statistical significance of p<0.05 by the t-test.

Mentions: After establishing that micro-patterned ESCs exhibit different signaling levels of Stat3 activation, we next sought to determine if this change in signaling activation had downstream consequences on known and predicted targets of Stat3. We hypothesized that Stat3 targets would be differentially expressed in small (D = 50 µm, P = 200 µm) colonies versus large (D = 200 µm, P = 400 µm) colonies because of the lower levels of pStat3 in small colonies. Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of several possible targets of Stat3 (Fig. 4). As expected the pluripotency genes decreased with smaller colony size. The expression of Jak-Stat pathway members decreased as well in small colonies, an observation which is to be expected by the auto-regulatory behavior of this pathway as revealed previously in non-patterned cultures[18]. Genes related to the epigenetic status of mESCs including Jmid1a, Dnmt1, and HP1 decreased as well. These findings provide further evidence that spatial control over Stat3 and its downstream transcriptional targets can be achieved in micro-patterned mESC cultures with implications to the pluripotency network and epigenetic state of the mESCs.


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)

Micro-patterning mESC cultures regulates transcription of known Stat3 targets.Quantitative real-time PCR results of mESCs cultured on D = 50 µm, P = 200 µm and D = 200 µm, P = 400 µm pattern cultured for 24 hours in serum-free media without LIF. The data is normalized to the house-keeping gene GAPDH and each gene is plotted relative to its expression found in the D = 200 µm, P = 400 µm pattern. Pluripotency-associated genes, Oct-4, Nanog, Klf4, and Sal4 decrease in expression with smaller colony size. Likewise, members of the Jak-Stat pathway, including LIF receptor (LIFR), gp130, Stat3, and Socs3 all decrease with smaller colony size demonstrating spatial control of this auto-regulatory pathway. C-myc, another target of pStat3 is also down-regulated in small colonies. Epigenetic modifiers of mESCs including HP1, Dmnt1, and Jmd1ja are also down-regulated in small colonies. Error bars represent the S.E.M for n = 3 biological replicates. Asterisks indicate statistical significance of p<0.05 by the t-test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2713412&req=5

pone-0006438-g004: Micro-patterning mESC cultures regulates transcription of known Stat3 targets.Quantitative real-time PCR results of mESCs cultured on D = 50 µm, P = 200 µm and D = 200 µm, P = 400 µm pattern cultured for 24 hours in serum-free media without LIF. The data is normalized to the house-keeping gene GAPDH and each gene is plotted relative to its expression found in the D = 200 µm, P = 400 µm pattern. Pluripotency-associated genes, Oct-4, Nanog, Klf4, and Sal4 decrease in expression with smaller colony size. Likewise, members of the Jak-Stat pathway, including LIF receptor (LIFR), gp130, Stat3, and Socs3 all decrease with smaller colony size demonstrating spatial control of this auto-regulatory pathway. C-myc, another target of pStat3 is also down-regulated in small colonies. Epigenetic modifiers of mESCs including HP1, Dmnt1, and Jmd1ja are also down-regulated in small colonies. Error bars represent the S.E.M for n = 3 biological replicates. Asterisks indicate statistical significance of p<0.05 by the t-test.
Mentions: After establishing that micro-patterned ESCs exhibit different signaling levels of Stat3 activation, we next sought to determine if this change in signaling activation had downstream consequences on known and predicted targets of Stat3. We hypothesized that Stat3 targets would be differentially expressed in small (D = 50 µm, P = 200 µm) colonies versus large (D = 200 µm, P = 400 µm) colonies because of the lower levels of pStat3 in small colonies. Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of several possible targets of Stat3 (Fig. 4). As expected the pluripotency genes decreased with smaller colony size. The expression of Jak-Stat pathway members decreased as well in small colonies, an observation which is to be expected by the auto-regulatory behavior of this pathway as revealed previously in non-patterned cultures[18]. Genes related to the epigenetic status of mESCs including Jmid1a, Dnmt1, and HP1 decreased as well. These findings provide further evidence that spatial control over Stat3 and its downstream transcriptional targets can be achieved in micro-patterned mESC cultures with implications to the pluripotency network and epigenetic state of the mESCs.

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