Limits...
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.

Show MeSH

Related in: MedlinePlus

Micro-patterning mESC cultures provides spatial control over endogeneous Jak-Stat activation.Using in silico models and experimental validation, we have demonstrated that endogenous activation of the Jak-Stat pathway can be regulated spatially by micro-patterning mESC cultures. Three parameters were explored: increasing colony diameter, decreasing colony pitch, and increasing the degree of clustering.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2713412&req=5

pone-0006438-g005: Micro-patterning mESC cultures provides spatial control over endogeneous Jak-Stat activation.Using in silico models and experimental validation, we have demonstrated that endogenous activation of the Jak-Stat pathway can be regulated spatially by micro-patterning mESC cultures. Three parameters were explored: increasing colony diameter, decreasing colony pitch, and increasing the degree of clustering.

Mentions: Local gradients of activated signaling molecules exist in ESC cultures and these gradients correlate with the expression of pluripotent stem cell markers such as Oct-4 and Nanog. These gradients can be correlated to the localized cell density in a single well as well as manipulated directly using micro-patterning technologies. Specifically, by altering spatial arrangement, we have shown for the first time that altering colony diameter, the distance between colonies, and the degree of clustering of a culture can modulate in a predictive manner the nuclear levels of pStat3 in mESCs (Fig. 5).


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 provides spatial control over endogeneous Jak-Stat activation.Using in silico models and experimental validation, we have demonstrated that endogenous activation of the Jak-Stat pathway can be regulated spatially by micro-patterning mESC cultures. Three parameters were explored: increasing colony diameter, decreasing colony pitch, and increasing the degree of clustering.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713412&req=5

pone-0006438-g005: Micro-patterning mESC cultures provides spatial control over endogeneous Jak-Stat activation.Using in silico models and experimental validation, we have demonstrated that endogenous activation of the Jak-Stat pathway can be regulated spatially by micro-patterning mESC cultures. Three parameters were explored: increasing colony diameter, decreasing colony pitch, and increasing the degree of clustering.
Mentions: Local gradients of activated signaling molecules exist in ESC cultures and these gradients correlate with the expression of pluripotent stem cell markers such as Oct-4 and Nanog. These gradients can be correlated to the localized cell density in a single well as well as manipulated directly using micro-patterning technologies. Specifically, by altering spatial arrangement, we have shown for the first time that altering colony diameter, the distance between colonies, and the degree of clustering of a culture can modulate in a predictive manner the nuclear levels of pStat3 in mESCs (Fig. 5).

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