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 (µCP) to investigate how niche size controls endogenous signaling thresholds. µCP 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 µm in diameter are too small to maximize endogenous Stat3 activation and that colonies separated by more than 400 µm 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.
Highlights
In vivo, embryogenesis is a highly orchestrated process involving the interaction of several signaling networks that produce local morphogenetic cues that determine cell fate and tissue organization[1]
We investigate whether micro-patterning mouse Embryonic stem cells (ESCs) (mESC) cultures directly modulates paracrine signaling through the Janus kinase – signal transducer and activator of transcription (Jak-Stat) pathway
The model described in this paper extends a stochastic model developed previously that predicts the fraction of autocrine and paracrine trajectories captured by a single cell in cell culture assays[31,32]
Summary
Embryogenesis is a highly orchestrated process involving the interaction of several signaling networks that produce local morphogenetic cues that determine cell fate and tissue organization[1]. Embryonic stem cells (ESCs) which are derived from the inner cell mass (ICM) of the embryo, are capable of recapitulating some of the early events of embryogenesis and have been shown to be capable of differentiation into many adult cell types in vitro[2,3,4] This promising capability of ESCs is often offset by the fact that ESC cultures can be highly heterogeneous, over resulting in low yields of target cell types upon differentiation. This in vitro situation can be contrasted to embryogenesis where cell fate and spatial location appear tightly regulated. Despite the increase in use of microscale approaches to stem cell bioengineering, parameters which govern the design of micropatterned stem cell cultures, namely colony size and separation, have not been investigated for their effects on endogenous signaling, a parameter that could be important for the control of cell specification and for interpreting the effects of test conditions on pluripotent cell fate
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