Modeling signaling-dependent pluripotency with Boolean logic to predict cell fate transitions.

Pluripotent stem cells (PSCs) exist in multiple stable states, each with specific cellular properties and molecular signatures. The mechanisms that maintain pluripotency, or that cause its destabilization to initiate development, are complex and incompletely understood. We have developed a model to predict stabilized PSC gene regulatory network (GRN) states in response to input signals. Our strategy used random asynchronous Boolean simulations (R-ABS) to simulate single-cell fate transitions and strongly connected components (SCCs) strategy to represent population heterogeneity. This framework was applied to a reverse-engineered and curated core GRN for mouse embryonic stem cells (mESCs) and used to simulate cellular responses to combinations of five signaling pathways. Our simulations predicted experimentally verified cell population compositions and input signal combinations controlling specific cell fate transitions. Extending the model to PSC differentiation, we predicted a combination of signaling activators and inhibitors that efficiently and robustly generated a Cdx2+Oct4- cells from naïve mESCs. Overall, this platform provides new strategies to simulate cell fate transitions and the heterogeneity that typically occurs during development and differentiation.

LIF signaling in stem cells and development.

Leukemia inhibitory factor (LIF) is a member of the interleukin-6 (IL-6) cytokine family. All members of this family activate signal transducer and activator of transcription 3 (STAT3), a transcription factor that influences stem and progenitor cell identity, proliferation and cytoprotection. The role of LIF in development was first identified when LIF was demonstrated to support the propagation of mouse embryonic stem cells. Subsequent studies of mice deficient for components of the LIF pathway have revealed important roles for LIF signaling during development and homeostasis. Here and in the accompanying poster, we provide a broad overview of JAK-STAT signaling during development, with a specific focus on LIF-mediated JAK-STAT3 activation.

Oct4 is required ~E7.5 for proliferation in the primitive streak.

Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ~E6.0-E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ~E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ~E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.

Predictive microfluidic control of regulatory ligand trajectories in individual pluripotent cells.

Local (cell-level) signaling environments, regulated by autocrine and paracrine signaling, and modulated by cell organization, are hypothesized to be fundamental stem cell fate control mechanisms used during development. It has, however, been challenging to demonstrate the impact of cell-level organization on stem cell fate control and to relate stem cell fate outcomes to autocrine and paracrine signaling. We address this fundamental problem using a combined in silico and experimental approach in which we directly manipulate, using laminar fluid flow, the local impact of endogenously secreted gp130-activating ligands and their activation of signal transducer and activator of transcription3 (STAT3) signaling in mouse embryonic stem cells (mESC). Our model analysis predicted that flow-dependent changes in autocrine and paracrine ligand binding would impact heterogeneity in cell- and colony-level STAT3 signaling activation and cause a gradient of cell fate determination along the direction of flow. Interestingly, analysis also predicted that local cell density would be inversely proportional to the degree to which endogenous secretion contributed to cell fate determination. Experimental validation using functional activation of STAT3 by secreted factors under microfluidic perfusion culture demonstrated that STAT3 activation and consequently mESC fate were manipulable by flow rate, position in the flow field, and local cell organization. As a unique demonstration of how quantitative control of autocrine and paracrine signaling can be integrated with spatial organization to elicit higher order cell fate effects, this work provides a general template to investigate organizing principles due to secreted factors.

Identification of a Conformational Equilibrium That Determines the Efficacy and Functional Selectivity of the µ-Opioid Receptor.

G-protein-coupled receptor (GPCR) ligands impart differing degrees of signaling in the G-protein and arrestin pathways, in phenomena called "biased signaling". However, the mechanism underlying the biased signaling of GPCRs is still unclear, although crystal structures of GPCRs bound to the G protein or arrestin are available. In this study, we observed the NMR signals from methionine residues of the µ-opioid receptor (µOR) in the balanced- and biased-ligand-bound states. We found that the intracellular cavity of µOR exists in an equilibrium between closed and multiple open conformations with coupled conformational changes on the transmembrane helices 3, 5, 6, and 7, and that the population of each open conformation determines the G-protein- and arrestin-mediated signaling levels in each ligand-bound state. These findings provide insight into the biased signaling of GPCRs and will be helpful for development of analgesics that stimulate µOR with reduced tolerance and dependence.

Synthetic peptide arrays for pathway-level protein monitoring by liquid chromatography-tandem mass spectrometry.

Effective methods to detect and quantify functionally linked regulatory proteins in complex biological samples are essential for investigating mammalian signaling pathways. Traditional immunoassays depend on proprietary reagents that are difficult to generate and multiplex, whereas global proteomic profiling can be tedious and can miss low abundance proteins. Here, we report a target-driven liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy for selectively examining the levels of multiple low abundance components of signaling pathways which are refractory to standard shotgun screening procedures and hence appear limited in current MS/MS repositories. Our stepwise approach consists of: (i) synthesizing microscale peptide arrays, including heavy isotope-labeled internal standards, for use as high quality references to (ii) build empirically validated high density LC-MS/MS detection assays with a retention time scheduling system that can be used to (iii) identify and quantify endogenous low abundance protein targets in complex biological mixtures with high accuracy by correlation to a spectral database using new software tools. The method offers a flexible, rapid, and cost-effective means for routine proteomic exploration of biological systems including "label-free" quantification, while minimizing spurious interferences. As proof-of-concept, we have examined the abundance of transcription factors and protein kinases mediating pluripotency and self-renewal in embryonic stem cell populations.

Functional immobilization of signaling proteins enables control of stem cell fate.

The mode of ligand presentation has a fundamental role in organizing cell fate throughout development. We report a rapid and simple approach for immobilizing signaling ligands to maleic anhydride copolymer thin-film coatings, enabling stable signaling ligand presentation at interfaces at defined concentrations. We demonstrate the utility of this platform technology using leukemia inhibitory factor (LIF) and stem cell factor (SCF). Immobilized LIF supported mouse embryonic stem cell (mESC) pluripotency for at least 2 weeks in the absence of added diffusible LIF. Immobilized LIF activated signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) signaling in a dose-dependent manner. The introduced method allows for the robust investigation of cell fate responses from interface-immobilized ligands.

LIF-mediated control of embryonic stem cell self-renewal emerges due to an autoregulatory loop.

Stem cells convert graded stimuli into all-or-nothing cell-fate responses. We investigated how embryonic stem cells (ESCs) convert leukemia inhibitory factor (LIF) concentration into an all-or-nothing cell-fate decision (self-renewal). Using a combined experimental/computational approach we demonstrate unexpected switch-like (on/off) signaling in response to LIF. This behavior emerges over time due to a positive feedback loop controlling transcriptional expression of LIF signaling pathway components. The autoregulatory loop maintains robust pathway responsiveness ("on") at sufficient concentrations of exogenous LIF, while autocrine signaling and low concentrations of exogenous LIF cause ESCs to adopt the weakly responsive ("off") state of differentiated cells. We demonstrate that loss of ligand responsiveness is reversible and precedes loss of the ESC transcription factors Oct4 and Nanog, suggesting an early step in the hierarchical control of differentiation. While endogenously produced ligands were insufficient to sustain the "on" state, they buffer it, influencing the timing of differentiation. These results demonstrate a novel switch-like behavior, which establishes the LIF threshold for ESC self-renewal.

Patterning of sharp cellular interfaces with a reconfigurable elastic substrate.

Micropatterned cocultures are a useful experimental tool for the study of cell-cell interactions. Patterning methods often rely on sequential seeding of different cell types or removal of a barrier separating two populations, but it is difficult to pattern sharp interfaces between pure populations with low cross-contamination when using these approaches. Patterning by the use of reconfigurable substrates can overcome these limitations, but such methods can be costly and challenging to employ in a typical biology laboratory. Here, we describe a low-cost and simple-to-use reconfigurable substrate comprised of a transparent elastic material that is partially cut to form a slit that opens when the device is stretched. The slit seals back up when released, allowing two initially separate, adherent cell populations to be brought together to form a contact interface. Fluorescent imaging of patterned cocultures demonstrates the early establishment of a sharp cellular interface. As a proof of principle, we demonstrate the use of this device to study competition at the interface of two stem cell populations.

Local BMP-SMAD1 signaling increases LIF receptor-dependent STAT3 responsiveness and primed-to-naive mouse pluripotent stem cell conversion frequency.

Conversion of EpiSCs to naive ESCs is a rare event that is driven by the reestablishment of the naive transcription factor network. In mice, STAT3 activation is sufficient to drive conversion of EpiSCs to the naive pluripotent stem cell (PSC) state. However, the lack of responsiveness of EpiSCs to LIF presents a bottleneck in this conversion process. Here, we demonstrate that local accumulation of BMP-SMAD1 signaling, in cooperation with GP130 ligands, enhances the recovery of LIF responsiveness by directly controlling transcription of the LIF receptor (Lif-r). Addition of BMP and LIF to EpiSCs increases both LIF responsiveness and conversion frequencies to naive PSCs. Mechanistically, we show that the transcriptional cofactor P300 plays a critical role by mediating complex formation between STAT3 and SMAD1. This demonstration of how the local microenvironment or stem cell niche reactivates dormant signaling responsiveness and developmental potential may be applicable to other stem cell niche-containing systems.

Microenvironment-mediated reversion of epiblast stem cells by reactivation of repressed JAK-STAT signaling.

Embryonic stem cells (ESC) and epiblast stem cells (EpiSC) are distinct pluripotent stem cell states that require different signaling pathways for their self-renewal. Forward transitions between ESC and EpiSC can be accomplished by changing culture conditions; however reverse transitions between EpiSC and ESC are rare events that require transgene insertion or culture on feeders. We demonstrate that transgene-free reversion of EpiSCs to ESCs can be enhanced by local microenvironmental control and the subsequent reactivation of dormant LIF-STAT3 signaling. Reactivation of LIF responsiveness occurs in regions of colony constraint (high local cell density) typical of culture on feeders, a condition that can be recapitulated using micropatterned (µP) colonies under defined conditions. This increased LIF responsiveness results in a subsequent increase in the frequency of EpiSC reversion. Importantly, the resulting revertant EpiSCs are functionally indistinguishable from naïve mESC. Our findings demonstrate that signaling pathway activation and repression create barriers to cell fate transitions that can be overcome by microenvironmental control.

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

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.