Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2.

Developmental cell fate specification is a unidirectional process that can be reverted in response to injury or experimental reprogramming. Whether differentiation and de-differentiation trajectories intersect mechanistically is unclear. Here, we performed comparative screening in lineage-related mouse naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), and identified the constitutively expressed zinc finger transcription factor (TF) Zfp281 as a bidirectional regulator of cell state interconversion. We showed that subtle chromatin binding changes in differentiated cells translate into activation of the histone H3 lysine 9 (H3K9) methyltransferase Ehmt1 and stabilization of the zinc finger TF Zic2 at enhancers and promoters. Genetic gain-of-function and loss-of-function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state and in restricting reprogramming of EpiSCs. Our study reveals that cell type-invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis-regulatory network underlying cellular plasticity.

Real-time fluorescence and deformability cytometry.

The throughput of cell mechanical characterization has recently approached that of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we developed an approach that combines real-time 1D-imaging fluorescence and deformability cytometry in one instrument (RT-FDC), thus opening many new research avenues. We demonstrated its utility by using subcellular fluorescence localization to identify mitotic cells and test for mechanical changes in those cells in an RNA interference screen.

The long noncoding RNA lncR492 inhibits neural differentiation of murine embryonic stem cells.

RNA interference (RNAi) screens have been shown to be valuable to study embryonic stem cell (ESC) self-renewal and they have been successfully applied to identify coding as well as noncoding genes required for maintaining pluripotency. Here, we used an RNAi library targeting >640 long noncoding RNAs (lncRNA) to probe for their role in early cell differentiation. Utilizing a Sox1-GFP ESC reporter cell line, we identified the lncRNA lncR492 as lineage-specific inhibitor of neuroectodermal differentiation. Molecular characterization showed that lncR492 interacts with the mRNA binding protein HuR and facilitates its inhibitory function by activation of Wnt signaling. Thus, lncRNAs modulate the fate decision of pluripotent stem cells.

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells.

V-ATPase inhibition increases cancer cell stiffness and blocks membrane related Ras signaling - a new option for HCC therapy.

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide and the third leading cause of cancer-related death. However, therapy options are limited leaving an urgent need to develop new strategies. Currently, targeting cancer cell lipid and cholesterol metabolism is gaining interest especially regarding HCC. High cholesterol levels support proliferation, membrane-related mitogenic signaling and increase cell softness, leading to tumor progression, malignancy and invasive potential. However, effective ways to target cholesterol metabolism for cancer therapy are still missing. The V-ATPase inhibitor archazolid was recently shown to interfere with cholesterol metabolism. In our study, we report a novel therapeutic potential of V-ATPase inhibition in HCC by altering the mechanical phenotype of cancer cells leading to reduced proliferative signaling. Archazolid causes cellular depletion of free cholesterol leading to an increase in cell stiffness and membrane polarity of cancer cells, while hepatocytes remain unaffected. The altered membrane composition decreases membrane fluidity and leads to an inhibition of membrane-related Ras signaling resulting decreased proliferation in vitro and in vivo. V-ATPase inhibition represents a novel link between cell biophysical properties and proliferative signaling selectively in malignant HCC cells, providing the basis for an attractive and innovative strategy against HCC.

Dissecting mechanisms of mouse embryonic stem cells heterogeneity through a model-based analysis of transcription factor dynamics.

Pluripotent mouse embryonic stem cells (mESCs) show heterogeneous expression levels of transcription factors (TFs) involved in pluripotency regulation, among them Nanog and Rex1. The expression of both TFs can change dynamically between states of high and low activity, correlating with the cells' capacity for self-renewal. Stochastic fluctuations as well as sustained oscillations in gene expression are possible mechanisms to explain this behaviour, but the lack of suitable data hampered their clear distinction. Here, we present a systems biology approach in which novel experimental data on TF heterogeneity is complemented by an agent-based model of mESC self-renewal. Because the model accounts for intracellular interactions, cell divisions and heredity structures, it allows for evaluating the consistency of the proposed mechanisms with data on population growth and on TF dynamics after cell sorting. Our model-based analysis revealed that a bistable, noise-driven network model fulfils the minimal requirements to consistently explain Nanog and Rex1 expression dynamics in heterogeneous and sorted mESC populations. Moreover, we studied the impact of TF-related proliferation capacities on the frequency of state transitions and demonstrate that cellular genealogies can provide insights into the heredity structures of mESCs.

Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells.

We combine a genome-scale RNAi screen in mouse epiblast stem cells (EpiSCs) with genetic interaction, protein localization, and "protein-level dependency" studies-a systematic technique that uncovers post-transcriptional regulation-to delineate the network of factors that control the expression of Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs versus embryonic stem cells (ESCs). Through multiparametric data analyses, we predict that Tox4 is associating with the Paf1C complex, which maintains cell identity in both cell types, and validate that this protein-protein interaction exists in ESCs and EpiSCs. We also identify numerous knockdowns that increase Oct4 expression in EpiSCs, indicating that, in stark contrast to ESCs, Oct4 is under active repressive control in EpiSCs. These studies provide a framework for better understanding pluripotency and for dissecting the molecular events that govern the transition from the pre-implantation to the post-implantation state.

Another brick in the wall: RNAi Screens identify new barriers in iPSC reprogramming.

Somatic cells can be reprogrammed to induced pluripotent stem cells via exogenous expression of a small set of transcription factors, but the regulatory mechanisms controlling this cell transition are poorly understood. Two recent reports demonstrate the value of RNAi screens as a tool to uncover roadblocks in this inefficient process.

EVEN-SKIPPED HOMEOBOX 1 controls human ES cell differentiation by directly repressing GOOSECOID expression.

TGFß signaling patterns the primitive streak, yet little is known about transcriptional effectors that mediate the cell fate choices during streak-like development in mammalian embryos and in embryonic stem (ES) cells. Here we demonstrate that cross-antagonistic actions of EVEN-SKIPPED HOMEOBOX 1 (EVX1) and GOOSECOID (GSC) regulate cell fate decisions in streak-like progenitors derived from human ES cells exposed to BMP4 and/or activin. We found that EVX1 repressed GSC expression and promoted formation of posterior streak-like progeny in response to BMP4, and conversely that GSC repressed EVX1 expression and was required for development of anterior streak-like progeny in response to activin. Chromatin immunoprecipitation assays showed that EVX1 bound to the GSC 5'-flanking region in BMP4 treated human ES cells, and band shift assays identified two EVX1 binding sites in the GSC 5'-region. Significantly, we found that intact EVX1 binding sites were required for BMP4-mediated repression of GSC reporter constructs. We conclude that BMP4-induced EVX1 repress GSC directly and the two genes form the core of a gene regulatory network (GRN) controlling cell fates in streak-like human ES cell progeny.

Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells.

Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17(+) endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17(+) cells more effectively than activin-mediated induction. Notably, activin induction of Gsc-GFP(+) cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17(+) cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.