β-catenin perturbations control differentiation programs in mouse embryonic stem cells.

The Wnt/β-catenin pathway is involved in development, cancer, and embryonic stem cell (ESC) maintenance; its dual role in stem cell self-renewal and differentiation is still controversial. Here, by applying an system enabling inducible gene expression control, we report that moderate induction of transcriptionally active exogenous β-catenin in β-catenin null mouse ESCs promotes epiblast-like cell (EpiLC) derivation . Instead, in wild-type cells, moderate chemical pre-activation of the Wnt/β-catenin pathway promotes EpiLC derivation.

A flow-through microfluidic chip for continuous dielectrophoretic separation of viable and non-viable human T-cells.

Effective methods for rapid sorting of cells according to their viability are critical in T cells based therapies to prevent any risk to patients. In this context, we present a novel microfluidic device that continuously separates viable and non-viable T-cells according to their dielectric properties. A dielectrophoresis (DEP) force is generated by an array of castellated microelectrodes embedded into a microfluidic channel with a single inlet and two outlets; cells subjected to positive DEP forces are drawn toward the electrodes array and leave from the top outlet, those subjected to negative DEP forces are repelled away from the electrodes and leave from the bottom outlet.

Cheetah: A Computational Toolkit for Cybergenetic Control.

Advances in microscopy, microfluidics, and optogenetics enable single-cell monitoring and environmental regulation and offer the means to control cellular phenotypes. The development of such systems is challenging and often results in bespoke setups that hinder reproducibility. To address this, we introduce Cheetah, a flexible computational toolkit that simplifies the integration of real-time microscopy analysis with algorithms for cellular control.

ChipSeg: An Automatic Tool to Segment Bacterial and Mammalian Cells Cultured in Microfluidic Devices.

Extracting quantitative measurements from time-lapse images is necessary in external feedback control applications, where segmentation results are used to inform control algorithms. We describe ChipSeg, a computational tool that segments bacterial and mammalian cells cultured in microfluidic devices and imaged by time-lapse microscopy, which can be used also in the context of external feedback control. The method is based on thresholding and uses the same core functions for both cell types.

A Microfluidic/Microscopy-Based Platform for on-Chip Controlled Gene Expression in Mammalian Cells.

Applications of control engineering to mammalian cell biology have been recently implemented for precise regulation of gene expression. In this chapter, we report the main experimental and computational methodologies to implement automatic feedback control of gene expression in mammalian cells using a microfluidics/microscopy platform.

Canonical Wnt Pathway Controls mESC Self-Renewal Through Inhibition of Spontaneous Differentiation via β-Catenin/TCF/LEF Functions.

The Wnt/β-catenin signaling pathway is a key regulator of embryonic stem cell (ESC) self-renewal and differentiation. Constitutive activation of this pathway has been shown to increase mouse ESC (mESC) self-renewal and pluripotency gene expression. In this study, we generated a novel β-catenin knockout model in mESCs to delete putatively functional N-terminally truncated isoforms observed in previous knockout models.

A dual druggable genome-wide siRNA and compound library screening approach identifies modulators of parkin recruitment to mitochondria.

Genetic and biochemical evidence points to an association between mitochondrial dysfunction and Parkinson's disease (PD). PD-associated mutations in several genes have been identified and include those encoding PTEN-induced putative kinase 1 (PINK1) and parkin. To identify genes, pathways, and pharmacological targets that modulate the clearance of damaged or old mitochondria (mitophagy), here we developed a high-content imaging-based assay of parkin recruitment to mitochondria and screened both a druggable genome-wide siRNA library and a small neuroactive compound library.

A tunable dual-input system for on-demand dynamic gene expression regulation.

Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function.

Role of β-Catenin Activation Levels and Fluctuations in Controlling Cell Fate.

Cells have developed numerous adaptation mechanisms to external cues by controlling signaling-pathway activity, both qualitatively and quantitatively. The Wnt/β-catenin pathway is a highly conserved signaling pathway involved in many biological processes, including cell proliferation, differentiation, somatic cell reprogramming, development, and cancer. The activity of the Wnt/β-catenin pathway and the temporal dynamics of its effector β-catenin are tightly controlled by complex regulations.

Regulation of Gene Expression and Signaling Pathway Activity in Mammalian Cells by Automated Microfluidics Feedback Control.

Gene networks and signaling pathways display complex topologies and, as a result, complex nonlinear behaviors. Accumulating evidence shows that both static (concentration) and dynamical (rate-of-change) features of transcription factors, ligands and environmental stimuli control downstream processes and ultimately cellular functions. Currently, however, methods to generate stimuli with the desired features to probe cell response are still lacking.

An extended model for culture-dependent heterogenous gene expression and proliferation dynamics in mouse embryonic stem cells.

During development, pluripotency is a transient state describing a cell's ability to give rise to all three germ layers and germline. Recent studies have shown that, in vitro, pluripotency is highly dynamic: exogenous stimuli provided to cultures of mouse embryonic stem cells, isolated from pre-implantation blastocysts, significantly affect the spectrum of pluripotency. 2i/LIF, a recently defined serum-free medium, forces mouse embryonic stem cells into a ground-state of pluripotency, while serum/LIF cultures promote the co-existence of ground-like and primed-like mouse embryonic stem cell subpopulations.

Modeling Dynamics and Function of Bone Marrow Cells in Mouse Liver Regeneration.

In rodents and humans, the liver can efficiently restore its mass after hepatectomy. This is largely attributed to the proliferation and cell cycle re-entry of hepatocytes. On the other hand, bone marrow cells (BMCs) migrate into the liver after resection.

β-catenin fluctuates in mouse ESCs and is essential for Nanog-mediated reprogramming of somatic cells to pluripotency.

The Wnt/β-catenin pathway and Nanog are key regulators of embryonic stem cell (ESC) pluripotency and the reprogramming of somatic cells. Here, we demonstrate that the repression of Dkk1 by Nanog, which leads indirectly to β-catenin activation, is essential for reprogramming after fusion of ESCs overexpressing Nanog. In addition, β-catenin is necessary in Nanog-dependent conversion of preinduced pluripotent stem cells (pre-iPSCs) into iPSCs.

T-cell factor 3 (Tcf3) deletion increases somatic cell reprogramming by inducing epigenome modifications.

The heterochromatin barrier must be overcome to generate induced pluripotent stem cells and cell fusion-mediated reprogrammed hybrids. Here, we show that the absence of T-cell factor 3 (Tcf3), a repressor of β-catenin target genes, strikingly and rapidly enhances the efficiency of neural precursor cell (NPC) reprogramming. Remarkably, Tcf3(-/-) ES cells showed a genome-wide increase in AcH3 and decrease in H3K9me3 and can reprogram NPCs after fusion greatly.

The Wnt/β-catenin signaling pathway tips the balance between apoptosis and reprograming of cell fusion hybrids.

Cell-cell fusion contributes to cell differentiation and developmental processes. We have previously showed that activation of Wnt/β-catenin enhances somatic cell reprograming after polyethylene glycol (PEG)-mediated fusion. Here, we show that neural stem cells and ESCs can fuse spontaneously in cocultures, although with very low efficiency (about 2%), as the hybrids undergo apoptosis.

Periodic activation of Wnt/beta-catenin signaling enhances somatic cell reprogramming mediated by cell fusion.

Reprogramming of nuclei allows the dedifferentiation of differentiated cells. Somatic cells can undergo epigenetic modifications and reprogramming through their fusion with embryonic stem cells (ESCs) or after overexpression of a specific blend of ESC transcription factor-encoding genes. We show here that cyclic activation of Wnt/beta-catenin signaling in ESCs with Wnt3a or the glycogen synthase kinase-3 (GSK-3) inhibitor 6-bromoindirubin-3'-oxime (BIO) strikingly enhances the ability of ESCs to reprogram somatic cells after fusion.

Diethylstilbestrol glutamate as a potential substrate for ADEPT.

The combination of systemic toxicity, water insolubility and a labile chemical structure has limited the clinical use of diethylstilbestrol (DES) 1 for the treatment of prostate cancer. To determine if DES could potentially be a prodrug substrate for the pre-targeting strategy known as antibody directed enzyme prodrug therapy (ADEPT), the DES-glutamate 5 was prepared. The synthesis required the activation of the bis-t-butyl glutamate ester 2 to the isocyanate 3 followed by addition of DES 1.

Site-specific PEGylation of native disulfide bonds in therapeutic proteins.

Native disulfide bonds in therapeutic proteins are crucial for tertiary structure and biological activity and are therefore considered unsuitable for chemical modification. We show that native disulfides in human interferon alpha-2b and in a fragment of an antibody to CD4(+) can be modified by site-specific bisalkylation of the two cysteine sulfur atoms to form a three-carbon PEGylated bridge. The yield of PEGylated protein is high, and tertiary structure and biological activity are retained.