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

The Wnt/ß-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 in vitro system enabling inducible gene expression control, we report that moderate induction of transcriptionally active exogenous ß-catenin in ß-catenin null mouse ESCs promotes epiblast-like cell (EpiLC) derivation in vitro. Instead, in wild-type cells, moderate chemical pre-activation of the Wnt/ß-catenin pathway promotes EpiLC in vitro derivation. Finally, we suggest that moderate ß-catenin levels in ß-catenin null mouse ESCs favor early stem cell commitment toward mesoderm if the exogenous protein is induced only in the "ground state" of pluripotency condition, or endoderm if the induction is maintained during the differentiation. Overall, our results confirm previous findings about the role of ß-catenin in pluripotency and differentiation, while indicating a role for its doses in promoting specific differentiation programs.

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. Computational fluid dynamics is used to predict the device separation efficacy, according to the applied alternative current (AC) frequency, at which the cells move from/to a negative/positive DEP region and the ionic strength of the suspension medium. The model is used to support the design of the operational conditions, confirming a separation efficiency, in terms of purity, of 96% under an applied AC frequency of 1.5 × 106  Hz and a flow rate of 20 µl/h. This work represents the first example of effective continuous sorting of viable and non-viable human T-cells in a single-inlet microfluidic chip, paving the way for lab-on-a-chip applications at the point of need.

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. Central to the platform is an image segmentation system based on the versatile U-Net convolutional neural network. This is supplemented with functionality to robustly count, characterize, and control cells over time. We demonstrate Cheetah's core capabilities by analyzing long-term bacterial and mammalian cell growth and by dynamically controlling protein expression in mammalian cells. In all cases, Cheetah's segmentation accuracy exceeds that of a commonly used thresholding-based method, allowing for more accurate control signals to be generated. Availability of this easy-to-use platform will make control engineering techniques more accessible and offer new ways to probe and manipulate living cells.

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. It allows us to segment individual cells in high cell density microfluidic devices, to quantify fluorescent protein expression over a time-lapse experiment, and to track individual mammalian cells. ChipSeg enables robust segmentation in external feedback control experiments and can be easily customized for other experimental settings and research aims.

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 ß-Catenin/TCF/LEF Functions.

The Wnt/ß-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 ß-catenin knockout model in mESCs to delete putatively functional N-terminally truncated isoforms observed in previous knockout models. We showed that aberrant N-terminally truncated isoforms are not functional in mESCs. In the generated knockout line, we observed that canonical Wnt signaling is not active, as ß-catenin ablation does not alter mESC transcriptional profile in serum/LIF culture conditions. In addition, we observed that Wnt signaling activation represses mESC spontaneous differentiation in a ß-catenin-dependent manner. Finally, ß-catenin (ΔC) isoforms can rescue ß-catenin knockout self-renewal defects in mESCs cultured in serum-free medium and, albeit transcriptionally silent, cooperate with TCF1 and LEF1 to inhibit mESC spontaneous differentiation in a GSK3-dependent manner.

The senotherapeutic drug ABT-737 disrupts aberrant p21 expression to restore liver regeneration in adult mice.

Young mammals possess a limited regenerative capacity in some tissues, which is lost upon maturation. We investigated whether cellular senescence might play a role in such loss during liver regeneration. We found that following partial hepatectomy, the senescence-associated genes p21, p16Ink4a, and p19Arf become dynamically expressed in different cell types when regenerative capacity decreases, but without a full senescent response. However, we show that treatment with a senescence-inhibiting drug improves regeneration, by disrupting aberrantly prolonged p21 expression. This work suggests that senescence may initially develop from heterogeneous cellular responses, and that senotherapeutic drugs might be useful in promoting organ regeneration.

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. We used a multiparameter principal component analysis and an unbiased parameter-agnostic machine-learning approach to analyze the siRNA-based screening data. The hits identified in this analysis included specific genes of the ubiquitin proteasome system, and inhibition of ubiquitin-conjugating enzyme 2 N (UBE2N) with a specific antagonist, Bay 11-7082, indicated that UBE2N modulates parkin recruitment and downstream events in the mitophagy pathway. Screening of the compound library identified kenpaullone, an inhibitor of cyclin-dependent kinases and glycogen synthase kinase 3, as a modulator of parkin recruitment. Validation studies revealed that kenpaullone augments the mitochondrial network and protects against the complex I inhibitor MPP+. Finally, we used a microfluidics platform to assess the timing of parkin recruitment to depolarized mitochondria and its modulation by kenpaullone in real time and with single-cell resolution. We demonstrate that the high-content imaging-based assay presented here is suitable for both genetic and pharmacological screening approaches, and we also provide evidence that pharmacological compounds modulate PINK1-dependent parkin recruitment.

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. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.

Role of ß-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/ß-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/ß-catenin pathway and the temporal dynamics of its effector ß-catenin are tightly controlled by complex regulations. The latter encompass feedback loops within the pathway (e.g., a negative feedback loop involving Axin2, a ß-catenin transcriptional target) and crosstalk interactions with other signaling pathways. Here, we provide a review shedding light on the coupling between Wnt/ß-catenin activation levels and fluctuations across processes and cellular systems; in particular, we focus on development, in vitro pluripotency maintenance, and cancer. Possible mechanisms originating Wnt/ß-catenin dynamic behaviors and consequently driving different cellular responses are also reviewed, and new avenues for future research are suggested.

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. Here, combining tools from Control Engineering and Synthetic Biology (cybergenetics), we propose a simple and cost-effective microfluidics-based platform to precisely regulate gene expression and signaling pathway activity in mammalian cells by means of real-time feedback control. We show that this platform allows (i) to automatically regulate gene expression from inducible promoters in different cell types, including mouse embryonic stem cells; (ii) to precisely regulate the activity of the mTOR signaling pathway in single cells; (iii) to build a biohybrid oscillator in single embryonic stem cells by interfacing biological parts with virtual in silico counterparts. Ultimately, this platform can be used to probe gene networks and signaling pathways to understand how they process static and dynamic features of specific stimuli, as well as for the rapid prototyping of synthetic circuits for biotechnology and biomedical purposes.

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. The latter heterogeneity correlates with temporal fluctuations of pluripotency markers, including the master regulator Nanog, in single cells. We propose a mathematical model of Nanog dynamics in both media, accounting for recent experimental data showing the persistence of a small Nanog Low subpopulation in ground-state pluripotency mouse embryonic stem cell cultures. The model integrates into the core pluripotency Gene Regulatory Network both inhibitors present in 2i/LIF (PD and Chiron), and feedback interactions with genes found to be differentially expressed in the two media. Our simulations and bifurcation analysis show that, in ground-state cultures, Nanog dynamics result from the combination of reduced noise in gene expression and the shift of the system towards a monostable, but still excitable, regulation. Experimental data and agent-based modelling simulations indicate that mouse embryonic stem cell proliferation dynamics vary in the two media, and cannot be reproduced by accounting only for Nanog-dependent cell-cycle regulation. We further demonstrate that both PD and Chiron play a key role in regulating heterogeneity in transcription factor expression and, ultimately, mouse embryonic stem cell fate decision.

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. Here, we find that a block of BMC recruitment into the liver severely impairs its regeneration after the surgery. Mobilized hematopoietic stem and progenitor cells (HSPCs) in the resected liver can fuse with hepatocytes, and the hybrids proliferate earlier than the hepatocytes. Genetic ablation of the hybrids severely impairs hepatocyte proliferation and liver mass regeneration. Mathematical modeling reveals a key role of bone marrow (BM)-derived hybrids to drive proliferation in the regeneration process, and predicts regeneration efficiency in experimentally non-testable conditions. In conclusion, BM-derived hybrids are essential to trigger efficient liver regeneration after hepatectomy.

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

The Wnt/ß-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 ß-catenin activation, is essential for reprogramming after fusion of ESCs overexpressing Nanog. In addition, ß-catenin is necessary in Nanog-dependent conversion of preinduced pluripotent stem cells (pre-iPSCs) into iPSCs. The activation of ß-catenin by Nanog causes fluctuations of ß-catenin in ESCs cultured in serum plus leukemia inhibitory factor (serum+LIF) medium, in which protein levels of key pluripotency factors are heterogeneous. In 2i+LIF medium, which favors propagation of ESCs in a ground state of pluripotency with many pluripotency genes losing mosaic expression, we show Nanog-independent ß-catenin fluctuations. Overall, we demonstrate Nanog and ß-catenin cooperation in establishing naive pluripotency during the reprogramming process and their correlated heterogeneity in ESCs primed toward differentiation.

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 ß-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. In addition, during reprogramming of NPCs into induced pluripotent stem cells, the silencing of Tcf3 increased AcH3 and decreased the number of H3K9me3-positive heterochromatin foci early and long before reactivation of the endogenous stem cell genes. In conclusion, our data suggest that Tcf3 functions as a repressor of the reprogramming potential of somatic cells.

The Wnt/ß-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/ß-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. In contrast, when Wnt/ß-catenin signaling is activated in ESCs and leads to accumulation of low amounts of ß-catenin in the nucleus, activated ESCs can reprogram somatic cells with very high efficiency after spontaneous fusion. Furthermore, we also show that different levels of ß-catenin accumulation in the ESC nuclei can modulate cell proliferation, although in our experimental setting, cell proliferation does not modulate the reprograming efficiency per se. Overall, the present study provides evidence that spontaneous fusion occurs, while the survival of the reprogramed clones is strictly dependent on induction of a Wnt-mediated reprograming pathway.

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. In addition, we show that reprogramming is triggered by a dose-dependent accumulation of active beta-catenin. Reprogrammed clones express ESC-specific genes, lose somatic differentiation markers, become demethylated on Oct4 and Nanog CpG islands, and can differentiate into cardiomyocytes in vitro and generate teratomas in vivo. Our data thus demonstrate that in ESCs, periodic beta-catenin accumulation via the Wnt/beta-catenin pathway provides a specific threshold that leads to the reprogramming of 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. The desired DES-glutamate 5 was water-soluble and upon incubation with carboxypeptidase G2 (CPG2) underwent carbamate cleavage to give DES 1. A control reaction in the absence of CPG2 demonstrated that the enzyme was necessary for rapid glutamate cleavage to give DES 1. HPLC analysis was required to follow the reaction of DES-glutamate 5 with CPG2. These preliminary results suggest that it may be possible to examine an ADEPT strategy for DES provided enzymatic kinetics can be measured.

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.