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.

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.

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. 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.Wnt pathway | induced pluripotent stem cell generation

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. The activation of β-catenin by Nanog causes fluctuations of β-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 β-catenin fluctuations. Overall, we demonstrate Nanog and β-catenin cooperation in establishing naive pluripotency during the reprogramming process and their correlated heterogeneity in ESCs primed toward differentiation.

Gene networks and signalling pathways display complex topologies and, as a result, complex nonlinear behaviours. 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 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 signalling 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 signalling pathway in single cells; (iii) to build a bio-hybrid 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 signalling 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.