Cyclic activation of the Wnt/β-catenin signaling pathway controls cell fusion-mediated somatic cell reprogramming. TCFs belong to a family of transcription factors that, in complex with β-catenin, bind and transcriptionally regulate Wnt target genes. Here, we show that Wnt/β-catenin signaling needs to be off during the early reprogramming phases of mouse embryonic fibroblasts (MEFs) into iPSCs. In MEFs undergoing reprogramming, senescence genes are repressed and mesenchymal-to-epithelial transition is favored. This is correlated with a repressive activity of TCF1, which contributes to the silencing of Wnt/β-catenin signaling at the onset of reprogramming. In contrast, the Wnt pathway needs to be active in the late reprogramming phases to achieve successful reprogramming. In conclusion, continued activation or inhibition of the Wnt/β-catenin signaling pathway is detrimental to the reprogramming of MEFs; instead, temporal perturbation of the pathway is essential for efficient reprogramming, and the "Wnt-off" state can be considered an early reprogramming marker.

Homotypic and heterotypic cell-to-cell fusion are key processes during development and tissue regeneration. Nevertheless, aberrant cell fusion can contribute to tumour initiation and metastasis. Additionally, a form of cell-in-cell structure called entosis has been observed in several human tumours. Here we investigate cell-to-cell interaction between mouse mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs). MSCs represent an important source of adult stem cells since they have great potential for regenerative medicine, even though they are also involved in cancer progression. We report that MSCs can either fuse forming heterokaryons, or be invaded by ESCs through entosis. While entosis-derived hybrids never share their genomes and induce degradation of the target cell, fusion-derived hybrids can convert into synkaryons. Importantly we show that hetero-to-synkaryon transition occurs through cell division and not by nuclear membrane fusion. Additionally, we also observe that the ROCK-actin/myosin pathway is required for both fusion and entosis in ESCs but only for entosis in MSCs. Overall, we show that MSCs can undergo fusion or entosis in culture by generating distinct functional cellular entities. These two processes are profoundly different and their outcomes should be considered given the beneficial or possible detrimental effects of MSC-based therapeutic applications.

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.

Understanding the mechanisms regulating cell cycle, proliferation and potency of pluripotent stem cells guarantees their safe use in the clinic. Embryonic stem cells (ESCs) present a fast cell cycle with a short G1 phase. This is due to the lack of expression of cell cycle inhibitors, which ultimately determines naïve pluripotency by holding back differentiation. The canonical Wnt/β-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/β-catenin controls the cell cycle of mESCs remains unknown. Here we show that the Wnt-effector Tcf1 is recruited to and triggers transcription of the Ink4/Arf tumor suppressor locus. Thereby, the activation of the Wnt pathway, a known mitogenic pathway in somatic tissues, restores G1 phase and drastically reduces proliferation of mESCs without perturbing pluripotency. Tcf1, but not Tcf3, is recruited to a palindromic motif enriched in the promoter of cell cycle repressor genes, such as p15^Ink4b, p16^Ink4a and p19^Arf, which mediate the Wnt-dependent anti-proliferative effect in mESCs. Consistently, ablation of β-catenin or Tcf1 expression impairs Wnt-dependent cell cycle regulation. All together, here we showed that Wnt signaling controls mESC pluripotency and proliferation through non-overlapping functions of distinct Tcf factors. Studying how to safely expand stem cells in culture is essential for regenerative medicine applications. Hence there is a clear need to decode how the cell cycle of mouse embryonic stem cells (mESCs) is regulated. Tcf3 and Tcf1 belong to the Tcf family of proteins. Tcf/Lef are effectors of the Wnt/β-catenin pathway and Tcf3 controls mESC pluripotency. Here we identified a recruitment site for Tcf1 embedded into a number of cell cycle repressor genes such as p15^Ink4b, p16^Ink4a and p19^Arf. Tcf1-mediated activation of these genes drastically slows down proliferation of mESCs. In conclusion, here we showed that the Wnt pathway, besides controlling mESC pluripotency via Tcf3, also regulates mESC cell cycle through the recruitment of Tcf1 to the regulatory sites of key cell cycle genes.

CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.