Wnt/ß-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) are pluripotent and can differentiate into cells belonging to the three germ layers of the embryo. However, mESC pluripotency and genome stability can be compromised in prolonged in vitro culture conditions. Several factors control mESC pluripotency, including Wnt/ß-catenin signaling pathway, which is essential for mESC differentiation and proliferation. Here we show that the activity of the Wnt/ß-catenin signaling pathway safeguards normal DNA methylation of mESCs. The activity of the pathway is progressively silenced during passages in culture and this results into a loss of the DNA methylation at many imprinting control regions (ICRs), loss of recruitment of chromatin repressors, and activation of retrotransposons, resulting into impaired mESC differentiation. Accordingly, sustained Wnt/ß-catenin signaling maintains normal ICR methylation and mESC homeostasis and is a key regulator of genome stability.

Reduced expression of Paternally Expressed Gene-3 enhances somatic cell reprogramming through mitochondrial activity perturbation.

Imprinted genes control several cellular and metabolic processes in embryonic and adult tissues. In particular, paternally expressed gene-3 (Peg3) is active in the adult stem cell population and during muscle and neuronal lineage development. Here we have investigated the role of Peg3 in mouse embryonic stem cells (ESCs) and during the process of somatic cell reprogramming towards pluripotency. Our data show that Peg3 knockdown increases expression of pluripotency genes in ESCs and enhances reprogramming efficiency of both mouse embryonic fibroblasts and neural stem cells. Interestingly, we observed that altered activity of Peg3 correlates with major perturbations of mitochondrial gene expression and mitochondrial function, which drive metabolic changes during somatic cell reprogramming. Overall, our study shows that Peg3 is a regulator of pluripotent stem cells and somatic cell reprogramming.

Mesenchymal stem cells generate distinct functional hybrids in vitro via cell fusion or entosis.

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.

Temporal perturbation of the Wnt signaling pathway in the control of cell reprogramming is modulated by TCF1.

Cyclic activation of the Wnt/ß-catenin signaling pathway controls cell fusion-mediated somatic cell reprogramming. TCFs belong to a family of transcription factors that, in complex with ß-catenin, bind and transcriptionally regulate Wnt target genes. Here, we show that Wnt/ß-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/ß-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/ß-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.

Remote control of induced dopaminergic neurons in parkinsonian rats.

Direct lineage reprogramming through genetic-based strategies enables the conversion of differentiated somatic cells into functional neurons and distinct neuronal subtypes. Induced dopaminergic (iDA) neurons can be generated by direct conversion of skin fibroblasts; however, their in vivo phenotypic and functional properties remain incompletely understood, leaving their impact on Parkinson's disease (PD) cell therapy and modeling uncertain. Here, we determined that iDA neurons retain a transgene-independent stable phenotype in culture and in animal models. Furthermore, transplanted iDA neurons functionally integrated into host neuronal tissue, exhibiting electrically excitable membranes, synaptic currents, dopamine release, and substantial reduction of motor symptoms in a PD animal model. Neuronal cell replacement approaches will benefit from a system that allows the activity of transplanted neurons to be controlled remotely and enables modulation depending on the physiological needs of the recipient; therefore, we adapted a DREADD (designer receptor exclusively activated by designer drug) technology for remote and real-time control of grafted iDA neuronal activity in living animals. Remote DREADD-dependent iDA neuron activation markedly enhanced the beneficial effects in transplanted PD animals. These data suggest that iDA neurons have therapeutic potential as a cell replacement approach for PD and highlight the applicability of pharmacogenetics for enhancing cellular signaling in reprogrammed cell-based approaches.

Rapid generation of functional dopaminergic neurons from human induced pluripotent stem cells through a single-step procedure using cell lineage transcription factors.

Current protocols for in vitro differentiation of human induced pluripotent stem cells (hiPSCs) to generate dopamine (DA) neurons are laborious and time-expensive. In order to accelerate the overall process, we have established a fast protocol by expressing the developmental transcription factors ASCL1, NURR1, and LMX1A. With this method, we were able to generate mature and functional dopaminergic neurons in as few as 21 days, skipping all the intermediate steps for inducting and selecting embryoid bodies and rosette-neural precursors. Strikingly, the resulting neuronal conversion process was very proficient, with an overall efficiency that was more than 93% of all the coinfected cells. hiPSC-derived DA neurons expressed all the critical molecular markers of the DA molecular machinery and exhibited sophisticated functional features including spontaneous electrical activity and dopamine release. This one-step protocol holds important implications for in vitro disease modeling and is particularly amenable for exploitation in high-throughput screening protocols.