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Las células madre embrionarias (ESCs) derivan de la masa cellular interna de los blastocistos y son capaces de generar las tres capas germinales. Muchos factores controlan la pluripotencia incluyendo la ruta Wnt/β-catenina. La implicación de esta ruta en la renovación celular no ha sido aún esclarecida, mientras que su función es indispensable para la diferenciación de ESCs. En esta tesis investigamos como Wnt puede afectar la stabilidad epigenética de las ESCs. Interesantemente, observamos que la reducción de señal Wnt/β-catenina provoca desmetilación del ADN y perdida de represores de la cromatina en varias regiones de control de la impronta genética (ICR). Al contrario, el mantenimiento de Wnt/β-catenina no afectó el estado de los ICR. Además, mostramos que β-catenina puede interaccionar directamente con KAP1 y ZFP57, Finalmente, demostramos que una baja dosis de Wnt/β-catenina es necesaria durante los eventos tempranos de reprogramación cellular, pero la inhibición continua de Wnt perjudica este proceso., Mouse embryonic stem cells (ESCs) derive from the inner cell mass of the blastocyst and they are able to generate the three germ layers. Many factors control ESC pluripotency including Wnt/β-catenin signaling pathway. The implication of Wnt/β-catenin pathway in self-renewal remains still unclear, whereas its function is indispensable for ESC differentiation. In this study we investigate the effect of Wnt activity on ESC epigenetic stability. Indeed we observed that loss of Wnt/β-catenin signaling causes loss of DNA methylation and chromatin repressors recruitment, such as H3K9me3 and ZFP57, at several imprinted control region (ICR). On the contrary, sustained Wnt/β-catenin signaling protected the ICR status. Moreover, we showed that β-catenin could directly interact with KAP1 and ZFP57 proteins. Finally, we demonstrated that a low dosage of Wnt/β-catenin signaling is necessary during the early stages of somatic cell reprogramming process. However continuous inhibition of Wnt/β-catenin pathway is detrimental to this process.

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.

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.