A study on the Death-Inducer Obliterator (Dido) function in somatic cell reprogramming to pluripotency

• Autores: Amaia Talavera Gutiérrez
• Directores de la Tesis: Carlos Martinez Alonso (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2022
• Idioma: inglés
• Número de páginas: 128
• Tribunal Calificador de la Tesis: Antonio Bernad Miana (presid.), María Abad Méndez (secret.), Alejandro Ocampo Mendez (voc.)
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Materias:
  • Ciencias de la vida
    • Biología celular
    • Genética
      • Embriología
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Throughout development, embryonic pluripotent stem cells (ESCs) acquire committed differentiated cell identities. By forced expression of four factors (reprogramming factors), it is possible to reinstate pluripotency in somatic cells; the cells obtained are called induced pluripotent stem cells (iPSCs).

    Differentiation and reprogramming can be understood as opposed processes. The Death Inducer Obliterator (Dido) gene is highly expressed in pluripotent cells. Lack of Dido exons 3 and 4 is associated with the human myelodysplastic/myeloproliferative syndrome (MDS/MPD). Embryonic development is not affected in Dido exons 3 and 4 depleted mice, while lack of Dido exon 16 (DidoE16) is embryonic lethal at the onset of gastrulation (d8.5). DidoE16 depleted ESCs self-renew, but their differentiation is impaired.

    Here, we study the role of Dido in the reprogramming to pluripotency process. We first developed a Cre-loxP conditional mouse with a floxed DidoE16 (to circumvent the embryonic lethality of the homozygotic DidoE16 deletion) and crossed it with a reprogrammable mouse that bears the reprogramming factors in a doxycycline-inducible cassette. We also generated reprogrammable mouse lacking Dido exons 3 and 4.

    We found that deletion of Dido exons 3 and 4 in heterozygosis or homozygosis have no impact in primary mouse embryonic fibroblasts (MEFs) reprogramming and generate bona-fide iPSC. However, hetero- and homozygotic deletion of DidoE16 in primary MEFs reduces the reprogramming efficiency.

    Therefore, DidoE16, and not Dido exons 3 and 4, have a role in the in vitro induction of pluripotency in MEFs. As possible molecular explanations for that reduction, we identified that DidoE16 depleted MEFs express the senescence-associated marker β-galactosidase, accumulate R-loops, present DNA damage and replication stress. Moreover, immortalization with the herpes virus E6 and E7 oncogenes (inactivates of p53 and Rb) of DidoE16 depleted MEFs not only recover the reprogramming efficiency but surpasses that one of the wild type MEFs. These findings, together with previous evidence of DidoE16 being needed during gastrulation and for a normal ESC differentiation, led us to postulate that DidoE16 facilitates cell identity changes, in particular processes that participate in gastrulation and reprogramming: epithelialto- mesenchymal and mesenchymal-to-epithelial transitions.

    Learning techniques and creating tools useful for studying the role of DIDO in humans was also an aim of this thesis. We generated bona-fide wild type human iPSC (hiPSC) from mesenchymal stem cells and confirmed their capacity to differentiate into myeloid progenitors. We designed the deletion of DIDOE16 with the CRISPR/Cas9 technique and validated it in HeLa cells. This methodological expertise can be applied for the knock-out of DIDO exons 3 and 4 in our wild type hiPSC, and for MDS/MPD disease modeling. Also, DIDOE16 can be deleted in the same pluripotent cells to study in vitro the role the exon in humans.