Induction of midbrain dopaminergic neurons from embryonic stem cells for application in cell therapy - generación de neuronas mesencefálicas dopaminérgicas a partir de células embrionarias pluripotenciales para su aplicación en terapia celular

• Autores: Antonio Jiménez Beristáin
• Directores de la Tesis: Carmen De Felipe Fernandez (dir. tes.)
• Lectura: En la Universidad Miguel Hernández de Elche ( España ) en 2021
• Idioma: español
• Tribunal Calificador de la Tesis: Stephen Hunt (presid.)
• Texto completo no disponible (Saber más ...)
• Resumen

  • Among neurodegenerative diseases Parkinson's disease is one of the main targets for cell therapy due to well-defined pathology, the loss of midbrain dopaminergic (mesDA) neurons followed by a reduction of dopamine (DA) in the striatum. Previous works has shown that foetal DA neurons can produce symptomatic relief in animal models as well as in clinical trails. However, foetal cell transplantation has important ethical, technical and practical difficulties, which limit it’s application in cellular replacement therapy. Embryonic stem (ES) cells are undifferentiated, pluripotent cell lines derived directly from the inner cell mass of preimplantation embryos. These cells have many desirable characteristics for an optimal cells source for cell therapy, such as their capacity for self–renewing, proliferation and differentiation towards a wide range of cellular phenotypes. Because ES cells are easily accessible for genetic modification without compromising pluripotency, they have been used for transgene expression studies during differentiation.

    Within the central nervous system (CNS), the paired-like homeobox transcriptional factor Pitx3 is expressed exclusively in the mesDA system, which include the substantia nigra (SN) and the ventral tegmental area (VTA). Unlike Nurr1 or Lmx1b, which display a broader expression pattern in the CNS, Pitx3 expression is confined only to mesDA neurons, which make it a specific marker for mesDA neurons. The general goal of this thesis was to generate neurons from embryonic stem cells displaying mesencephalic dopaminergic phenotype, and to study the effect of cell replacement therapy in a model of PD in mice.

    The first aim was to generate an ES cell line targeted with the enhanced green fluorescence protein (eGFP) into the Pitx3 locus. This ES cell line was differentiated by the stromal cell-derived inducing activity (SDIA) method to Pitx3+ cells. The Pitx3+ cells expressed the neuronal proteins β Tubulin III, MAPII, Tuc4, the DA neuron-related markers TH and DAT, and the midbrain-related markers Wnt1 and Nurr1, as well as established synapses in vitro. Cytotoxicity assays revealed that the Pitx3+ cells display vulnerability to the DA neurotoxins 6- hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in similar fashion than their counterpart in the brain. Additionally, the Pitx3+ cells produce and release dopamine as assessed by HPLC.

    The second aim was to investigate the effect of an intrastriatal graft of Pitx3+ cells in the functional recovery of a PD animal model. SDIA-induced Pitx3+ dopaminergic neurons were transplanted into the 6-OHDA-lesioned mouse striatum. Direct eGFP fluorescence visualization proved the existence of cell deposits in the grafted animals, indicating cell survival, for at least 5 weeks post-transplantation. There was no evidence of tumour formation. Immunocytochemical staining showed that the transplanted cells remain positive for TH, DAT and Girk2 expression. The apomorphine-induced rotational behaviour test and elevated body swing test showed a behavioural relief in Pitx3+ cells-grafted animals compared to the control groups.