Potential of dopamine neurons derived from human induced pluripotent stem cells reprogrammed by mRNA for Parkinson’s disease
Résumé
Parkinson’s disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compact (SNpc), leading to a loss of dopamine in the target brain region, the striatum. Cell replacement therapy in PD aim to restore the DA neurotransmission by transplanting new midbrain DA neurons precursors. Human induced pluripotent stem cells (hiPSC) is one of the most promising source of cells for autologous transplantation. Indeed, they are an ethical and unlimited source of transplantable cells. We used adult human dermal fibroblasts to reprogram to a pluripotent state, by means of mixture of mRNA reprogramming factors. The mRNA technology allows the generation of hiPSC under feeder-free conditions and eliminates the risk of genomic integration. After pluripotency characterization of hiPSC, we induced in vitro differentiation into dopaminergic neurons using a 3-step system. During differentiation, the cells were first induced into midbrain-specified floor plate progenitor cells, before expansion and differentiation into mature dopaminergic neurons. After 25 days of differentiation, the cells expressed DA precursor markers Nurr1 (38,7%) and FoxA2 (71%). After 51 days of differentiation, 43,6% of the cells are NeuN+. Among these neurons, around 70% are TH+. We grafted hiPSC derived DA precursors into the 6-OHDA lesioned SN in an animal model of PD. Eight months after transplantation, 46% of grafted cells are NeuN+ and 35,5% of neurons express DA marker TH. Among DA neurons, 46,3% are GIRK2+, a marker of SNpc DA neurons. Immunolabelling of hNCAM+ revealed graft-derived fibers along the nigrostriatal pathway into the medial forebrain bundle, and in the target structure of A9 DA neurons: the dorsal striatum. In addition, grafted animals showed a significant recovery in amphetamin-induced rotation from 3 months post-transplantation, and this effect persisted after 9 months. We have shown the possibility to obtain DA neurons of SNpc subtype from mRNA-reprogrammed hiPSC compatible with future clinical applications. More importantly, intranigral grafted neurons express midbrain specific DA markers and allow anatomical and functional reconstruction of degenerated nigrostriatal pathway in an animal model of PD.