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 dopamine neurons precursors. Human induced pluripotent stem cells (hiPSC) is one of the most promising source of cells for autologous transplantation as these cells are theoretically able to differentiate into any cell type of any organ. Besides, they are an ethical and unlimited source of transplantable cells.
Here, we used adult human dermal fibroblasts to reprogram to a pluripotent state, using following mRNAs reprogramming factors OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28. The mRNA technology permits the generation of hiPSC under non-hypoxic, feeder-free conditions and eliminates the risk of genomic integration. After characterization of the pluripotency of hiPSC, we induced the in vitro differentiation into dopaminergic neurons using a three-step system. During differentiation, the cells were first induced into midbrain-specified floor plate progenitor cells. After expansion, the cells were differentiated into mature dopaminergic neurons.
After 25 days of differentiation, the cells expressed DA precursor markers Nurr1 (38,7 ± 9%) and FoxA2 (71 ± 6,1%). After 51 days of differentiation, 43,6 ± 13% of the cells are NeuN positives. Among these neurons, around 70% are TH positives. We grafted hiPSC derived DA precursors (day 25 of differentiation) 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 tyrosine hydroxylase (TH). Among DA neurons, 46,3% are GIRK2+, a marker of SNpc DA neurons. Immunolabelling showed hNCAM+ fibers along the nigrostriatal pathway into the medial forebrain bundle, and in the target structure of A9 dopaminergic neurons: the dorsal striatum. Axonal projections of grafted neurons in the dorsal striatum co-expressed hNCAM and TH. In addition, grafted animals shown functional recovery in the apomorphin-induced rotation test, 6 months after transplantation. This recovery is correlated with the number of TH neurons present in the graft (Spearman r = 0,8214, p<0,02).
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 the reconstruction of degenerated nigrostriatal pathway in an animal model of PD.