ABSTRACT
Parkinson's disease (PD) caused by SNCA gene triplication (3XSNCA) leads to early onset, rapid progression, and often dementia. Understanding the impact of 3XSNCA and its absence is crucial. This study investigates the differentiation of human induced pluripotent stem cell (hiPSC)-derived floor-plate progenitors into dopaminergic neurons. Three different genotypes were evaluated in this study: patient-derived hiPSCs with 3XSNCA, a gene-edited isogenic line with a frame-shift mutation on all SNCA alleles (SNCA 4KO), and a normal wild-type control. Our aim was to assess how the substantia nigra pars compacta (SNpc) microenvironment, damaged by 6-hydroxydopamine (6-OHDA), influences tyrosine hydroxylase-positive (Th+) neuron differentiation in these genetic variations. This study confirms successful in vitro differentiation into neuronal lineage in all cell lines. However, the SNCA 4KO line showed unusual LIM homeobox transcription factor 1 alpha (Lmx1a) extranuclear distribution. Crucially, both 3XSNCA and SNCA 4KO lines had reduced Th+ neuron expression, despite initial successful neuronal differentiation after two months post-transplantation. This indicates that while the SNpc environment supports early neuronal survival, SNCA gene alterations-either amplification or knock-out-negatively impact Th+ dopaminergic neuron maturation. These findings highlight SNCA's critical role in PD and underscore the value of hiPSC models in studying neurodegenerative diseases.
ABSTRACT
Transplantation of immature dopaminergic neurons or neural precursors derived from embryonic stem cells (ESCs) into the substantia nigra pars compacta (SNpc) is a potential therapeutic approach for functional restitution of the nigrostriatal pathway in Parkinson's disease (PD). However, further studies are needed to understand the effects of the local microenvironment on the transplanted cells to improve survival and specific differentiation in situ. We have previously reported that the adult SNpc sustains a neurogenic microenvironment. Non-neuralized embryoid body cells (EBCs) from mouse ESCs (mESCs) overexpressing the dopaminergic transcription factor Lmx1a gave rise to many tyrosine hydroxylase (Th+) cells in the intact and damaged adult SNpc, although only for a short-term period. Here, we extended our study by transplanting EBCs from genetically engineered naive human ESC (hESC), overexpressing the dopaminergic transcription factors LMX1A, FOXA2, and OTX2 (hESC-LFO), in the SNpc. Unexpectedly, no graft survival was observed in wild-type hESC EBCs transplants, whereas hESC-LFO EBCs showed viability in the SNpc. Interestingly, neural rosettes, a developmental hallmark of neuroepithelial tissue, emerged at 7- and 15-days post-transplantation (dpt) from the hESC-LFO EBCs. Neural rosettes expressed specification dopaminergic markers (Lmx1a, Otx2), which gave rise to several Th+ cells at 30 dpt. Our results suggest that the SNpc enables the robust initiation of neural differentiation of transplanted human EBCs prompted to differentiate toward the midbrain dopaminergic phenotype.
