Embryoid Body Cells from Human Embryonic Stem Cells Overexpressing Dopaminergic Transcription Factors Survive and Initiate Neurogenesis via Neural Rosettes in the Substantia Nigra

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.

Alpha-Synuclein Gene Alterations Modulate Tyrosine Hydroxylase in Human iPSC-Derived Neurons in a Parkinson’s Disease Animal Model

“…We previously demonstrated that the 6-OHDA-lesioned SNpc favors the Th+ fate of grafted embryoid body cells (EBCs) derived from mouse ESCs and human EBCs overexpressing dopaminergic transcription factors [ 39 , 40 ]. To assess the Th+ lineage maturation potential of 3X SNCA and SNCA 4KO floor-plate progenitors in the SNpc, we examined the Th expression compared to the wild-type line.…”

“…These results strongly suggest the feasibility of the survival and effective differentiation of dopaminergic neurons derived from dopaminergic progenitor cells in the SNpc. Our data demonstrate that the intact rat SNpc is a permissive neurogenic environment for neuronal development [ 38 , 39 , 40 ]. Yet, it does not inherently promote the mesencephalic dopaminergic phenotype without the overexpression of key dopaminergic transcription factors like Lmx1a [ 39 , 40 ].…”

“…Our data demonstrate that the intact rat SNpc is a permissive neurogenic environment for neuronal development [ 38 , 39 , 40 ]. Yet, it does not inherently promote the mesencephalic dopaminergic phenotype without the overexpression of key dopaminergic transcription factors like Lmx1a [ 39 , 40 ]. This emphasizes the importance of understanding the microenvironmental influences on transplanted cells.…”

“…A PD model was generated through unilateral administration of the neurotoxin 6-hydroxydopamine (6-OHDA), which effectively targets and kills catecholaminergic neurons [ 74 ]. Following our previous work [ 39 , 40 ], rats were given intraperitoneal anesthesia using a mixture of xylazine (8 mg/kg, PiSA, Mexico City, Mexico) and ketamine (100 mg/kg, PiSA) before surgical procedures. Of the total of 39 anaesthetized animals, 24 (6-OHDA group) and 15 (Sham group) were secured in a stereotaxic device and injected with a total volume of 0.5 mL of a 0.9% saline solution supplemented with 40 μg of 6-OHDA (Sigma-Aldrich) and 32 μg/mL of L-ascorbate (JT Baker, Phillipsburg, NJ, USA) into the left SNpc at a rate of 0.125 mL/min [ 39 , 40 ].…”

“…This is crucial for gaining insights into the prodromal development of the disease, which could also contribute to the development of rational cell replacement therapies, particularly those based on SNCA knock-out autologous hiPSC transplants. Furthermore, whether 3X SNCA or SNCA -deleted hiPSCs can differentiate into a tyrosine hydroxylase-positive (Th+) lineage within the SNpc at the floor-plate stage requires further research [ 38 , 39 , 40 ].…”