Human embryonic stem cells-derived dopaminergic neurons transplanted in parkinsonian monkeys recover dopamine levels and motor behavior

Abstract

<jats:title>Abstract</jats:title><jats:p>Human embryonic stem cells (hESCs) differentiate into specialized cells, including midbrain dopaminergic neurons (DAN). Non-human primates (NHPs) injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine develop some alterations observed in Parkinson’s disease (PD) patients. We obtained DAN from hESCs and confirmed that they express dopaminergic markers, generate action potentials, and release dopamine (DA) <jats:italic>in vitro</jats:italic>. DAN were transplanted bilaterally in the putamen of parkinsonian NHPs. After grafting, animals improved motor behavior, evaluated by the HALLWAY task, and in agreement with this recovery, DA release was detected by microdialysis. Imaging techniques revealed changes in fractional anisotropy and mean diffusivity in magnetic resonance imaging and higher 11C-DTBZ binding in positron-emission tomography scans, associated with grafts. <jats:italic>Postmortem</jats:italic> analysis showed that transplanted DAN survived over ten months in the putamen, without developing tumors, in the immunosuppressed NHPs. These results indicate that cell replacement therapy with hESCs-derived DAN causes long-term biochemical, anatomical, and physiological improvements in this model of PD.</jats:p>

Dynamic landscape of chromatin accessibility and transcriptomic changes during differentiation of human embryonic stem cells into dopaminergic neurons

“…Dopaminergic differentiation can be monitored by mRNA expression: H9 cells at D0 express high levels of OCT4 and SOX2 , but not LMX1A , FOXA2 or TH , by RT-qPCR; at D14, LMX1A and FOXA2 were detected, and its expression progressively increased. Transcripts for TH were initially detected at D21 and significantly increased afterwards 13 . Here we performed RNA-Seq at D0, D14 and D28 to determine transcriptional profiles underlying early DA induction.…”

“…1 d) are present at D28. It is important to note that at D14, TH is not expressed; this enzyme is detected by immunocytochemistry in 15% of neurons at D24, reaching 50% of TH + neurons at D28 and this percentage increases to 80% at D42 13 . Immunoblot experiments in pluripotent cells and NPC show that they do not express TH, and this enzyme shows increasing levels at D21 and D28 (Fig.…”

“…Thus, the times selected for this study allow a clear distinction between pluripotent cells, neural precursors, and early mDA. Extended periods of differentiation yield mDA that fire action potentials and release DA in vitro at D60 and D70, respectively 13 .

Figure 1 Dopaminergic differentiation of human ESC is progressive, showing three defined stages.

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“…H9 human embryonic stem cells (WA09) of female origin were purchased from WiCell (Madison, WI, USA), transduced with lentiviral vectors to express GFP as described 18 and cultured until 75% confluency. These GFP-expressing cells are useful for transplantation studies 13 and behave as its parental H9 hESC. At day zero (D0), cells were considered pluripotent.…”

“…These neurons have electrophysiological activities that resemble mDA. Furthermore, ESC derived neurons release dopamine, survive after grafting in parkinsonian non-human mammalian models, and improve the behavioral test performance of these animals, when compared with non-grafted condition 13 . Therefore, differentiation of human ESCs to mDA provides an important model for studying the correlation between the dynamic chromatin accessibility landscape and gene expression changes.…”