Human midbrain precursors activate the expected developmental genetic program and differentiate long-term to functional A9 dopamine neurons in vitro. Enhancement by Bcl-XL

Abstract

Understanding the molecular programs of the generation of human dopaminergic neurons (DAn) from their ventral mesencephalic (VM) precursors is of key importance for basic studies, progress in cell therapy, drug screening and pharmacology in the context of Parkinson's disease. The nature of human DAn precursors in vitro is poorly understood, their properties unstable, and their availability highly limited. Here we present positive evidence that human VM precursors retaining their genuine properties and long-term capacity to generate A9 type Substantia nigra human DAn (hVMl model cell line) can be propagated in culture. During a one month differentiation, these cells activate all key genes needed to progress from pro-neural and prodopaminergic precursors to mature and functional DAn. For the first time, we demonstrate that gene cascades are correctly activated during differentiation, resulting in the generation of mature DAn. These DAn have morphological and functional properties undistinguishable from those generated by VM primary neuronal cultures. In addition, we have found that the forced expression of Bcl-X L induces an increase in the expression of key developmental genes (MSX1, Abbreviations: A9-DAn, (dopaminergic neuron from the A9 group); 6-OHDA, (6-hidroxydopamine); DA, (dopamine); DAn, (dopaminergic neuron); FB, (forebrain); FP, (floor plate); hESC, (human Embryonic stem cells); hNSC, (human neural stem cells); hVM, (human ventral mesencephalon); SNpc, (substantia nigra pars compacta); TH, (tyrosine hydroxylase); VM, (ventral mesencephalon); VM DAn, (dopaminergic neuron from ventral mesencephalon); VM hNSC, (human neural stem cells from ventral mesencephalon) NGN2), maintenance of PITX3 expression temporal profile, and also enhances genes involved in DAn long-term function, maintenance and survival (ENl, LMXIB, NURRl and PITX3). As a result, Bcl-X L anticipates and enhances DAn generation.

Optimization of the magnetic labeling of human neural stem cells and MRI visualization in the hemiparkinsonian rat brain

“…The percentage of total cells expressing markers characteristic of differentiated VM hNSCs, like β-III-tubulin, GFAP and TH, was compared among samples ( Figure 4B, C). No significant differences between control and MNPs-labeled cells were observed, demonstrating that MNPs do not affect the ability of hVM cells to differentiate into glial cells, neurons and, specifically, dopaminergic neurons, obtaining similar results for THand β-III-tubulin-positive cells (17% and 25% respectively) to those previously described in [7]. Thus, these results demonstrate that labeling VM hNSCs cells with optimal doses of MNPs does not affect their stemness and differentiation potential.…”

“…Recent pre-clinical research has demonstrated that immortalized human NSCs, derived from VM (hVM1 cell line) and modified for the elevated expression of Bcl-XL (hVM1-highBcl-XL cells), have the potential to differentiate into DAn in vivo at a high rate [5][6][7][8][9]. After transplantation in hemiparkinsonian rats, these hVM cells survive, integrate, and differentiate into DAn, alleviating behavioral motor asymmetry and skilled paw use [5,9,10].…”

Influence of Oxygen Tension on Dopaminergic Differentiation of Human Fetal Stem Cells of Midbrain and Forebrain Origin

“…Almost all cells expressed this general marker of neural precursor cells, which was in accordance with previous studies showing that both the midbrain stem cell line and the forebrain stem cell line express nestin even after long-term in vitro propagation [34], [35], [37], [38], [47]. We also found GFAP expression during propagation but mainly in forebrain-derived cultures.…”

“…This could reflect differences in neuronal and glial differentiation capacity or region-specific requirements of the NSCs. In favour of this hypothesis is the fact that we developed our dopaminergic differentiation protocol using the midbrain-derived cell line [18] and with the aim of generating authentic and functional dopaminergic neurons with A9 (substantia nigra) characteristics [39], [47]. Thus, the applied protocol may not be optimal, when it comes to guiding NSCs from other brain regions towards the A9 dopaminergic phenotype.…”

“…In addition, the midbrain-derived TH-ir cells had a very mature neuronal appearance whereas the forebrain-derived TH-ir cells displayed an immature neuronal morphology. One explanation of these differences could be that even though these cells are multipotent [18], [37], [47] and respond to overlapping growth and differentiation factors, they are not equivalent to each other and maintain certain region-specific properties. Consequently they respond differently to local environmental factors released from the surrounding host tissue microenvironment.…”