In Vitro and in Vivo Enhanced Generation of Human A9 Dopamine Neurons from Neural Stem Cells by Bcl-XL

Center of Molecular Biology Severo Ochoa (Consejo Superior de Investigaciones Científicas-UAM), Department of Molecular Biology, Autonomous University of Madrid, 28049 Madrid, Spain.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2010; 285(13):9881-97. DOI: 10.1074/jbc.M109.054312
Source: PubMed


Human neural stem cells derived from the ventral mesencephalon (VM) are powerful research tools and candidates for cell therapies in Parkinson disease. Previous studies with VM dopaminergic neuron (DAn) precursors indicated poor growth potential and unstable phenotypical properties. Using the model cell line hVM1 (human ventral mesencephalic neural stem cell line 1; a new human fetal VM stem cell line), we have found that Bcl-X(L) enhances the generation of DAn from VM human neural stem cells. Mechanistically, Bcl-X(L) not only exerts the expected antiapoptotic effect but also induces proneural (NGN2 and NEUROD1) and dopamine-related transcription factors, resulting in a high yield of DAn with the correct phenotype of substantia nigra pars compacta (SNpc). The expression of key genes directly involved in VM/SNpc dopaminergic patterning, differentiation, and maturation (EN1, LMX1B, PITX3, NURR1, VMAT2, GIRK2, and dopamine transporter) is thus enhanced by Bcl-X(L). These effects on neurogenesis occur in parallel to a decrease in glia generation. These in vitro Bcl-X(L) effects are paralleled in vivo, after transplantation in hemiparkinsonian rats, where hVM1-Bcl-X(L) cells survive, integrate, and differentiate into DAn, alleviating behavioral motor asymmetry. Bcl-X(L) then allows for human fetal VM stem cells to stably generate mature SNpc DAn both in vitro and in vivo and is thus proposed as a helpful factor for the development of cell therapies for neurodegenerative conditions, Parkinson disease in particular.

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    • "Only very few cells in the cultures had spontaneously differentiated into neurons (β-tub III-ir) of which few were found to co-express the catecholaminergic marker TH. Thus, the very low content of differentiated cells in both types of cultures showed that our cell lines were maintained in an undifferentiated state, corresponding well with other studies showing that neural precursors can undergo long-term expansion in vitro using EGF and bFGF [21], [36]–[39], [50]. "
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    ABSTRACT: Neural stem cells (NSCs) constitute a promising source of cells for transplantation in Parkinson's disease (PD), but protocols for controlled dopaminergic differentiation are not yet available. Here we investigated the influence of oxygen on dopaminergic differentiation of human fetal NSCs derived from the midbrain and forebrain. Cells were differentiated for 10 days in vitro at low, physiological (3%) versus high, atmospheric (20%) oxygen tension. Low oxygen resulted in upregulation of vascular endothelial growth factor and increased the proportion of tyrosine hydroxylase-immunoreactive (TH-ir) cells in both types of cultures (midbrain: 9.1±0.5 and 17.1±0.4 (P<0.001); forebrain: 1.9±0.4 and 3.9±0.6 (P<0.01) percent of total cells). Regardless of oxygen levels, the content of TH-ir cells with mature neuronal morphologies was higher for midbrain as compared to forebrain cultures. Proliferative Ki67-ir cells were found in both types of cultures, but the relative proportion of these cells was significantly higher for forebrain NSCs cultured at low, as compared to high, oxygen tension. No such difference was detected for midbrain-derived cells. Western blot analysis revealed that low oxygen enhanced β-tubulin III and GFAP expression in both cultures. Up-regulation of β-tubulin III was most pronounced for midbrain cells, whereas GFAP expression was higher in forebrain as compared to midbrain cells. NSCs from both brain regions displayed less cell death when cultured at low oxygen tension. Following mictrotransplantation into mouse striatal slice cultures predifferentiated midbrain NSCs were found to proliferate and differentiate into substantial numbers of TH-ir neurons with mature neuronal morphologies, particularly at low oxygen. In contrast, predifferentiated forebrain NSCs microtransplanted using identical conditions displayed little proliferation and contained few TH-ir cells, all of which had an immature appearance. Our data may reflect differences in dopaminergic differentiation capacity and region-specific requirements of NSCs, with the dopamine-depleted striatum cultured at low oxygen offering an attractive micro-environment for midbrain NSCs.
    PLoS ONE 05/2014; 9(5):e96465. DOI:10.1371/journal.pone.0096465 · 3.23 Impact Factor
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    • "Furthermore, this clone #30 is clearly superior to its parental cell line (control hVM1 cells, refs. [19], [20]). "
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    ABSTRACT: A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH(+) neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experiments.
    PLoS ONE 12/2012; 7(12):e52714. DOI:10.1371/journal.pone.0052714 · 3.23 Impact Factor
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    • "In these studies cells were typically expanded in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor 2 (bFGF) (Hovakimyan et al., 2006; Sanchez-Pernaute et al., 2001; Storch et al., 2001) and differentiated by removal of mitogens and addition of neurotrophins such as brain derived neurotrophic factor (BDNF) (Maciaczyk et al., 2008) and glial cell-line derived neurotrophic factor (GDNF) (Jin et al., 2005; Storch et al., 2001), ascorbic acid, cyclic adenosine monophosphate (cAMP) (Sanchez-Pernaute et al., 2001) or cytokines (Jin et al., 2005; Storch et al., 2001). Immortalized human mesencephalic cell lines have also been established (Donato et al., 2007; Lotharius et al., 2002; Villa et al., 2009), however DA neurons could only be generated and maintained after stable overexpression of Bcl-X L , an anti-apoptotic gene (Courtois et al., 2010). Surprisingly, none of the studies up to date have examined the use of region-specific developmentally appropriate morphogens for the expansion and differentiation of hVM cells. "
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    ABSTRACT: Human fetal midbrain tissue grafting has provided proof-of-concept for dopamine cell replacement therapy (CRT) in Parkinson's disease (PD). However, limited tissue availability has hindered the development and widespread use of this experimental therapy. Here we present a method for generating large numbers of midbrain dopaminergic (DA) neurons based on expanding and differentiating neural stem/progenitor cells present in the human ventral midbrain (hVM) tissue. Our results show that hVM neurospheres (hVMN) with low cell numbers, unlike their rodent counterparts, expand the total number of cells 3-fold, whilst retaining their capacity to differentiate into midbrain DA neurons. Moreover, Wnt5a promoted DA differentiation of expanded cells resulting in improved morphological maturation, midbrain DA marker expression, DA release and electrophysiological properties. This method results in cell preparations that, after expansion and differentiation, can contain 6-fold more midbrain DA neurons than the starting VM preparation. Thus, our results provide evidence that by improving expansion and differentiation of progenitors present in the hVM it is possible to greatly enrich cell preparations for DA neurons. This method could substantially reduce the amount of human fetal midbrain tissue necessary for CRT in patients with PD, which could have major implications for the widespread adoption of this approach.
    Neurobiology of Disease 08/2012; 49C(1):118-127. DOI:10.1016/j.nbd.2012.08.006 · 5.08 Impact Factor
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