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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

ABSTRACT 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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    • "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.
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    • "In any case, hVM1-Bcl-X L grafted animals seem to achieve a better performance in some behavioral tests (drug-induced rotation, paw reaching, T-maze rewarded alternation tests) from the second month than control cells grafted ones. This could be due to either a faster or enhanced maturation of the transplant in the case of hVM1-Bcl-X L cells or to a higher level of vesicular dopamine release from the TH + , DA-ergic neurons present in the transplant [2]. Future studies will be focused on determining the therapeutic effect on both biochemical and behavioural grounds of hVM1-Bcl- X L cells at longer time points (up to 12 months) and using a nude rat model to avoid possible side effects of the administration of CsA. "
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    • "38 were dopaminergic as determined by dopamine E.I.A. These results suggest that the transcription factors, Pitx3 and Nurr1, not only function as a dopaminergic promoters in chick, mouse, or human embryonic cells [41] [68] [71] [76], but also can participate in dopamine production in adult human olfactory-derived progenitors. Based on previous studies which focused on the regulatory function of Pitx3 and Nurr1 in dopaminergic neuron promotion [63] [68] [70] [72] [74] [77] and the studies described in this manuscript, we hypothesized that Pitx3 and Nurr1 may collaborate to induce a higher efficiency of dopamine production in midbrain DA neuron maturation. "
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