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.
    Neurobiology of Disease 08/2012; 49C:118-127. DOI:10.1016/j.nbd.2012.08.006 · 5.20 Impact Factor
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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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    ABSTRACT: Parkinson's disease (PD) motor symptoms are caused by the progressive degeneration of ventral mesencephalic (VM) dopaminergic neurons (DAn) in the Substantia Nigra pars compacta (SNpc). Cell replacement therapy for PD is based on the concept that the implantation of DAn in the striatum can functionally restore the dopamine levels lost in the disease. In the current study we have used an immortalized human VM neural stem cell line (hVM1) that generates DAn with the A9 phenotype. We have previously found that the forced expression of Bcl-X(L) in these cells enhances DAn generation and improves, short-term, d-amphetamine-induced rotation after transplantation in the 6-OH-DA rat model of PD 2-month post-grafting. Since functional maturation of human A9 DAn in vivo requires long survival times, in the present study we investigated the behavioral amelioration induced by the transplantation of these precursors (naïve and Bcl-X(L)-modified) in the striatum of Parkinsonian rats for up to 5 months. The main findings observed are an improvement on drug-induced behaviour and importantly, in spontaneous behavior tests for both cell-transplanted groups. Finally, we have also tested whether the grafts could ameliorate cognitive performance in PD, in addition to motor deficits. Significant difference was observed for T-maze alternation test in the cell-transplanted animals as compared to sham operated ones. To our knowledge, this is the first report showing an amelioration in spontaneous motor behavior and in cognitive performance in Parkinsonian animals after receiving human VM neural stem cell grafts. Histological studies confirmed that the grafts generated mature dopaminergic cells.
    Behavioural brain research 04/2012; 232(1):225-32. DOI:10.1016/j.bbr.2012.04.020 · 3.39 Impact Factor
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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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    ABSTRACT: Human adult olfactory epithelium contains neural progenitors (hONPs) which replace damaged cellular components throughout life. Methods to isolate and expand the hONPs which form neurospheres in vitro have been developed in our laboratory. In response to morphogens, the hONPs differentiate along several neural lineages. This study optimized conditions for the differentiation of hONPs towards dopaminergic neurons. The hONPs were treated with Sonic hedgehog (Shh), in the presence or absence of retinoic acid (RA) and/or forskolin (FN). Transcription factors (nurr1, pitx3 and lmx1a) that promote embryonic mouse or chicken dopaminergic development were employed to determine if they would modulate lineage restriction of these adult human progenitors. Four expression vectors (pIRES-pitx3-nurr1, pLN-CX2-pitx3, pLNCX2-nurr1 and pLNCX2-lmx1a) were transfected into the hONPs, respectively. Transcription factor expression and the rate-limiting enzyme in dopamine synthesis tyrosine hydroxylase (TH) were detected in the transfected cells after 4 month-selection with G418, indicating transfected hONPs were stably restricted towards a dopaminergic lineage. Furthermore, a dopamine enzyme immunoassay (EIA) was employed to detect the synthesis and release of dopamine. The most efficient transfection paradigm was determined. Several neurotrophic factors were detected in the pre-transfected hONPs which have potential roles in the maintenance, survival and proliferation of dopaminergic neurons. Therefore the effect of transfection on the neurotrophin synthesis was examined. Transfection did not alter synthesis. The use of olfactory progenitors as a cell-based therapy for Parkinson’s disease (PD) would allow harvest without invasive surgery, provide an autologous cell population, eliminate need for immunosuppression and avoid the ethical concerns associated with embryonic tissues. This study suggests that specific transcription factors and treatment with morphogens can restrict human adult olfactory-derived progenitors to a dopaminergic neuronal lineage. Future studies will evaluate the utility of these unique cells in cell-replacement paradigms for the treatment of PD like animal models.
    Stem Cell Discovery 10/2011; 1(3):29-43. DOI:10.4236/scd.2011.13004
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