EnhancedIn VitroMidbrain Dopamine Neuron Differentiation, Dopaminergic Function, Neurite Outgrowth, and 1-Methyl-4-Phenylpyridium Resistance in Mouse Embryonic Stem Cells Overexpressing Bcl-XL

Abstract

Embryonic stem (ES) cells provide a potentially unlimited source of specialized cells for regenerative medicine. The ease of inducing stable genetic modifications in ES cells allows for in vitro manipulations to enhance differentiation into specific cell types and to optimize in vivo function of differentiated progeny in animal models of disease. We have generated mouse ES cells that constitutively express Bcl-XL, an antiapoptotic protein of Bcl-2 family. In vitro differentiation of Bcl-XL overexpressing ES (Bcl-ES) cells resulted in higher expression of genes related to midbrain dopamine (DA) neuron development and increased the number of ES-derived neurons expressing midbrain DA markers compared with differentiation of wild-type ES cells. Moreover, DA neurons derived from Bcl-ES cells were less susceptible to 1-methyl-4-phenylpyridium, a neurotoxin for DA neurons. On transplantation into parkinsonian rats, the Bcl-ES-derived DA neurons exhibited more extensive fiber outgrowth and led to a more pronounced reversal of behavioral symptoms than wild-type ES-derived DA neurons. These data suggest a role for Bcl-XL during in vitro midbrain DA neuron differentiation and provide an improved system for cell transplantation in a preclinical animal model of Parkinson's disease.

Bcl-XL modulates the differentiation of immortalized human neural stem cells

“…19,20 The analysis of the proliferation of neural precursor cells (Nestin þ -BrdU þ ), glia progenitor/precursor cells (GFAP þ -BrdU þ and vimentin þ -BrdU þ ), and neuronal progenitors (b-III-tubulin þ -BrdU þ ) showed: Firstly, the quantification of BrdU þ or Nestin þ cells confirmed the expected parallel temporal profile for both markers and Bcl-X L increased the numbers of stained cells during the whole of the differentiation period. These findings are consistent with results from studies using mES cells, 7 and bax-bak-deficient mice which displayed an increased pool of progenitor cells in the subventricular zone. 31 Second, quantification of double BrdU þ -Nestin þ cells indicated that most of the proliferating cells (BrdU þ ) were Nestin þ , while approximately one half of the Nestin þ cells incorporated BrdU.…”

“…In conclusion, in the context of hNSCs research, amounting data indicate that Bcl-X L plays a major role in modulating the generation of neurons from both rodent-and human-derived stem cells, [7][8][9][10] and present work. Here, we contribute data indicating a more general role of Bcl-X L , controlling the balance between the generation of neurons and glia from differentiating immortalized and non-immortalized hNSCs.…”

“…Similarly, mouse ES cells overexpressing Bcl-X L showed a better reinnervation of the striatum of 6-OH-DA rats. 7 In the present work, it is important to note that Bcl-X L did enhance hNSCs survival and neuronal differentiation, when implanted in a non-neurogenic site and, furthermore, impoverished, due to aging, for the supply of trophic factors such as brain-derived neurotrophic factor. 35 Therefore, Bcl-X L seems to be able to override some of the tissue-imposed limitations for in vivo neuron generation by hNSCs.…”

“…[3][4][5][6] In previous studies, we and others have demonstrated that Bcl-X L overexpression has a major impact on the generation of dopaminergic neurons from human neural stem cells (hNSCs) and mouse embryonic stem cells (mES). 7,8 Also, recent data from conditional Bcl-X L mutant mice add support to these observations. 9,10 In the present study, we are extending these observations and analyzing in detail the effects of Bcl-X L on hNSCs differentiation, focusing on precursor cell fate choices, cell death, and precursor proliferation.…”

Generation of Functional Dopamine Neurons from Neural Precursor Cells Isolated from the Subventricular Zone and White Matter of the Adult Rat Brain Using Nurr1 Overexpression

“…Generation of 6-hydroxydopamine-lesioned PD model rats and an amphetamine-induced rotation test were performed as described previously [24]. Adult rat SVZ NP cells under bFGF-proliferation were harvested 2 days after Nurr1 transduction and dissociated into single cells in CMF-HBSS as described above.…”