Transplantation of genetically engineered cells into the CNS offers immense

Progress in stem cell biology research is enhancing our ability to generate specific neuron types for basic and applied studies and to design new treatments for neurodegenerative diseases. In the case of Parkinson's disease (PD), alternative human dopaminergic (DAergic) neurons other than primary fetal tissue do not yet exist. One possible source could be human neural stem cells (hNSCs), although the yield in DAergic neurons and their survival are very limited.In this study, we found that Bcl-X L enhances (one-to-two orders of magnitude) the capacity for spontaneous dopaminergic differentiation of hNSCs, which then exceeds that of cultured human ventral mesencephalic tissue. Bcl-X L also enhanced total neuron generation by hNSCs, but to a lower extent. Neuronal phenotypes other than DA were not affected by Bcl-X L , indicating an exquisitely specific effect on DAergic neurons. In vivo, grafts of Bcl-X L -overexpressing hNSCs do generate surviving human TH ϩ neurons in the adult rat 6-OHdopamine lesioned striatum, something never seen when naive hNSCs were transplanted. Most of the data obtained here in terms of the effects of Bcl-X L are consistent with an enhanced survival type of mechanism and not supportive of induction, specification, or proliferation of DAergic precursors.From this in vitro and in vivo evidence, we conclude that enhancing Bcl-X L expression is important to obtain human DAergic neurons from hNSCs. These findings may facilitate the development of drug-screening and cell-replacement activities to discover new therapeutic strategies for PD.

Understanding basic processes of human neural stem cell (hNSC) biology and differentiation is crucial for the development of cell replacement therapies. Bcl-X L has been reported to enhance dopaminergic neuron generation from hNSCs and mouse embryonic stem cells. In this work, we wanted to study, at the cellular level, the effects that Bcl-X L may exert on cell death during differentiation of hNSCs, and also on cell fate decisions and differentiation. To this end, we have used both v-myc immortalized (hNS1 cell line) and non-immortalized neurosphere cultures of hNSCs. In culture, using different experimental settings, we have consistently found that Bcl-X L enhances neuron generation while precluding glia generation. These effects do not arise from a glia-to-neuron shift (changes in fate decisions taken by precursors) or by only cell death counteraction, but, rather, data point to Bcl-X L increasing proliferation of neuronal progenitors, and inhibiting the differentiation of glial precursors. In vivo, after transplantation into the aged rat striatum, Bcl-X L overexpressing hNS1 cells generated more neurons and less glia than the control ones, confirming the results obtained in vitro. These results indicate an action of Bcl-X L modulating hNSCs differentiation, and may be thus important for the future development of cell therapy strategies for the diseased mammalian brain. The efficient generation of human neurons and glia from stem cells is the object of intense investigation, in the context of basic and preclinical cell replacement research. 1,2 Different means of genetic and epigenetic manipulations of stem cell cultures are being tested, aiming at enhancing their ability to generate the desired cell types. [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.Bcl-X L is the most potent antiapoptotic protein among Bcl-2 family members, both in vitro and in vivo. 11-14 More specifically, Bcl-X L is essential for neuronal survival during brain development and in the adult central nervous system (CNS) (knockout mice and gene-expression studies 15,16 ). In addition to its antiapoptotic role, recent studies have described new roles of Bcl-X L in cell physiology -effects on Ca 2 þ homeostasis and gene expression 17 and synaptic transmission regulation 18 -under not necessarily apoptotic conditions. Several other studies have also described the role of Bcl-X L in the control of cell cycle, 19,20 a process well known to be tightly linked to progression of precursor cells toward neuronal and glia differentiation. 21,22 In this work, we aimed...

Calcium release in response to ATP or forced Bcl-XL expression increases the length of the cell cycle and increases the intermediate progenitors and the neuron population. The effect of calcium signaling on cell cycle control is mediated by the p53–p21 pathway.