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

Emma G Seiz,Elise T Courtois,Carlos Bueno,Isabel Liste,Alberto Martínez-Serrano,Milagros Ramos,Claudia G Castillo

doi:10.1074/jbc.m109.054312

Emma G Seiz, Elise T Courtois + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m109.054312

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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.

Highlights

Human neural stem cells derived from the ventral mesencephalon (VM) are powerful research tools and candidates for cell therapies in Parkinson disease
Previous transplantation studies established that the generation of functional substantia nigra pars compacta (SNpc) dopaminergic neuron (DAn) in vivo was highly dependent on the regional tissue origin, the VM being the optimal region [6], and that only DAn with SNpc properties were able to reinervate the striatum and induce a therapeutic effect [7]
Serial sections were used for immunohistochemistry with polyclonal antibodies against TH (1:1000; Chemicon), DAT (1:1000; Chemicon), and Dcx (1:1000; Santa Cruz Biotechnology) and monoclonal antibodies against human GFAP (1:1000; Sternberger), human neuron-specific enolase (1:1000; Chemicon), or human nuclei (1:100; Chemicon)

Summary

Introduction

Human neural stem cells derived from the ventral mesencephalon (VM) are powerful research tools and candidates for cell therapies in Parkinson disease. 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 enhanced by Bcl-XL. These effects on neurogenesis occur in parallel to a decrease in glia generation. Human neural stem cells (hNSCs) derived from the developing and adult central nervous system were initially used, but they were inefficient for DAn generation [3] Another stem cell source, the embryonic stem cells, required long and difficult differentiation protocols as well as neuronal and DAn progenitor selection to obtain high amounts of DAn [4, 5]. Bcl-XL (basal cell lymphoma-extra large) belongs to the Bcl-2 (B-cell lymphoma 2) protein family, playing an important antiapoptotic role in mammals [16], during central nervous system development [17], and modulating neuronal differentiation (18 –21)

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