RESUMEN
Neural stem cells (NSCs) have the potential to be used for the treatment of Parkinson's disease (PD), as they can be expanded, manipulated and differentiated in vitro to generate dopaminergic neurones which are suitable for transplantation. Since NSCs have a tendency to follow an astrocytic lineage after differentiation in vitro, researchers are investigating ways to induce a neuronal phenotype in these cells. In this study, the human immunodeficiency virus 1 (HIV-1) transactivator of transcription (TAT) protein transduction domain (PTD) system was used in an attempt to promote neuronal differentiation in rodent NSCs. A fusion protein that incorporated both the TAT PTD and the Pax6 protein (a determinant of neurogenesis) was created and added to the differentiation phase of embryonic day (E) 12 rat ventral mesencephalic (VM) neurosphere cultures. Subsequently, application of dopaminergic growth factors (GFs) was used in an attempt to induce the newly-generated neuronal progenitors to adopt a dopaminergic phenotype. In addition, a technique involving the differentiation of intact neurospheres (instead of the differentiation of neurosphere-derived dissociated cells) was investigated for its ability to promote dopaminergic neurogenesis. Immunocytochemical analysis of the differentiated neurosphere cultures indicated that both of these techniques had a significant effect on the emergence of dopaminergic neurones. Moreover, upon combination of these techniques, a further increase in dopaminergic neuronal generation was observed. Based on the findings of the present study, it is clear that NSCs are greatly influenced by their environment and that optimised in vitro conditions can support the potential of these cells to differentiate into dopaminergic neurones.
Asunto(s)
Dopamina/metabolismo , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Neuronas/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Células Madre/citología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Embrión de Mamíferos , Proteínas del Ojo/genética , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Mesencéfalo/citología , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Embarazo , Ratas , Ratas Sprague-Dawley , Proteínas Represoras/genética , Células Madre/efectos de los fármacos , Transcripción Genética/fisiología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Neural stem cells (NSCs) have been the focus of an intensive effort to direct their differentiation in vitro towards desired neuronal phenotypes for cell replacement therapies. It is thought that NSCs derived from older embryos have limited neurogenic capacity and are restricted towards an astroglial fate. This idea is largely based on studies that typically analysed NSC-derived progeny following one week of in vitro differentiation. In this report, the neurogenic capacity of older ventral midbrain (VM) NSCs was assessed. When the older NSCs were differentiated for three weeks, there were significant increases in the numbers of newly born neurons at 14 and 21 days, as assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. Therefore this study demonstrates that older NSCs retain significantly more neurogenic potential than was previously thought. These data have implications for NSC preparatory protocols and the choice of donor age for cell transplantation studies, and contributes to the understanding of NSC behaviour in vitro.