TAT-PAX6 protein transduction in neural progenitor cells: a novel approach for generation of dopaminergic neurones in vitro.
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.
Spitere
Katie
K
Department of Neuroscience/Anatomy, Biosciences Research Institute, National University of Ireland Cork, College Road, Cork, Ireland.
Toulouse
André
A
O'Sullivan
David B
DB
Sullivan
Aideen M
AM
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D004298:Dopamine / Q000378:metabolism*; D004622:Embryo, Mammalian; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D008636:Mesencephalon / Q000166:cytology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects; D014158:Transcription, Genetic / Q000502:physiology; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000502:physiology*
D005136:Eye Proteins; D018398:Homeodomain Proteins; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605733:Pax6 protein, rat; D012097:Repressor Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine