RESUMO
The differentiation of human pluripotent stem cells into ventral mesencephalic dopaminergic (DA) fate is relevant for the treatment of Parkinson's disease. Shortcuts to obtaining DA cells through direct reprogramming often include forced expression of the transcription factor LMX1A. Although reprogramming with LMX1A can generate tyrosine hydroxylase (TH)-positive cells, their regional identity remains elusive. Using an in vitro model of early human neural tube patterning, we report that forced LMX1A expression induced a ventral-to-dorsal fate shift along the entire neuroaxis with the emergence of roof plate fates despite the presence of ventralizing molecules. The LMX1A-expressing progenitors gave rise to grafts containing roof plate-derived choroid plexus cysts as well as ectopically induced TH-positive neurons of a forebrain identity. Early activation of LMX1A prior to floor plate specification was necessary for the dorsalizing effect. Our work suggests using caution in employing LMX1A for the induction of DA fate, as this factor may generate roof plate rather than midbrain fates.
Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos , Células-Tronco Embrionárias Humanas , Proteínas com Homeodomínio LIM , Mesencéfalo , Fatores de Transcrição , Humanos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/citologia , Proteínas com Homeodomínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Padronização Corporal/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Animais , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The study of brain development in humans is limited by the lack of tissue samples and suitable in vitro models. Here, we model early human neural tube development using human embryonic stem cells cultured in a microfluidic device. The approach, named microfluidic-controlled stem cell regionalization (MiSTR), exposes pluripotent stem cells to signaling gradients that mimic developmental patterning. Using a WNT-activating gradient, we generated a neural tissue exhibiting progressive caudalization from forebrain to midbrain to hindbrain, including formation of isthmic organizer characteristics. Single-cell transcriptomics revealed that rostro-caudal organization was already established at 24 h of differentiation, and that the first markers of a neural-specific transcription program emerged in the rostral cells at 48 h. The transcriptomic hallmarks of rostro-caudal organization recapitulated gene expression patterns of the early rostro-caudal neural plate in mouse embryos. Thus, MiSTR will facilitate research on the factors and processes underlying rostro-caudal neural tube patterning.