RESUMEN
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.
Asunto(s)
Diferenciación Celular , Neuronas Dopaminérgicas , Células Madre Embrionarias Humanas , Proteínas con Homeodominio LIM , Mesencéfalo , Factores de Transcripción , Humanos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/citología , Proteínas con Homeodominio LIM/metabolismo , Proteínas con Homeodominio LIM/genética , Mesencéfalo/citología , Mesencéfalo/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Tipificación del Cuerpo/genética , Tirosina 3-Monooxigenasa/metabolismo , Tirosina 3-Monooxigenasa/genética , Animales , Regulación del Desarrollo de la Expresión GénicaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
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.
