Direct generation of functional dopaminergic neurons from mouse and human fibroblasts

Abstract

Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors--Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a--that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.

Specification of functional neurons and glia from human pluripotent stem cells

“…In addition, although both adult and fetal neural stem cells in theory are able to be amplified using mitogens such as FGF2 or EGF, these neural stem cells always undergo undesired differentiation and tend to lose their potential to generate the appropriate type of cells for cell replacement therapy (Wright et al, 2006). Functional neurons (Vierbuchen et al, 2010;Ambasudhan et al, 2011;Marro et al, 2011;Pang et al, 2011;Qiang et al, 2011;Yoo et al, 2011), and lately subtype-specific neurons such as dopaminergic neurons (Caiazzo et al, 2011;Pfisterer et al, 2011) and motor neurons (Son et al, 2011) have been converted directly from other type of somatic cells of mouse and human by introducing pro-neuronal transcription factors or micro-RNA. The neurons generated through these procedures were called induced neurons (iNs) and the process was termed transdifferentiation.…”

Pioneer transcription factors in cell reprogramming

“…Of these factors, Ascl1 plays a central role to initiate transdifferentiation because Ascl1 alone is sufficient to induce immature iN cells, but Brn2 and Myt1l are not. Soon after, dopaminergic and motor neurons were generated from fibroblasts using different sets of transcription factors, but both sets included Ascl1 (Caiazzo et al 2011;Son et al 2011). Recently, Wernig and colleagues (Wapinski et al 2013) revealed a hierarchical mechanism governing the early stage of transdifferentiation to iNs: Ascl1 acts as a pioneer transcription factor to bind closed chromatin and recruit Brn2 to Ascl1 target sites (Table 1), and Brn2 is primarily required for the later stage of transdifferentiation by contributing to iN maturation.…”

Vers une régénération induite du système nerveux

“…Pour l'heure, la génération de neurones à partir de cellules pluripotentes induites, basée majoritairement, mais pas uniquement, sur l'utilisation de facteurs extrinsèques, reste plus efficace que la reprogrammation directe de fibroblastes en neurones, et permet d'obtenir une plus large gamme de sous-populations neuronales, notamment des neurones corticaux [15,16]. Le domaine plus récent de la génération directe de neurones à partir de fibroblastes, basée sur l'utilisation de facteurs intrinsèques (facteurs de transcription en majorité, mais aussi microARN), a néanmoins permis l'obtention de sous-types neuronaux d'intérêt thérapeutique à partir de cellules de patients [17][18][19]. L'identification d'un plus grand nombre de ces facteurs de transcription clés (ou de combinaisons de ces facteurs) d'une part, et des mécanismes moléculaires, notamment épigé-nétiques, qui sous-tendent ou à l'inverse bloquent le processus de reprogrammation directe d'autre part [20], permettront à terme d'élargir le panel de sous-types neuronaux dérivés de fibroblastes.…”