Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons

Abstract

Nurr1 is a transcription factor critical for the development of midbrain dopaminergic (DA) neurons. This study modified mouse embryonic stem (ES) cells to constitutively express Nurr1 under the elongation factor-1alpha promoter. The Nurr1-expression in ES cells lead to up-regulation of all DA neuronal markers tested, resulting in about a 4- to 5-fold increase in the proportion of DA neurons. In contrast, other neuronal and glial markers were not significantly changed by Nurr1 expression. It was also observed that there was an additional 4-fold increase in the number of DA neurons in Nurr1-expressing clones following treatment with Shh, FGF8 and ascorbic acid. Several lines of evidence suggest that these neurons may represent midbrain DA neuronal phenotypes; firstly, they coexpress midbrain DA markers such as aromatic L-amino acid decarboxylase, calretinin, and dopamine transporter, in addition to tyrosine hydroxylase and secondly, they do not coexpress other neurotransmitters such as GABA or serotonin. Finally, consistent with an increased number of DA neurons, the Nurr1 transduction enhanced the ability of these neurons to produce and release DA in response to membrane depolarization. This study demonstrates an efficient genetic manipulation of ES cells that facilitates differentiation to midbrain DA neurons, and it will serve as a framework of genetic engineering of ES cells by key transcription factor to regulate their cell fate.

ES cell-derived renewable and functional midbrain dopaminergic progenitors

“…1 B-D). To test whether these mDA NPs can be selected from ESCs, we used an embryoid body (EB)-based five-stage in vitro differentiation procedure (8) (Fig. S1A), which is known to well recapitulate early embryonic development.…”

Nodal-dependant Cripto signaling in ES cells: from stem cells to tumor biology

“…However, this issue is still debated; in fact, besides exogenous inhibitory factors (either soluble or paracrine), recent data suggest that cell adhesion may also have a key role in neural differentiation of ES cells (Watanabe et al, 2005). Interestingly, it has been recently described that Cripto À/À ES cells show enhanced neurogenesis compared to wild-type ES cells (Sonntag et al, 2005) following the five stage-neuronal differentiation protocol, a method described for efficient generation of dopaminergic and serotonergic neurons from ES cells (Lee et al, 2000;Chung et al, 2002). However, multilineage cell types have also been found in Cripto À/À ES cells, thus suggesting that loss of function of Cripto is not sufficient to result in a 'default' neural pathway, at least in this in vitro paradigm (Sonntag et al, 2005).…”

Des cellules souches embryonnaires humaines pour le traitement de la maladie de Parkinson ?

“…Cette activité membranaire est généralement sous le contrôle du cytosquelette d'actine, lui-même réglé par les petites protéines de la famille des Rho GTPases [2,3]. Deux études récemment publiées dans Science, l'une menée par le groupe de C. Watts [4] et la seconde réalisée dans notre laboratoire [5], ont permis de mieux comprendre le rôle de la régulation du cytosquelette d'actine au cours des différentes phases de la pré-sentation antigénique. L'équipe de C. Watts vient de montrer que l'activation des TLR à la surface des cellules dendritiques immatures conduit à une augmentation de leur capacité de macropinocytose des antigènes et, par conséquent, à une augmentation de la présentation antigénique par les molé-cules du CMH.…”

“…Les cellules ES d'origine humaine constituent donc une source idéale de greffons pour des applications cliniques de thérapie cellulaire, mais seulement si l'on sait diriger efficacement leur croissance et leur différencia-tion vers les types cellulaires souhaités. Depuis quelques années, plusieurs équipes ont réussi à différencier des cellules ES murines, génétiquement modifiées ou non, en neurones dopaminergiques [2][3][4][5]. Ces neurones ont, chez le rat parkinsonien, un potentiel thérapeu-tique proche de celui de tissus foetaux [6,7].…”