The Hippo/YAP pathway serves as a major integrator of cell surface-mediated signals and regulates key processes during development and tumorigenesis. The neural crest is an embryonic tissue known to respond to multiple environmental cues in order to acquire appropriate cell fate and migration properties. Using multiple in vitro models of human neural development (pluripotent stem cell-derived neural stem cells; LUHMES, NTERA2 and SH-SY5Y cell lines), we investigated the role of Hippo/YAP signaling in neural differentiation and neural crest development. We report that the activity of YAP promotes an early neural crest phenotype and migration, and provide the first evidence for an interaction between Hippo/YAP and retinoic acid signaling in this system.

Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV)-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC) 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72%) and glial cells (9% astrocytes, 6% oligodendrocytes). Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts). Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial) pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside the cells at all time points.

Reprogramming of somatic cells to induced pluripotent stem cells (iPSC) and subsequent differentiation opened up the opportunity of deriving cell types in vitro which (like neurons) had a very restricted accessibility in the past. However, cell culture protocols for iPSC reprogramming, neural induction and differentiation tend to be labor and time intensive, costly and commonly depend on viral vector delivery. Single-step reprogramming to induced neural stem cells (iNSC) avoids many of the necessary intermediate steps of the aforementioned method but yields a cell type that proliferates over longer time spans and readily differentiates to mature neurons when required. Here we describe a plasmid based reprogramming protocol employing defined, commercially available components for induction and proliferation of iNSC, followed by a defined, small molecule based differentiation step toward mature neurons. The described method might be of particular interest for groups with limited resources and/or restricted access to higher biosafety level facilities required for viral transduction, but also for groups requiring a high throughput for dealing with large numbers of cell lines.

Interneurons comprise a minority of the striatal neuronal population of roughly 5%. However, this heterogeneous population is of particular interest as it fulfills an important relay function in modulating the output of the only type of striatal projection neurons, i.e., the medium spiny neuron (MSN).One subtype of this heterogenous group, the cholinergic interneuron, is of particular scientific interest as there is a relevant body of evidence from animal models supporting its special significance in the disease process. The development of protocols for directed differentiation of human pluripotent stem cells (PSC) into striatal interneurons provides a unique opportunity to derive in vitro those cell types that are most severely affected in dystonia.In this review we first aim to give a concise overview about the normal function of striatal interneurons and their dysfunction in dystonia in order to identify the most relevant interneuronal subtype for the pathogenesis of dystonia. Secondly we demonstrate how knowledge about the embryonic development of striatal interneurons is of particular help for the development of differentiation protocols from PSC and by this depict potential ways of deriving in vitro disease models of dystonia. We furthermore address the question as to whether cell replacement therapies might represent a beneficial approach for the treatment of dystonia.

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