Generating Human iPSC-Derived Astrocytes with Chemically Defined Medium for In Vitro Disease Modeling.

To better understand and model neurological, in particular neurodegenerative diseases, human induced pluripotent stem cells (hiPSCs) offer a great source for generation of neural cells. We provide a protocol for the differentiation of hiPSc-derived astrocytes in vitro. This protocol not only is chemically defined, that is, it does not use serum, but also allows for the expansion of astrocyte progenitor cells and mature astrocytes. Large batches of hiPSc-derived astrocytes can be stored and used for defined in vitro disease models.

A high-content screening assay for the Nogo receptor based on cellular Rho activation.

Rho family proteins can coordinate multiple signaling pathways through their ability to regulate both gene transcription and the actin cytoskeleton. With respect to the neuronal Nogo receptor (NgR), recent data assign a key role for the GTPase Rho in the control of cellular responses leading to actin cytoskeletal rearrangements and finally resulting in axonal outgrowth inhibition and growth cone collapse in the adult human central nervous system. In order to evaluate potential NgR antagonists, human embryonic kidney 293 cells stably overexpressing RhoA in the absence or presence of NgR have been generated. RhoA activation induced by stimulation with the alkaline phosphatase-tagged NgR ligand Nogo66 (AP-Nogo66) was confirmed by affinity-precipitation of the GTPase with the Rho-binding domain from Rhotekin. As this pull-down assay is not applicable to a higher-throughput format, a cellular Rho GTPase activation assay strategy based on the ability of Rho to regulate the actin cytoskeleton was developed. Stimulation with L-alpha-lysophosphatidic acid (LPA), a Rho activator acting through the ubiquitiously expressed LPA receptors, induced significant cytoskeletal rearrangement resulting in cell contraction in all RhoA-overexpressing cell lines. In contrast, stimulation with AP-Nogo66 resulted in Rho-dependent cell contraction with a similar time course only in the NgR-expressing cell line. Moreover, the NgR-induced Rho-dependent morphological changes could be analyzed and quantified with customary image analysis software. In conclusion, the cytoskeletal rearrangement assay is amenable to automated high-content screening and has the potential to eliminate major technical bottlenecks of the pull-down assay. The increased throughput of the cellular GTPase activation assay compared with the biochemical method should facilitate the evaluation of compounds that modulate the actin cytoskeleton through Rho.