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  • Protocol for the Standardized Generation of Forward Programmed Cryopreservable Excitatory and Inhibitory Forebrain Neurons.

Protocol for the Standardized Generation of Forward Programmed Cryopreservable Excitatory and Inhibitory Forebrain Neurons.

STAR protocols (2020-10-29)
Michael Peitz, Tamara Krutenko, Oliver Brüstle
ABSTRACT

This protocol describes a highly standardized pipeline for transcription factor-mediated forward programming of human pluripotent stem cells into highly enriched glutamatergic or GABAergic neurons followed by a cryopreservation step that enables the generation of large quality-controlled batches. This approach is particularly useful for reducing interexperimental variability in the context of collaborative studies across different locations and time points. For complete details on the use and execution of this protocol, please refer to Meijer et al. (2019) and Rhee et al. (2019).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Sigma-Aldrich
Poly-L-ornithine hydrobromide, mol wt 30,000-70,000
Sigma-Aldrich
Cytosine β-D-arabinofuranoside, crystalline, ≥90% (HPLC)
Sigma-Aldrich
D-(+)-Trehalose dihydrate, ≥99% (HPLC), from Saccharomyces cerevisiae
Sigma-Aldrich
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, 1-2 mg/mL in Tris-buffered saline, 0.2 μm filtered, BioReagent, suitable for cell culture