This article describes a protocol for differentiating human pluripotent stem cells (hPSCs) into neural progenitor cells (NPCs) using a transgene under an antibiotic-inducible promoter. The process involves the use of small molecules to promote cell proliferation and differentiation.
Take a multi-well plate coated with an extracellular matrix, or ECM.
Into separate wells, introduce non-transgenic and transgenic human pluripotent stem cells, or hPSCs, in a culture medium.
Transgenic hPSCs carry a transgene under an antibiotic-inducible promoter. The transgene encodes a transcription factor that induces neural differentiation.
Incubate to facilitate cell adhesion to the ECM. Small molecules in the medium prevent apoptosis, promoting cell proliferation.
Add a detachment solution to dissociate the cells; add the medium to stop dissociation, and then transfer them to another ECM-coated microplate to allow cell proliferation.
Introduce a neural differentiation medium containing small molecules onto the non-transgenic cells and the inducer antibiotic onto the transgenic cells.
The small molecules induce non-transgenic cells to differentiate into neural progenitor cells or NPCs. In the transgenic cells, the antibiotic-induced expression of the transcription factor activates the genes required for differentiation into NPCs.
Maintain the NPCs in culture for downstream applications.
For the generation of non-transgenic neuronal progenitors and neurons, or hNeurons, see day 14 hPSC-derived neural progenitors into ECM-coated six-well plates with 2 milliliters of neural medium and Y27632 per well.
If working with doxycycline-inducible iNeurons, add neural medium containing doxycycline when the cells are about 35% confluent to induce differentiation and activate gene expression. After two days, feed transgenic and non-transgenic lines with 2 milliliters of neural media per well per day. Continue until the cells are ready to lift at 70% confluency.
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