Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays
Transcription
Take transduced human-induced pluripotent stem cells or hiPSCs, overexpressing neurogenin-2, driven by the antibiotic doxycycline-inducible system.
Transfer them to the adhesion protein-coated active electrode areas in the wells of a multi-well microelectrode array.
Allow hiPSCs to attach to the coated active electrode areas.
Add media supplemented with doxycycline, which induces neurogenin-2 overexpression, a neural-specific transcription factor.
Neurogenin-2 triggers molecular pathways, and leads to hiPSC differentiation into neural precursor cells, while media constituents aid cell survival.
Introduce astrocytes to the micro-electrode array wells, where they adhere to the electrode's active area.
Replace media with neurobasal media containing neurogenic growth factors, doxycycline, and a cell growth inhibitor.
The inhibitor prevents astrocyte proliferation and eliminates undifferentiated hiPSCs.
Over time, sustained neurogenin-2 expression and growth factors drive neural precursor cell differentiation into neurons.
Further, replace media with neurobasal media containing serum for astrocyte survival.
Astrocytes secrete growth factors promoting neuron maturation and synapse formation, forming a neuronal network.
