This study outlines a method for differentiating human-induced pluripotent stem cells (hiPSCs) into neurons using neurogenin-2. The process involves the use of microelectrode arrays to facilitate cell attachment and differentiation.
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.
For the six-well MEAs, dilute the cells to obtain a cell suspension of 7.5 x 105 cells per milliliter. Aspirate the diluted laminin from the six-well MEAs. For the six-well MEAs, plate the cells by adding 100 microliters of cell suspension to the active electrode area in each well.
Next, place the six-well MEAs in a humidified 37 degrees Celsius incubator. After two hours, carefully add 500 microliters of the prepared E8 medium to each well of the six-well MEAs. Subsequently, place the six-well MEAs overnight in a humidified 37 degrees Celsius incubator.
On day three, warm 0.05% Trypsin-EDTA to room temperature. Warm DPBS in DMEM/F-12 with 1% penicillin-streptomycin to 37 degrees Celsius. Then, aspirate the spent medium of the rat astrocyte culture. Wash the culture by adding 5 milliliters of DPBS, and swish it around gently.
Afterward, aspirate DPBS and add 5 milliliters of 0.05% Trypsin-EDTA. Swish the Trypsin-EDTA around gently. Then, incubate the cells in a humidified 37 degrees Celsius incubator for 5 to 10 minutes. Subsequently, check under the microscope to examine whether the cells are detached.
Detach the last cells by hitting the flask a few times. Then, add 5 milliliters of DMEM/F-12 to the flask. Triturate the cells gently inside the flask with a 10-milliliter pipette. Afterward, collect the cell suspension in a 15-milliliter tube. Spin the tube at 200 times g for eight minutes.
After that, aspirate the supernatant and resuspend the cells in 1 milliliter of DMEM/F-12. Determine the number of cells per milliliter using a hemocytometer.
Next, add 7.5 x 104 astrocytes to each well of the six-well MEAs, and add 2 x 104 astrocytes to each well of the 24-well plate. Incubate the cells overnight in a humidified 37 degrees Celsius incubator.
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