The possibility of generating different subtypes of motor neurons is particularly relevant for modeling modern motor neuron diseases such as amyotrophic lateral sclerosis. This method allows the acquisition of cell populations that are highly rigid for motor neurons with a spinal or cranial identity in a short time frame and without the purification step. The generation of human motor neurons from induced pluripotent stem cells or iPSC under simplified culture condition may facilitate drug screening for motor neuron diseases.
The inducible expression of programming transcription factor can be used to generate another cell types such as neuronal, skeletal muscle, and glial cells from an iPSC repository. For NIL and NIP iPSC line generation, rinse the human iPSC culture with calcium-and magnesium-free PBS before treating the cells with cell dissociation reagent for five to 10 minutes at 37 degrees Celsius. When the cells have begun to detach from the culture container, use a P1000 pipette to gently manually dissociate the cells three to four times.
Transfer the cells to a 15 milliliter tube and bring the final volume up to 10 milliliters with fresh PBS without calcium and magnesium for counting. Collect one times 10 to the sixth cells by centrifugation and resuspend the pellet in 100 microliters of buffer R from the electroporation kit. Add 4.5 micrograms of transposable vector plasma DNA and 0.5 micrograms of the piggyBac transposase plasma DNA for transfection.
Transfect with the cell electroporation system according to the manufacturer's instructions with the indicated parameters. Then, seed the cells in human iPSC medium supplemented with 10 micromolar ROCK inhibitor Y-27632 in a six millimeter matrix-coated dish. Two days after the transfection, add 5 micrograms per milliliter of blasticidin to the culture medium keeping the cells in the antibiotic for at least seven to 10 days to counterselect the cells that have not integrated the transgenes.
At the end of the incubation, maintain the stably transfected cells as a mixed population composed of cells with different numbers of transgenes and different integration sites or isolate single clones. Prepare an additional dish to allow the effective expression of the transgenes upon one microgram per milliliter of doxycycline induction to be assessed by RT-PCR with transgene-specific primers for neurogenin-2 as previously described. At this stage, stocks of the novel NIL and NIP iPSC lines should also be frozen in freezing medium for human iPSCs.
For motor neuron differentiation, dissociate the stably transfected cell cultures with dissociation reagent as demonstrated to allow the cells to be collected in a 15 milliliter tube containing five volumes of DMEM/F12 medium. After centrifugation, resuspend the cells in human iPSC medium supplemented with micromolar ROCK inhibitor for counting. Seed the cells on matrix-coated dishes at a 6.25 times 10 the fourth cells per centimeter squared density.
For the next two days, replace the supernatants with fresh differentiation medium supplemented with doxycycline. On day two, change the medium to neurobasal B27 medium supplemented with gamma secretase inhibitor, vascular endothelial growth factor receptor inhibitor, and doxycyline. On day five, dissociate the cells as demonstrated and resuspend the detached cells in four milliliters of DMEM/F12 medium for counting.
Pipetting is important for a complete dissociation of the cells. Freeze an aliquot of the motor neuron progenitors in cell-freezing medium according to the manufacturer's instructions and collect the rest of the cells by centrifugation. Resuspend the pellet in neuronal medium supplemented with 10 micromolar ROCK inhibitor.
Seed the cells on polyornithine laminin-coated microslide supports with polymer cover slips at a one times 10 to the fifth cells per centimeter squared density. On day six, carefully replace the medium with fresh neuronal medium without inhibitor for culture until downstream analysis without detaching the cells from the support surface. A uniform expression of the pluripotency marker octamer-binding transcription factor four or OTC4 can be observed in differentiating cell cultures on day zero in the absence of pan neuronal marker neuron-specific class three beta-tubulin TUJ1 positivity.
On day three, a strong decrease in the number of OCT4-positive cells is evident. That is mirrored by the expression of TUJ1 in a subset of differentiating iPSCs. At day five, no expression of OCT4 is observed in the population which shows a consistent expression of TUJ1 and an acquired neuronal morphology.
Immunostaining analysis seven days post replating of the motor neuron progenitor cells reveals a uniform expression of TUJ1 and the mature motor neuron marker choline acetyltransferase. iPSC NIL-derived neurons successfully display voltage-dependent sodium currents and voltage-dependent potassium currents. 80%of the iPSC NIL-derived motor neurons clamped in current clamp modality are able to trigger spike trains when injected with a current pulse of plus 60 picoamps or more.
The minimum current required to elicit repetitive firing in more than 50%of the recorded cells is plus 40 picoamps with a spike threshold of approximately minus 38 millivolts and an average firing frequency at plus 40 picoamps of about eight hertz, suggesting that iPSC NIL-derived spinal motor neurons exhibit functional properties typical of mature neurons. Spinal and cranial motor neurons can be derived from controlled iPSCs or iPSCs carrying pathological mutations and that these cells can be used as thesis models or for drug screening. Our method can be used to help in understanding why different motor neuron units are differentially affected by ALS.
