The overall goal of this protocol for the 3D human iPSC serum free embryoid bodies, is to provide an in-vitro system that approximates human early cortical development, which can be used for drug discovery. This method can help answer key questions in the stem cell field such as, how neurons derive from iPSCs, form functional networks in 3D tissue. The main advantage of this technique is that it allows us to model aspects of cortical development in a dish, from human tissue containing the genetics of diseased individuals, thus the system serves as a cell based platform for drugs screening that is scalable and can be used to study many different neurobiological disorders.
Although this method can provide insight into neurobiological or neuro development disorders, it can also be applied to other systems, such as those that focus on models of neuroimmune function or neural information. For this protocol, begin with establishing fetal cells. To establish this culture, plate 600, 000 fetal cells into each well of a six well plate, with 300 milliliters of medium per well.
After culturing the fetal cells for 48 hours, replace the medium. Incubate the plates for an hour, while preparing the stem cells. Thaw the stem cells in a 37 degrees celsius water bath.
Once thawed, transfer the cells to a 15 milliliter conical tube and fill the tube to five milliliters with culture medium, added drop wise. Next, gently centrifuge the cells for four minutes, aspirate the supernatant and gently re-suspend the pellet in one milliliter of stem cell medium with ROCK inhibitor. After quantifying the cell density, plate 300, 000 stem cells onto the feeder cells.
Then grow and expand the cultures, periodically selecting for healthy cells, after expansion and freezing cells for backup. Grow the stem cell colonies on standard plates until they reach 50%to 70%confluence. Then, use a mild enzymatic treatment and gentle trituration with the one milliliter pipette tip to harvest the hiPSCs for neuro precursor cell differentiation.
Gently centrifuge the suspension, aspirate the supernatant and re-suspend the pellet in five milliliters human iPSC medium. Then, transfer the cell suspension to a 0.1%gelatin coated five centimeter cell culture dish, and incubate the plate for one hour. After an hour, transfer the non-adherent cells to a 15 milliliter centrifuge tube.
Then gently rinse the plate with three milliliters of medium and transfer it to the same tube. Next, repeat the re-suspension step with gentle centrifugation, then, determine the cell density and plate the cells into a 96 well, low adhesion V bottom plate at 9, 000 cells per well. Now, spin the plate for three minutes and start culturing the cells.
Over the next 14 days, every other day replace half a medium with fresh differentiating medium one. After two weeks of culturing the cells, prepare six well plates with 40 micron cell culture inserts and one milliliter of DM1 medium. Incubate these plates for at least four hours before adding the cultured cells.
Next, using a 200 microliter wide mouth pipette tip, collect two week old cell aggregates worth about 20 microliters of medium. Transfer four to six aggregates onto each insert with minimal solution, remove any excess. During this step, it's important to work quickly and with a steady hand, while removing medium from around SFEBs, you may see the SFEBs move across the solution due to surface tension.
It's okay to reposition them after the medium is removed. Once all the aggregates are loaded onto inserts, change the medium to one milliliter of DM2 and continue culturing the cells every other day. Replace three fourths of the medium with fresh medium.
After 14 days of culture, start adding DM3 medium to the cultures. Continue the culturing for 16 more days to grow the SFEBs. Now gently detach the SFEBs from the insert, using a 200 microliter wide mouth pipette tip.
Transfer the SFEBs to 12 well plates, loading four to eight per well. Then, stain them as outline in the text protocol. To prepare the 12 well and the eight plates, first wash them three times with sterile water.
Then, wash them once using 75%ethanol and once more with 100%ethanol. Then, bake the plates at 50 degrees celsius for four to five hours upside down, to complete the sterilization process. After baking, add 500 microliters of 0.2%PEI solution to each well and let the plate sit at room temperature for an hour.
Then, aspirate the PEI and wash the wells four times with the sterile distilled water and air dry them overnight. The next day, prepare fresh laminant in L15 medium and add 10 microliters to the center of each well. Then, add distilled water to the surrounding reservoirs to prevent evaporation and incubate the plate for one hour at 37 degrees celsius.
Be sure to beam the laminant in the center of electrodes, being careful not to exceed the space of the grounding electrodes, if this space exceeded, neurons will grow over the grounding electrodes, causing noise artifacts and affecting fidelity of the recording. Now, add one prepared SFEB into each well, using gentle suction with a wide bore pipette. Then, add 200 microliters of DM2 to each well and culture the plate overnight.
Later, to record neural activity, put a plate into the plate reader. Preheat it to 37 degrees celsius. Then, use the software to record 10 minutes of activity from each well.
SFEB is grown using the described method, yielded tissue with morphological characteristics that resemble an early developing cortical sub-ventricular zone replete with extensive Tuj1 positive neurons, as well as neuro progenitors. Numerous developing cortical rosettes were observed in the outer layers and in the inner layers of the bodies. The outer edge of the bodies resembles a developing cortical plate containing postmitotic neurons, this is supported by the expression of Brn2, a marker for neuro progenitors and the most ventralised outer cortical layers.
The outer layers also contain Reelin positive cells that may be casual gratzel cells or their progenitors. The bodies also expressed markers consistent with the mixed codal and medial ganglionic immenants origin. Based on the expression of cooked TF2 and Nkx2.1 markers.
The cultured SFEBs yield at both CalR and CalB positive inter neurons. As well as, VGLUT positive excitatory neurons and Tbr1 positive glutamatergic neural progenitors. Consistent with other reports, 61%of the cells from the bodies were VGLUT positive and 43%were Tbr1 positive.
MEM recordings made from the bodies, can be taken for long periods of time and the recordings showed the development of cortical network level bursting. After watching this video, you should have a good understanding of how to grow SFEBs from human iPSCs, characterize the neurula content of punitive developing cortical rosettes with NSFEBs. and perform neurophysiological assays on maturing SFEBs.
Generally, individuals new to this method might struggle because it takes time and practice to develop the necessary scientific field for the behavior of iPSCs and the iPSC to arrive neurons in culture. The cultured cells should be monitored daily. Following this procedure, other methods like calcium imaging can be performed in order to answer additional questions, such as, what drug treatments might do to cortical network oscillations.
