The overall goal of this procedure is to generate cephalic neurons from human embryonic stem cells or H ESCs by going through checkpoints which are similar to those observed during human development. This is accomplished by first allowing HSCs to spontaneously differentiate in suspension in HESC medium, and then transferring them into neural induction medium to differentiate more selectively. Next embryo bodies or PSC aggregates are allowed to attach onto tissue culture treated plates where the cells expand and differentiate into a monolayer.
With the centers forming neuro epithelial cells morphologically similar to kilometer cells. Next, the neuro epithelial cells are isolated and placed in suspension cultures where they form neurospheres. Finally, the neurospheres are plated onto cover slips where they can further differentiate.
Ultimately, results can be obtained that show the specific types of neurons formed through immuno staining or electrophysiology. The implications of this technique extend towards a wide variety of genetically inherited diseases because patient specific neurons can also be generated from induced pluripotent stem cells where the mechanism of action as well as potential therapies can be elucidated. Though this method can provide insight into the development of talon cephalic neuro subtypes from pluripotent stem cells.
It can also be applied to other systems such as human diseases related to talon cephalic neurons To generate human pluripotent stem cell aggregates begin by culturing human pluripotent stem cells on mouse embryonic fibroblasts feeders in HESC medium supplemented with basic FGF for five to seven days, the colonies will be big, but still undifferentiated. When the colonies have reached the correct stage in a 50 milliliter tube. Prepare a one milligram per milliliter solution of dis bays in D-M-E-M-F 12 medium according to the text protocol.
Aspirate the medium from the cells and then rinse them once with D-M-E-M-F 12. After removing the wash, add one milliliter of dis bays to each well and incubate at 37 degrees Celsius for three to five minutes. At the three minute mark, check under the microscope for curled up edges that look a bit darker.
If necessary, incubate the cells for longer. When the cells are ready, remove the dase solution and wash the cells once with D-M-E-M-F 12 before replacing the wash with 1.5 milliliters of HESC medium to detach and break up the cells. Position the tip of a 10 milliliter glass pipette on the cells towards the bottom right hand wall of a well, and with a small up and down motion, move across the well to the other side before returning in the opposite direction.
Once all of the cells are in suspension, place them in 15 milliliter tubes and centrifuge them at 200 G for two minutes before removing the medium from the pellet. Using HESC medium, transfer the cells to a flask for culturing. For example, for cells from six wells of HSCs, use one T 75 flask with 50 milliliters of HESC.Medium.
Incubate the cells at 37 degrees Celsius for 12 hours. Next to remove any cell debris, transfer the cells and medium to a 50 milliliter tube and wash the cells once before transferring them with 50 milliliters of fresh medium into a new flask. Continue to feed the cells in this manner until day five.
On day five, spin down the cells and transfer them to a new T 75 flask. With 50 milliliters of neural induction medium or NIM, incubate them for two to three days replacing half of the NIM each day after the incubation plate, the cells using either laminin or FBS to help them attach to the plates to plate. Using FBS, add 1.5 milliliters of medium with cells to each.
Well gently shake the plate to spread out the aggregates and allow the cells to attach overnight. At 37 degrees Celsius the following day, replace all of the medium with two milliliters of NIM per well. After a day or two of incubation, each aggregate will spread out to form a monolayer and within a couple of days, the centers of most of these cells will morphologically appear similar to columer cells.
Around days 14 to 17 columer cells will appear more compact. Sometimes the cells form ridges or rings with a visible lumen inside. Before isolating the cells, examine them for the presence of any non-EU epithelial cells and use a pipette tip to scrape them off.
To isolate the neuro epithelial cells that are found in the center of the colonies. Use a micro pipette and a sterile one milliliter filtered pipette to apply pressure to the center of the colony so that it lifts off. Take care not to let the outer portion of the colony detach as well.
Transfer two plates worth of isolated cells to a 15 milliliter tube and spin at 200 G for two minutes. After removing the medium, transfer the cells to a non tissue culture treated T 25 flask containing 10 milliliters of NIM. With the addition of B 27 incubate overnight the following day, the cells should have a sphere like appearance.
Sometimes the peripheral cells will also be isolated and they usually will attach to the bottom of the flask. If this happens, transfer the spherical cells into a new flask, leaving the attached cells behind culture. The neurospheres and suspension for one week in NIMB 27 to promote cell endurance and proliferation.
Add cyclic A MP and insulin-like growth factor one. At the end of the week, the neurospheres will contain cephalic progenitors and are ready to be plated onto cover slips for terminal differentiation. To prepare polyurethane laminin coated cover slips in 24, well plates begin by coating clean cover slips with a 0.1 milligram per milliliter solution of polyurethane at 37 degrees Celsius overnight to coat the cover slips with laminin at 50 microliters of 20 microgram per milliliter of laminin in neural differentiation.
Medium to each polyurethane treated cover slip. Being careful to only have the liquid touching the cover slip itself. Incubate them at 37 degrees Celsius for one to two hours.
After the laminin incubation, collect and wash the neurospheres in D-M-E-M-F 12. If the cells are too large to attach properly, add around two milliliters of Accutane and incubate the cells at 37 degrees Celsius for three minutes. Next, add an equal volume of trypsin inhibitor and after centrifusion the cells at 200 G for three minutes, replace the solution with NDM and use a P 200 to break up the cells for small clusters.
Place them onto pre-coated cover slips, aspirate most of the medium and transfer the cells into a six centimeter Petri dish in a few drops of the medium so that selecting them becomes easier without removing the laminin from the pre-coated cover slips. Transfer around four to five neurospheres to each one. Allow the neurospheres to attach for at least two to four hours before adding 0.5 milliliters of neuro differentiation medium supplemented with B 27 cyclic.
A MP and neurotrophic factors replace half of the medium every other day. The cells can be maintained for several months as shown here. This protocol describes how to differentiate human PSCs into talon cephalic glutamatergic neurons through several critical steps.
The formation of PSC aggregates the induction of neuro epithelial cells, the generation of talon cephalic progenitors, and the terminal differentiation of these progenitors into talon cephalic neurons. This system is robust and efficien in the generation of cephalic, progenitors and glutamatergic neurons, for example. Without the addition of izing factors, the HSCs differentiated into the neural lineage and 24 days post differentiation.
The majority of neural precursors were positive for Fox G one, A transcription factor expressed by the cephalon, but negative for HOKs B four, A marker for hind brain and spinal cord cells suggesting the telon cephalic progenitors were successfully generated. These telon cephalic progenitors possess a dorsal phenotype as indicated by the expression of PAC six. A marker expressed by dorsal progenitors, but not NK X 2.1 A marker for ventral progenitors.
Following further differentiation, these cephalic dorsal progenitors differentiated into glutamatergic neurons, which were positive for the glutamatergic marker. TBR one neurons that sustained positive for TBR one were also positive for neuronal markers beta three tubulin or map two. These cells also expressed vesicular glutamate transporter one or V glut one, a marker for mature glutamatergic neurons suggesting the efficient generation of talon cephalic glutamatergic neurons in the culture.
Following this procedure, other methods like electrophysiology can be performed in order to answer additional questions like whether these neurons can make synaptic connections with other cells. After watching this video, you should have a good understanding. It had to differentiate human pluripotent stem cells and into cephalic glutamatergic neurons through several critical steps.
The formation of pluripotent stem cell aggregates the induction of neuro epithelial cells, the generation of cephalic progenitors, and the terminal differentiation of these progenitors into cephalic neurons.
