The overall goal of this procedure is to generate neural crest progenitor cells from human pluripotent stem cells in a short reproducible and defined protocol. These cells can then be used for either the study of neural crest related diseases in vitro or the continued differentiation of these cells into progeny cells. This is accomplished by first plating a high density of human pluripotent stem cells in a monolayer on matrigel.
The second step is to differentiate the cells into neuro epithelial cells using a defined sequence and combination of small molecules that affect key signaling pathways. Next, the neuro epithelial cells are replated and further differentiated into neural crest progenitor cells. The final step is to isolate the neuro crest cells using fluorescence activated cell sorting.
Ultimately, immunofluorescence and functional assays such as the cell's ability to migrate are used to show that proper neural crest cells were generated. The main advantage of this technique over existing protocols for the derivation of neuro crest cells from human pluripotent stem cells is that it is shorter. It's only 18 days.
It is more reproducible due to the lack of MS five feeder cells. It's easy to upscale it and it's highly reproducible among human pluripotent stem cell lines. The implications of this technique extend towards studies of mechanisms of human diseases related to the neurore lineage and show potential for improving detail in vitro modeling and drug screening of these diseases.
Prior to starting this protocol ensured that the colonies of human pluripotent stem cells are large enough to be seen by eye and still have sharp edges with few differentiating cells at their borders. Then when the H PSCs are ready to be split, aspirate the medium wash once with five milliliters of one XPBS and add four milliliters of 0.05%trips in EDTA to the cells Next vigorously. Shake the dish horizontally for two minutes until the mouse embryonic fibroblasts are seen under the microscope lifting off the plate as single cells.
The HPSC colonies will remain attached to the dish as colonies, then immediately and thoroughly aspirate the trypsin and leave the plate at room temperature for two to four minutes. Add two milliliters of HES medium to the plate and use a P 1000 pipette to detach the cells. If the cells do not lift off easily, then incubate the plate for an additional two to three minutes.
At room temperature, transfer the cell suspension to a 15 milliliter conical tube containing eight milliliters of HES medium, supplemented with 10 micromolar Y 2 7 6 3 2 dihydrochloride. Then aspirate the matrigel from a previously treated 10 centimeter dish and plate the cells at a one-to-one or one to two ratio onto the matrigel treated plate. Incubate the cells overnight at 37 degrees Celsius in a 5%carbon dioxide incubator.
Feed the cells daily with HES medium until they are ready for differentiation. Then begin the neural differentiation when the cells are 90%to 100%confluent. This is day zero.
Feed the cells daily from day zero to day three with 10 milliliters of KSR differentiation, medium containing 0.1 micromolar, LDN 1, 9 3, 8, 9, and 10 MICROMOLAR SB 4 3 1 5 42. Then on day four and five, feed the cells with 10 milliliters of media containing 75%KSR differentiation, medium 25%and two, differentiation medium, 0.1, micromolar LDN 1, 9 3, 1, 8, 9, and 10 micromolar SB 4 3 1 5 42. On day six and seven, feed the cells with media adjusted to contain 50%KSR differentiation, medium and 50%And two, differentiation medium in addition to being supplemented with 0.1 micromolar LDN, 1, 9 3, 1, 8, 9, and 10 micromolar SB 4 3 1 5 2.
For day eight to nine, feed the cells with 25%KSR differentiation, medium and 75%and two, differentiation medium, both containing 0.1 micromolar LDN 1 9 3 1 8, 9, and 10 micromolar SB 4 3 5 2. Then on day nine and 10, prepare culture dishes with poly L orhan laminin one and fibronectin as described in the text protocol, which will be used for replating The cells on day 11, feed the cells with N two differentiation medium, supplemented with 0.1 micromolar L DN 1 9 3, 109, and 10 micromolar SB 4 31 5 42. On day 10 on day 11, aspirate the laminin one and fibronectin solution from the previously prepared culture dishes and let them dry at room temperature in a tissue culture hood for 20 to 30 minutes.
Next, remove the medium from the differentiating cells and wash the cells once with one XPBS. Then add eight milliliters of acuate to each 10 centimeter plate and incubate the plates for 20 minutes at 37 degrees Celsius. After the incubation, use a cell lifter to detach the cells and then resuspend the cells in the Accutane solution.
With a five milliliter pipette, transfer the cell suspension to a 15 milliliter conical tube. Next, add five milliliters of one XPBS to the tube and spin the cells for five minutes at 115 Gs.Discard the supernatant and resuspend the cell pellet in 10 milliliters of one XPBS. Then filter the cell suspension through a 40 micron cells trainer to ensure a single cell suspension.
Count the cells using a hemo cytometer. Spin the cells down again for five minutes at 115 Gs and resuspend the cells to a concentration of 100, 000 to 150, 000 cells per 10 microliters of supplemented N two differentiation medium. Use a repeater pipette to plate 10 microliter droplets of the cell suspension onto 15 centimeter culture dishes that have been pretreated with poly L orhan.
Laminin one and fibronectin. Allow the droplets to stand at room temperature for 15 to 20 minutes. Then very carefully at 30 milliliters of the supplemented N two differentiation medium to the plate without disturbing the droplets.
Incubate the cells overnight at 37 degrees Celsius in a 5%carbon dioxide incubator. On day 12, carefully feed the cells with 20 milliliters of N two differentiation medium containing 200 micromolar as soic acid. 20 nanograms per milliliter of brain derived neurotrophic factor 100 nanograms per milliliter of fiberblast growth factor eight and 20 nanograms per milliliter of sonic hedgehog per 15 centimeter dish.
Then from day 14 to day 17, feed the cells every two to three days with N two differentiation medium containing only 200 micromolar as oric acid. 20 nanograms per milliliter of brain-derived neurotrophic factor and 100 nanograms per milliliter of fiberblast growth. Factor eight, prepare plates with poly L orin laminin one and fibronectin on day 16 and 17 to be used for replating of the neural crest cells.
After fax analysis on day 18, after the cells have been sorted by fluorescence activated cell sorting, count the neural crest cells and then spin them down at 115 Gs for five minutes. Re suspend the cell pellet in N two. Differentiation medium supplemented with 10 nanograms per milliliter.
FGF two 20 nanograms per milliliter EGF and 10 micromolar Y 2 7 6 3 2 di hydrochloride at a concentration of 30, 000 cells per 10 microliters. Thoroughly dry culture dishes that were previously prepared with poly L orhan laminin one and fibronectin. Then plate approximately 50 to 70 droplets of the cell suspension per 10 centimeter dish.
Incubate the cells at room temperature for 15 to 20 minutes at 20 milliliters of N two. Differentiation medium supplemented with 10 nanograms per milliliter. FGF two 20 nanograms per milliliter EGF and 10 micromolar Y 2 7 6 3 2 dihydrochloride to each 10 centimeter dish without disturbing the droplets.
Then incubate the cells overnight at 37 degrees Celsius in a 5%carbon dioxide incubator. Following the incubation, maintain and passage the neural crest cells, according to the accompanying text protocol, neural crest cells were derived from human pluripotent stem cells in 18 days. Using this new feeder free in vitro differentiation protocol.
The fax analysis on day 18 identified cells that were positive for both HNK one and P 75, which confirmed the presence of neural crest cells. In both the feeder free differentiation protocol and feeder cell dependent protocol, the neural crest cells passed through a neural roset stage. In addition, the fact sorted cells have identical morphology and expressed the neural crest markers, H and K one and a P two comparison of the global gene expression of neural crest cells derived from these two differentiation protocols found that both sets of cells cluster closely together.
Analysis of the migration capacity of the neural crest cells derived using the feeder free protocol showed the cells migrated successfully into the scratch after 48 hours. These cells also have the potential to be differentiated into cells from the autonomic nervous system as confirmed by positive staining for mash one and touch one after an additional four days of differentiation. After watching this video, you should have a good understanding of how to neuralize human pluripotent stem cells and to derive neuro crest progenic cells in vitro.
