Long-term research focus is finding potential therapeutics for neurological disorders in Down syndrome. And our current focus is Down syndrome impaired neurogenesis, a major cause of intellectual disability. We found down syndrome impaired neurogenesis is caused by biphasic cell cycle defect, contrary to popular belief that it is only due to senescence of Down syndrome neural progenitor cells.
Identification of biphasic cell cycle defect using human IPSC based protocol described in this manuscript will drive developmental potential therapeutic strategies considering both cell cycle state that is reduced proliferation at early phase, and failure to exit cell cycle at late phase. To begin, add 10 micromolar of rock inhibitor into complete human iPCS's media, DPBS, cell detachment solution, and feeder-conditioned medium supplemented with 25 nanograms per milliliter of basic fibroblast growth factor. Warm all reagents to 37 degrees Celsius.
Take a six-well plate of iPSCs on feeders. Pipette out the differentiated colonies, and add fresh complete human iPSC medium. Place the plate at 37 degrees Celsius in carbon dioxide incubator for two hours.
After incubation, wash the wells once with calcium-and magnesium-free DPBS. Then, add one milliliter of cell detachment solution to each well. Place the plate at 37 degrees Celsius in a carbon dioxide incubator for 12 to 14 minutes.
Observe the cells under an inverted microscope. If most cells are detaching, but if some remain adhered, tap the plate gently from the sides. Add three milliliters of DPBS supplemented with calcium and magnesium into each well to dilute the cell detachment solution.
With a five-milliliter pipette, perform gentle pipetting two to three times to break clumps, while avoiding bubbles. Pass the cells through a 40-micrometer strainer into a 50-milliliter centrifuge 2. Centrifuge the suspension at room temperature for five minutes at 200 G.Use a vacuum aspiration system to aspirate the media gently.
Now, re-suspend the cells in 10 milliliters of human iPSC medium. Transfer all the cells in suspension onto a 15-centimeter dish coated with 0.1%gelatin, and incubate in a carbon dioxide incubator. Collect the media with cells into a centrifuge 2.
Centrifuge at room temperature for five minutes at 200 G.Then aspirate the media gently with the vacuum aspiration system. Re-suspend the cells in one milliliter of feeder-conditioned medium, supplemented with 25 nanograms per milliliter of BFGF, and 10 micromolar of rock inhibitor. After cell counting, prepare a suspension of 30, 000 to 50, 000 cells per milliliter.
Seed the suspension into a well plate. After two days, replace the feeder-conditioned medium with neural progenitor cell, or NPC medium. Change media on days two and 18 as given.
On day 28, replace the medium in the plate with NPC medium supplemented with 10 micromolar of rock inhibitor. Then incubate the plate at 37 degrees Celsius in a carbon dioxide incubator for two hours. After incubation, aspirate the supernatant, and wash the wells once with calcium-and magnesium-free DPBS.
Add one milliliter of cell detachment solution into each well. Then observe the cells under an inverted microscope at 4x magnification. Next, pipette three milliliters of calcium-and magnesium-supplemented DPBS into each well of the plate.
With a five-milliliter pipette, gently pipette the suspension, and break any clumps. Strain the cell suspension through a 40-micrometer strainer placed over a 50 milliliter centrifuge 2. Centrifuge, and aspirate as demonstrated earlier, then re-suspend the pellet in one milliliter of defined differentiation medium supplemented with B 27 with vitamin A.Count live cells using Trypan blue and a hemocytometer.
Then seed 50, 000 cells into each well of a qualified matrix-coated 48 well plate. On day 33, replace the medium in the culture wells with neural differentiation medium. A significant reduction in TUBB3-positive neurons was observed in Down syndrome cultures, compared to isogenic euploid cells, with approximately a twofold decrease evident at day 85.
A significantly lower proportion of KI67 positive cells were observed in Down syndrome cultures during the early neurogenic stage, compared to isogenic euploid cells, showing approximately a fourfold reduction. In the late neurogenic stage, most isogenic euploid cells exited the cell cycle with minimal KI67 staining, while the majority of Down syndrome cells remained KI67-positive, showing a fivefold increase. A second pair of Sown syndrome and isogenic euploid human iPSCs confirmed reduced TUBB3-positive neuron levels and elevated PAX6-positive neural progenitor cells in Down syndrome cultures at the end of neural differentiation, with a twofold reduction in TUBB3-positive neurons, and a more than twofold increase in PAX6-positive cells.
