Y-27632

Y-27632 primes human induced pluripotent stem cells (hIPSCs) to selectively differentiate towards mesendodermal lineage via epithelial-mesenchymal transition-like modulation.^[2]

The hiPSC line is cultured on six-well plates coated with 1% Matrigel. Pluripotent stem cells are maintained in mTeSR1 (Stem Cell) medium supplemented with 100 µg/mL Normocin coated with 1% Matrigel. For differentiation, hiPSC colonies are detached from the culture dish using Accutase Cell Dissociation Reagent. Then, cells are collected as a single-cell suspension in Essential 8 Flex medium containing 20 µm Y27632. Cells are distributed at a density of 3500 cells in 100 µL of medium per well into 96-well U-bottom plates. After 24 h of incubation at 37 ℃, fresh E8 medium without Y27632 is added to a total volume of 200 µL per well, and the plates are incubated at 37℃ continuously. Next, cell aggregates are cultured in E6-based differentiation medium containing 2% Matrigel, 10 µm SB431542, 4 ng mL 1 basic FGF, and 2.5 ng mL 1 BMP4. On day 3 of differentiation, 200 ng mL 1 LDN-193189 and 50 g mL 1 FGF are added to induce cranial neural crest cell formation. On day 12, all cell aggregates are transferred to 24-well low-attachment plates in 500 µL of organoid maturation mediumcontaining 1% Matrigel to induce self-assembly of the epidermis. Half of the medium is replaced after 16 d after differentiation. On day 17, the organoids are collected for the subsequent experiments.

Y27632, a specific inhibitor of ROCK, supresses ROCK as well as altered pathways and leads to a significant enhancement in corneal nerve regeneration.^[3]