Inhibition of ROCK–Myosin II Signaling Pathway Enables Culturing of Human Pluripotent Stem Cells on Microcarriers Without Extracellular Matrix Coating

Large quantities of human pluripotent stem cells (hPSCs) needed for therapeutic applications can be grown in scalable suspended microcarrier cultures. These microcarriers are coated with animal or human extracellular matrix (ECM) proteins to promote cell growth and maintain pluripotency. However, the coating is costly for large-scale cultures and it presents safety risks. This study demonstrates that hPSCs can be propagated on noncoated positively charged cellulose microcarriers in a serum-free medium containing the ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin. In the presence of these two inhibitors, myosin phosphatase 1 and myosin light chain 2 were dephosphorylated suggesting that reduced myosin contractility is responsible for hPSC survival and growth on ECM coating-free microcarriers. Cells propagated on the noncoated microcarriers for 12 passages maintained their pluripotency and karyotype stability. Scalability was demonstrated by achieving a cell concentration of 2.3×106 cells/mL wit...