title: Inhibition of ROCK-myosin II signaling pathway enables culturing of human pluripotent stem cells on microcarriers without extracellular matrix coating.
abstract: 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 with 11.5-fold expansion (HES-3) in a 100-mL spinner flask. The differentiation capability of these cells toward three primary lineages is demonstrated via in vitro embryoid bodies and in vivo teratoma formations. Moreover, the directed differentiation to polysialylated neuronal cell adhesion molecule-positive (PSA-NCAM+) neural progenitors produced high cell concentrations (9.1±1.2×10⁶ cells/mL) with a cell yield of 412±77 neural progenitor cells per seeded HES-3 and a PSA-NCAM expression level of 91±1.1%. This defined serum- and coating-free scalable microcarrier culturing system is a safer and less expensive method for generating large amounts of hPSCs for cell therapies.
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR) , Singapore, Singapore .
authors.forename: Allen Kuan-Liang
authors.initials: AK
authors.lastname: Chen
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Chen
authors.forename: Yu Ming
authors.initials: YM
authors.lastname: Lim
authors.forename: Shaul
authors.initials: S
authors.lastname: Reuveny
authors.forename: Steve Kah Weng
authors.initials: SK
authors.lastname: Oh
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D020099:Coated Materials, Biocompatible / Q000494:pharmacology*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects; D005109:Extracellular Matrix / Q000378:metabolism*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D008863:Microspheres; D024462:Myosin Type II / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D039904:Pluripotent Stem Cells / Q000166:cytology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D020099:Coated Materials, Biocompatible; D006576:Heterocyclic Compounds, 4 or More Rings; D047428:Protein Kinase Inhibitors; C472645:blebbistatin; D054460:rho-Associated Kinases; D024462:Myosin Type II