title: Alternative cultures for human pluripotent stem cell production, maintenance, and genetic analysis.
abstract: Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.
authors: NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health; cheng@mail.nih.gov.
authors: Kevin G
authors: KG
authors: Chen
authors: NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health.
authors: Rebecca S
authors: RS
authors: Hamilton
authors: Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health.
authors: Pamela G
authors: PG
authors: Robey
authors: NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health.
authors: Barbara S
authors: BS
authors: Mallon
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D003584:Cytological Techniques / Q000379:methods*; D004146:Dioxanes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology; D007797:Laminin / Q000494:pharmacology; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014162:Transfection; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000577:Amides; D004146:Dioxanes; D004791:Enzyme Inhibitors; D007797:Laminin; D011725:Pyridines; D011743:Pyrimidines; D013844:Thiazoles; C520365:Y-39983; C545214:thiazovivin; C108830:Y 27632; D054460:rho-Associated Kinases