title: Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.
abstract: A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, Karolinska Institute, Stockholm, Sweden.
authors.forename: Sergey
authors.initials: S
authors.lastname: Rodin
authors.affiliation: Department of Clinical Sciences, Division of Obstetrics and Gynecology, Intervention and Technology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Liselotte
authors.initials: L
authors.lastname: Antonsson
authors.affiliation: Department of Clinical Sciences, Division of Obstetrics and Gynecology, Intervention and Technology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Outi
authors.initials: O
authors.lastname: Hovatta
authors.affiliation: 1] Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, Karolinska Institute, Stockholm, Sweden. [2] Cardiovascular and Metabolic Disorders Program, Duke-NUS (National University of Singapore), Singapore, Singapore.
authors.forename: Karl
authors.initials: K
authors.lastname: Tryggvason
mesh_terms: D015820:Cadherins / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002454:Cell Differentiation; D019976:Cloning, Organism; D006801:Humans; D007797:Laminin / Q000737:chemistry / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology
chemical_list: D015820:Cadherins; D007797:Laminin; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human