© 2014 Nature America, Inc. All rights reserved. PROTOCOL 2354 | VOL.9 NO.10 | 2014 | NATURE PROTOCOLS INTRODUCTION hPS cells have unlimited proliferation potential and a capability to differentiate into any cell type of the three primary germ layers, i.e., ectoderm, endoderm and mesoderm. Therefore, they are an attractive source of cells for stem cell research and regenerative medicine. Several types of hPS cells have been described in the literature 1,2 , but in this article we use the acronym hPS cells to designate both human embryonic stem (hES) 3 cells and human- induced pluripotent stem (iPS) 4 cells, which are the two best stud- ied classes. For applications to regenerative medicine, hES cells are of particular interest if they can be maintained in a genetically stable state. A major drawback to initial hPS cell culture and dif- ferentiation protocols was that they were not chemically defined or fully xeno-free. Poorly reproducible results are obtained if components with wide batch-to-batch variation and chemi- cally undefined components, such as feeder cells 3 or Matrigel 5 , are used for maintaining hES cell lines. Arbitrary criteria used in the procedures also cause variable results, and components of animal origin may lead to immunogenicity of the cells 6 . Largely because of those problems, only a few clinical trials have been approved for hPS cell–based therapies. Thus, there is a great need to develop robust xeno-free and chemically defined protocols for hES cell cultures and differentiation methods under Good Manufacturing Practice (GMP)-compliant quality standards. An additional important issue has been the poor clonogenicity of conventionally cultured hPS cells. Clonal growth of stable hPS cells may facilitate manipulation of their genomes and applica- tions based on the use of FACS. Development of hES cell culture systems The first hES cell culture system described consisted of a layer of feeder cells, usually mitotically inactivated mouse or human fibroblasts, and a chemically undefined medium 3 . The feeder cells themselves produce a large amount of proteins that are different from batch to batch, they represent an undefined substratum and environment by nature and they can potentially transmit viral or bacterial contaminations. hPS cells cultured on feeders grow in thick multilayer colonies. Representative colonies of hES cells grown on feeders are shown in Figure 1a,b. Usually, in the middle and sometimes on the rim of the colonies, there are areas of dif- ferentiated cells. Therefore, there is a need for active manual selec- tion of undifferentiated parts, which requires the skills of specially trained personnel. Generally, mechanical passaging is a laborious procedure based on arbitrary criteria defining the undifferenti- ated parts of colonies and determining the size of pieces that should be taken. This leads to variability and irreproducibility of the experiments. Thus, the classic feeder-based culturing of hPS cells is neither robust, nor xeno-free, nor chemically defined and required replacement. The first chemically defined medium formulation TeSR1 (ref. 5) substantially improved the culture of hES cells. However, the sub- stratum Matrigel 5,7 , which was initially used with this medium, is not a defined cell culture dish coating. Matrigel is a whole mouse sarcoma tissue extract, containing extracellular matrix and cellular proteins and variable amounts of growth factors and hormones, and thus it varies extensively from batch to batch. Several reports have described xeno-free, chemically defined substrata, which can serve as a functional substitution for Matrigel. However, the majority of these substitutes do not allow passage in single-cell suspensions 8–10 . Passage of cell aggregates implies the generation of cellular clumps of different sizes, which is an arbitrary crite- rion. Indeed, if the aggregates used are too large, overcrowding and subsequent death or differentiation of cells in the middle of the new colonies occurs. In contrast, aggregates that are too small die on such substrata because of anoikis. Figure 1c,d shows hPS cell cultures 24 h after passaging of large clumps and appropriately sized clumps of cells on Vitronectin 10 , respectively. Therefore, although passaging in cell aggregates can support self-renewal of hPS cells under xeno-free and chemically defined conditions, Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions Sergey Rodin 1 , Liselotte Antonsson 2 , Outi Hovatta 2 & Karl Tryggvason 1,3 1 Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, Karolinska Institute, Stockholm, Sweden. 2 Department of Clinical Sciences, Division of Obstetrics and Gynecology, Intervention and Technology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden. 3 Cardiovascular and Metabolic Disorders Program, Duke-NUS (National University of Singapore), Singapore, Singapore. Correspondence should be addressed to K.T. (karl.tryggvason@ki.se). Published online 11 September 2014; doi:10.1038/nprot.2014.159 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 cm 2 , 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.