Research Protocols
Below are links to a number of different online resources for embryonic stem (ES) cell protocols developed by leaders in human ES cell research. With help from the research community, as this site develops we plan to establish a forum for human ES cell methods where researchers can discuss problems and improve established protocols as well as describe new methodologies.
Derivation of Human ES Cells
Derivation of Human Embryonic Stem Cell Lines PDF of Chapter 4 from “Human Embryonic Stem Cells: The Practical Handbook” (2007, Wiley Publishing) describes methods used by Harvard University researchers to establish human ES cell linesNote: This is a large (1.62 MB) PDF document.
Derivation of Human Embryonic Stem Cells from Single Blastomeres (2007) This protocol details the derivation of hESC lines without embryo destruction by removal of a single blastomere from an eight-cell stage embryoThe technique was developed by Advanced Cell Technology, Inc(Nature Protocols 2:1963-72)
Derivation and maintenance of human embryonic stem cells from poor-quality in vitro fertilization embryos (2008) This article details the protocols developed by the laboratory of Geoge Daley (Children's Hospital, Boston, MA) for the derivation and culture of hESC lines from poor-quality IVF embryos.
Derivation of Induced Pluripotent Stem (iPS) Cells
Generation of Human-Induced Pluripotent Stem Cells (2008) Protocol for generation of iPS cells from adult fibroblasts by ectopic expression of six factors. From the laboratory of George Daley, Children's Hosptial, Boston.
Induction of pluripotent stem cells from fibroblast cultures (2007) Detailed protocols for the derivation and growth of mouse iPS cells by the laboratory of Shinya Yamanaka, the first to demonstrate reprogramming.
Basic Techniques for Growth of Human ES Cells
Culture Protocols from the NIH Stem Cell Unit Details human ES cell culture on feeder and feeder-free as well as subcloning, EB differentiation, marker staining and FACS analysis.
HUES (Human Embryonic Stem) Cell Collection Instruction Manual From the laboratory of Douglas Melton and the human ES cell facility, Harvard UniversityThis manual describes the derivation of the “HUES” cell lines and provides detailed information about thawing, maintenance and passaging of the human ES cells as well as MEF protocolsThe manual also has detailed media and plating instructions with specific reagents listed.
WiCell Protocols Detailed set of PDFs with figures covering MEF feeder derivation and culture and the thawing, splitting, selection and maintenance of human ES cells.
Bresagen Protocols This PDF (2004) covers topics such as: derivation, growth and use of MEFs, bulk and microdissection passaging of human ES cells and preparation of human ES cells for karyotyping.
Stem Cell Society Singapore Links to the methodology manual from ES Cell International and others as well as methods for isolation of adult stem cells and gene delivery into stem cells.
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Directed Differentiation of Human ES Cells
Directed differentiation protocols for human ES cells are available primarily in the scientific literature and in handbooks of human ES cell methodologyBelow are links to some manuscripts that describe differentiation of human ES cells into specific cell typesThis list is not comprehensive at this point, but is intended to provide some examples of these methods.
Adipocytes
Xiong Chen, Xie Chang-Qing, Zhang Li, Zhang Jifeng, Xu Kefeng, Fu Mingui, Thompson Winston E, Yang Li-Jun, Chen Yuqing EDerivation of adipocytes from human embryonic stem cells Stem Cells Dev2005 Dec;14(6):671-5.
Cardiomyocytes
Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein LHuman embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes J Clin Invest2001 Aug;108(3):407-14.
Dendritic Cells
Slukvin Igor I, Vodyanik Maxim A, Thomson James A, Gumenyuk Maryna E, Choi Kyung-Dal Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway J Immunol2006 Mar 1;176(5):2924-32.
Hepatocytes
Cai Jun, Zhao Yang, Liu Yanxia, Ye Fei, Song Zhihua, Qin Han, Meng Sha, Chen Yuezhou, Zhou Rudan, Song Xijun, Guo Yushan, Ding Mingxiao, Deng HongkuiDirected differentiation of human embryonic stem cells into functional hepatic cells Hepatology2007 May;45(5):1229-39.
Mesenchymal Cells
Barberi Tiziano, Willis Lucy M, Socci Nicholas D, Studer LorenzDerivation of multipotent mesenchymal precursors from human embryonic stem cells PLoS Med2005 Jun;2(6):e161Epub 2005 Jun 28.
Motor Neurons
Lee Hyojin, Shamy George Al, Elkabetz Yechiel, Schofield Claude M, Harrsion Neil L, Panagiotakos Georgia, Socci Nicholas D, Tabar Viviane, Studer LorenzDirected differentiation and transplantation of human embryonic stem cell-derived motoneurons Stem Cells2007 Aug;25(8):1931-9Epub 2007 May 3.
Neurons
Schulz Thomas C, Palmarini Gail M, Noggle Scott A, Weiler Deborah A, Mitalipova Maisam M, Condie Brian GDirected neuronal differentiation of human embryonic stem cells BMC Neurosci2003 Oct 22;4:27.
Yan Yiping, Yang Dali, Zarnowska Ewa D, Du Zhongwei, Werbel Brian, Valliere Chuck, Pearce Robert A, Thomson James A, Zhang Su-ChunDirected differentiation of dopaminergic neuronal subtypes from human embryonic stem cells Stem Cells2005 Jun-Jul;23(6):781-90.
Phillips Blaine W, Hentze Hannes, Rust William L, Chen Qi-Ping, Chipperfield Hiram, Tan Ee-Kim, Abraham Suman, Sadasivam Akila, Soong Poh Loong, Wang Siew Tein, Lim Ricky, Sun William, Colman Alan, Dunn N RayDirected differentiation of human embryonic stem cells into the pancreatic endocrine lineage Stem Cells Dev2007 Aug;16(4):561-78.
Shim J H, Kim S E, Woo D H, Kim S K, Oh C H, McKay R, Kim J HDirected differentiation of human embryonic stem cells towards a pancreatic cell fate Diabetologia2007 Jun;50(6):1228-38Epub 2007 Apr 18.
Pancreatic Cells
Phillips Blaine W, Hentze Hannes, Rust William L, Chen Qi-Ping, Chipperfield Hiram, Tan Ee-Kim, Abraham Suman, Sadasivam Akila, Soong Poh Loong, Wang Siew Tein, Lim Ricky, Sun William, Colman Alan, Dunn N RayDirected differentiation of human embryonic stem cells into the pancreatic endocrine lineage Stem Cells Dev2007 Aug;16(4):561-78.
Shim J H, Kim S E, Woo D H, Kim S K, Oh C H, McKay R, Kim J HDirected differentiation of human embryonic stem cells towards a pancreatic cell fate Diabetologia2007 Jun;50(6):1228-38Epub 2007 Apr 18.
Osteoblasts
Sottile Virginie, Thomson Alison, McWhir JimIn vitro osteogenic differentiation of human ES cells Cloning Stem Cells2003;5(2):149-55.
Smooth Muscle
Huang Huarong, Zhao Xiaoli, Chen Liangbiao, Xu Chao, Yao Xing, Lu Yongliang, Dai Licheng, Zhang MingDifferentiation of human embryonic stem cells into smooth muscle cells in adherent monolayer culture Biochem Biophys Res Commun2006 Dec 15;351(2):321-7Epub 2006 Oct 17.
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Stem Cell Handbooks
These are a sampling of manuals that you can purchase that provide comprehensive protocols for all aspects of producing maintaining and differentiating human ES cells.
Human Stem Cell Technology and Biology: A Research Guide and Laboratory Manual Gary S. Stein (Editor), Maria Borowski (Editor), Mai X. Luong (Editor), Meng-Jiao Shi (Editor), Kelly P. Smith (Editor), Priscilla Vazquez (Editor), 440 pages, Wiley-Blackwell. (January 2011)
Human embryonic stem cells: The Practical Handbook Stephen Sullivan (Editor), Chad A Cowan (Co-Editor), Kevin Eggan (Co-Editor) 424 pagesJohn Wiley and Sons, Inc (August 2007)
Human embryonic stem cell protocols Kursad Turksen (Editor) 408 pagesHumana Press (March 2006)