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 lines. Note: 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 embryo. The 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
Protocols from the Laboratory of James Thomson, University of Wisconsin. Describes the growth of MEF feeders, thawing and freezing of human ES cells, as well as growth on feeders and Matrigel.
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 University. This 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 protocols. The 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.
UCSF Protocols. UCSF Embryonic Stem Cell Research Center has online methods for MEFs and cell line specific methods for passaging of H9, HSF1 and HSF6 ES cells. Freezing and thawing protocols are also available. Note the freezing protocol as frozen cells obtained from UCSF have been reported to survive freezing much better than lines obtained from other sources.
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 Research 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 methodology. Below are links to some manuscripts that describe differentiation of human ES cells into specific cell types. This 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 E. Derivation of adipocytes from human embryonic stem cells Stem Cells Dev. 2005 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 L. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes J Clin Invest. 2001 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 Immunol. 2006 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 Hongkui. Directed differentiation of human embryonic stem cells into functional hepatic cells Hepatology. 2007 May;45(5):1229-39.
Mesenchymal Cells
Barberi Tiziano, Willis Lucy M, Socci Nicholas D, Studer Lorenz. Derivation of multipotent mesenchymal precursors from human embryonic stem cells PLoS Med. 2005 Jun;2(6):e161. Epub 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 Lorenz. Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons Stem Cells. 2007 Aug;25(8):1931-9. Epub 2007 May 3.
Neurons
Schulz Thomas C, Palmarini Gail M, Noggle Scott A, Weiler Deborah A, Mitalipova Maisam M, Condie Brian G. Directed neuronal differentiation of human embryonic stem cells BMC Neurosci. 2003 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-Chun. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells Stem Cells. 2005 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 Ray. Directed differentiation of human embryonic stem cells into the pancreatic endocrine lineage Stem Cells Dev. 2007 Aug;16(4):561-78.
Shim J H, Kim S E, Woo D H, Kim S K, Oh C H, McKay R, Kim J H. Directed differentiation of human embryonic stem cells towards a pancreatic cell fate Diabetologia. 2007 Jun;50(6):1228-38. Epub 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 Ray. Directed differentiation of human embryonic stem cells into the pancreatic endocrine lineage Stem Cells Dev. 2007 Aug;16(4):561-78.
Shim J H, Kim S E, Woo D H, Kim S K, Oh C H, McKay R, Kim J H. Directed differentiation of human embryonic stem cells towards a pancreatic cell fate Diabetologia. 2007 Jun;50(6):1228-38. Epub 2007 Apr 18.
Osteoblasts
Sottile Virginie, Thomson Alison, McWhir Jim. In vitro osteogenic differentiation of human ES cells Cloning Stem Cells. 2003;5(2):149-55.
Smooth Muscle
Huang Huarong, Zhao Xiaoli, Chen Liangbiao, Xu Chao, Yao Xing, Lu Yongliang, Dai Licheng, Zhang Ming. Differentiation of human embryonic stem cells into smooth muscle cells in adherent monolayer culture Biochem Biophys Res Commun. 2006 Dec 15;351(2):321-7. Epub 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 embryonic stem cells: The Practical Handbook Stephen Sullivan (Editor), Chad A Cowan (Co-Editor), Kevin Eggan (Co-Editor) 424 pages. John Wiley and Sons, Inc. (August 2007)
Human embryonic stem cell protocols Kursad Turksen (Editor) 408 pages. Humana Press (March 2006)