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Isolation of Human Embryonic Stem Cell–Derived Teratomas for the Assessment of Pluripotency

Karin Gertow1,2,  Stefan Przyborski3,  Jeanne F. Loring4,  Jonathan M. Auerbach5,  Olga Epifano5,  Timo Otonkoski6,  Ivan Damjanov7,  Lars Ährlund‐Richter1,8

1Department of Laboratory Medicine, Clinical Research Center, Unit for Molecular Embryology, Karolinska Institute, Sweden
2Monash Immunology and Stem Cell Laboratories, Monash University, Australia
3School of Biological and Biomedical Science, University of Durham, Durham, United Kingdom
4Burnham Institute for Medical Research, LaJolla, California
5GlobalStem Inc., Rockville, Maryland
6Hospital for Children and Adolescents and the Biomedicum Stem Cell Center, University of Helsinki, Finland
7Department of Pathology, The University of Kansas, School of Medicine, Kansas City
8Department of Woman and Child Health, Karolinska Institute, Stocholm, Sweden.








Publication Name: 
Current Protocols in Stem Cell Biology
Unit Number: 
Unit 1B.4
DOI: 
10.1002/9780470151808.sc01b04s3
Online Posting Date: 
March, 2009
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Abstract

This unit describes protocols on how to assess the developmental potency of human embryonic stem cells (hESCs) by performing xenografting into immunodeficient mice to induce teratoma formation. hESCs can be injected under the testis capsule, or alternatively into the kidney or subcutaneously. Teratomas that develop from grafted hESCs are surgically removed, fixed in formaldehyde, and paraffin embedded. The tissues in the teratoma are analyzed histologically to determine whether the hESCs are pluripotent and form tissues derived from of all three embryonic germ layers (ectoderm, mesoderm, and endoderm). Teratomas can also be fixed in Bouin's or cryosectioned for analysis, and they can be analyzed by immunohistochemistry for tissue markers. Methods for these procedures are included in this unit. Curr. Protoc. Stem Cell Biol. 3:1B.4.1-1B.4.29. © 2007 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; pluripotency; teratoma; immunodeficient mice

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Injection of hESC Under the Testis Capsule in Immunodeficient Mice
  • Alternate Protocol 1: Injection of hESC Under the Kidney Capsule
  • Alternate Protocol 2: Subcutaneous Injection of hESC
  • Support Protocol 1: Preparation of hESC for Injection
  • Support Protocol 2: Anesthesia for the Mouse
  • Basic Protocol 2: Excision and Fixation for Paraffin Embedding of the hESC Teratoma
  • Alternate Protocol 3: Bouin's Fixation of Teratomas
  • Alternate Protocol 4: Tissue Cryopreservation and Preparation for Cryo–Microtome Sectioning
  • Basic Protocol 3: Evaluation of Tissue Formation and Demonstration of the Presence of Embryonic Germ Layers in the hESC Teratoma
  • Basic Protocol 4: Paraformaldehyde Fixation and Preparation of Tissues for Immunohistochemistry
  • Basic Protocol 5: Preparation of Tissues for mRNA Expression Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Injection of hESC Under the Testis Capsule in Immunodeficient Mice

 Materials
  • hESCs (see Support Protocol 1)
  • Mice (immunodeficient; either from immunosuppressive treatment, or genetic mutation; see Strategic Planning)
  • 70% ethanol
  • 0.015 mg/ml Temgesic (Buprenorphinum)
  • Stereomicroscope for harvesting hESCs in the animal surgery room (optional depending on procedure used for the harvest of hESCs; see Support Protocol 1)
  • Sterile paper tissue
  • Electric clippers
  • Sterile drapes
  • 2 curved forceps
  • Small surgical scissors
  • Dissecting microscope
  • 1-ml syringe (e.g., U-100 Micro-Fine 12.7-mm; Becton Dickinson), or a Hamilton syringe
  • Needle holder
  • Culture dish
  • Resorbable sutures (e.g., Ethicon, Vicryl V422 4-0)
  • 9-mm stainless steel wound clips (autoclips from MikRon Precision)
  • Clip applier
  • Additional reagents and equipment for anesthetizing the recipient mouse (Support Protocol 2)

NOTE: Protocols for surgical opening of the abdomen require the use of sterile instruments, surgical gloves, and aseptic procedures to minimize the risk of post-surgical infection.

Alternate Protocol 2: Subcutaneous Injection of hESC

 Additional Materials (see Basic Protocol 1)
  • Phosphate buffered saline, calcium- and magnesium-free (CMF-PBS)
  • 21-G needle

NOTE: Subcutaneous injection of cells does not require anesthesia.

Support Protocol 1: Preparation of hESC for Injection

 Materials
  • Cultures of hESC
  • Enzyme for harvesting
  • Dissecting microscope or stereomicroscope
  • 1-ml insulin syringe
  • Mouth pipet
  • Thick needle
  • Equipment for enzymatic splitting
  • Stereomicroscope
  • Centrifuge

Support Protocol 2: Anesthesia for the Mouse

 Materials
  • Recipient animals
  • Anesthetics (in compliance with local guidelines for major surgery): e.g., isoflurane (1-chloro-2.2.2-trifluoroethyl difluoromethyl ether)
  • Ophthalmic ointment or artificial tears
  • Hypnorm (fentanyl/fluanisone)
  • Anesthesia unit: induction chamber (0.8-liter) and maintenance mask (e.g., Univentor 400; http://www.univentor.com)
  • Surgical tape

Basic Protocol 2: Excision and Fixation for Paraffin Embedding of the hESC Teratoma

 Materials
  • Mouse with teratoma
  • 70% ethanol
  • Sodium pentobarbitone (for perfusion fixation experiments)
  • 4% (w/v) buffered formaldehyde or paraformaldehyde (PFA) in saline
  • Paraffin wax
  • Sterile paper tissue
  • Scissors
  • Forceps curved and straight
  • 10-cm dish
  • Razor blade
  • 50-ml tubes

CAUTION: Formaldehyde is an irritant; avoid skin contact and inhalation of its vapors.

Alternate Protocol 3: Bouin's Fixation of Teratomas

 Additional Materials (also see Basic Protocol 2)
  • Bouin's fixative: 70% (v/v) saturated picric acid (Sigma); 25% (v/v) of 37% to 40% formaldehyde; 5% (v/v) glacial acetic acid (Sigma)
  • Additional reagents and equipment for fixation, embedding, and sectioning tissues (http://home.primus.com.au/royellis/histo.html) and cryosectioning (Hofman, 2002)

Alternate Protocol 4: Tissue Cryopreservation and Preparation for Cryo–Microtome Sectioning

 Materials
  • Freshly excised teratoma tissue
  • Cryomount ( e.g., TissueTek OCT; Sakura)
  • Liquid nitrogen
  • Razor blade
  • Specimen holder/Cryomould
  • Long forceps
  • Cryotube
  • Cryomicrotome
  • SuperFrost+ slides

CAUTION: Use protective gloves and glasses when handling liquid nitrogen. Use long forceps to place holders/cryotubes in liquid nitrogen.

Basic Protocol 4: Paraformaldehyde Fixation and Preparation of Tissues for Immunohistochemistry

 Materials
  • hESC-derived teratomas
  • 4% (w/v) paraformaldehyde (Sigma; 4% (w/v) formaldehyde may also be used)
  • Phosphate-buffered saline (PBS, Sigma)
  • 60%, 70%, 90%, and 95% ethanol
  • Absolute ethanol
  • Histoclear (Sigma) or xylene
  • Paraffin wax and appropriate molds
  • 10 mM citrate buffer (Sigma), pH 6
  • Blocking/washing solution: 1% (w/v) bovine serum albumin (BSA, Sigma)/0.2% (v/v) Triton-X-100 (Sigma)/5% (v/v) normal goat serum (Sigma) in PBS
  • Primary antibody
  • Secondary labeled (e.g., FITC-conjugated) antibody
  • Mounting medium: DPX (Sigma) or aqueous mountant (Vectorlabs)
  • Rotary microtome
  • Microscope slides and coverslips (electrostatically charged for improved section adhesion, Sigma)
  • Microwave oven
  • Fluorescence microscope
  • Digital camera and associated imaging software

Basic Protocol 5: Preparation of Tissues for mRNA Expression Analysis

 Materials
  • hESC-derived teratoma, surgically removed
  • Liquid nitrogen (alternatively dry ice/ethanol bath)
  • RNA purification kit (Ambion)
  • Reverse transcriptase
  • Labeled nucleotide
  • Primer composed of oligo(dT) fused to a bacteriophage T7 promoter
  • T7 polymerase
  • Uridine triphosphate (UTP) and Biotin-16-UTP (e.g., Perkin Elmer Life and Analytical Sciences)
  • RNA amplification kit (e.g., the Illumina RNA Amplification kit; Ambion)
  • Amersham Fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences)
  • Cryostat
  • 1.5-ml nuclease-free microcentrifuge tubes
  • Microarray chip (e.g., the Refseq 6 BeadChip; Illumina, Inc)
  • Confocal scanner and software (e.g., Illumina BeadArray Reader confocal scanner and software (Illumina BeadArray)
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Figures

  • Figure 1B.4.1
    Instrument set up and schematic procedure for the intra testicular implantation. (A) The anesthesia unit comprised of the (1) Univentor × 2, (2) induction chamber, and (3) maintenance mask. (B) Position of the mouse in the maintenance mask and the incision along linea alba (LA). (C,D) hESC injection under the testis capsule. T = testis; F = fat tissue; E = epididymis (E) Effect from injecting too large a volume under the testis capsule.

    View Image
  • Figure 1B.4.2
    Undifferentiated hESC colonies, each containing ~3000 cells.

    View Image
  • Figure 1B.4.3
    Hematoxylin and eosin (HE) histology from formaldehyde-fixed teratoma tissues derived from human ES cells transplanted into the testis of SCID-beige mice and grown for 8 weeks. In (A) two different ectodermal epithelia are shown, (ep) of peridermal character and (p*) a pigmented epithelium possibly of retinal origin; (bn* and bn) intramembraneous bone formation and, (ca) cartilage are also seen. The structures (p*) and (bn*) from (A) are shown in higher magnification in (B). (C) Endodermal epithelia (en-ep) of intestinal character next to a fluid filled cystic (cy) region. (D) Striated muscle (mu) of mesodermal origin and a vessel (ve). Scale bars: (A) 150 µm; (B, C, D) 100 µm.

    View Image
  • Figure 1B.4.4
    Histological analysis of Bouin's fixed teratoma tissues derived from human ES cells transplanted into the testis of severe combined immunodeficient mice and grown for 6 to 8 weeks. The images show an example of tissues representative of each germ layer. (A) Early stage of endochondral ossification, where cartilage (ca) transforms into bone. (B) Structure resembling wall of intestinal tract including surface epithelium (ep) lining crypt-like structures, underlying sub-mucosa (mu), smooth muscle layer (sm), and neural ganglia (ng). (C) Neural ganglion (ng) with associated nerve fiber (nf). (D) Intestinal epithelium viewed under oil immersion using the ×100 objective lens showing high level of cellular detail, including a goblet cell (gc). Histological staining: Weigert's (A,B) and hematoxylin and eosin (C,D). Scale bars: (A,C) 100 µm; (B) 150 µm; (D) 10 µm.

    View Image
  • Figure 1B.4.5
    Immunohistochemical analysis of formaldehyde-fixed teratoma tissue. In these examples, proteins expressed in ectodermal tissue were localized in tumors developed from human ES cells transplanted into the testis of severe combined immunodeficient mice. Teratomas were grown for a period of 6 to 8 weeks. Serial tissue sections (A,B) were taken through a region of neural differentiation, showing the typical morphology of neuroepithelium organized as neural rosettes (nr; A, stained with HE). Neuroprogenitor cells are thought to reside within the rosette, whereas more mature neural tissues are located around the periphery of these proliferative centers, as indicated by the staining of -tubulin-III (B), a marker of more mature neural cells. Other types of epithelia identified within the teratoma that lined surfaces and cavities were not neural in nature and possessed a distinctly different morphology (C). These epithelia stained positive for human epidermal keratin (D) but not markers of the neural lineage. Scale bars: (A,B) 150 µm; (C,D) 75 µm.

    View Image

Literature Cited

Literature Cited
    Allison, D.B., Cui, X., Page, G.P., and Sabripour, M. 2006. Microarray data analysis: from disarray to consolidation and consensus. Nat. Rev. Genet. 7:55-65.
    Ang, S.L., Wierda, A., Wong, D., Stevens, K.A., Cascio, S., Rossant, J., and Zaret, K.S. 1993. The formation and maintenance of the definitive endoderm lineage in the mouse: Involvement of HNF3/forkhead proteins. Development 119:1301-1315.
    Bosma, G.C., Custer, R.P., and Bosma, M.J. 1983. A severe combined immunodeficiency mutation in the mouse. Nature 301:527-530.
    Cai, J., Chen, J., Liu, Y., Miura, T., Luo, Y., Loring, J.F., Freed, W.J., Rao, M.S., and Zeng, X. 2006. Assessing self-renewal and differentiation in human embryonic stem cell lines. Stem Cells 24(3):516-530.
    Candia, A.F. and Wright, C.V. 1996. Differential localization of Mox-1 and Mox-2 proteins indicates distinct roles during development. Int. J. Dev. Biol. 40:1179-1184.
    Candia, A.F., Hu, J., Crosby, J., Lalley, P.A., Noden, D., Nadeau, J.H., and Wright, C.V. 1992. Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during early mesodermal patterning in mouse embryos. Development 116:1123-1136.
    Conlon, F.L., Lyons, K.M., Takaesu, N., Barth, K.S., Kispert, A., Herrmann, B., and Robertson, E.J. 1994. A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. Development 120:1919-1928.
    Cooke, M.J., Stojkovic, M., and Przyborski, S.A. 2006. Growth of teratomas derived from human pluripotent stem cells is influenced by the graft site. Stem Cells Dev. 15:254-259.
    Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1-1.8.4.
    Ellis, P., Fagan, M., Magness, S.T., Hutton, S., Taranova, D., Hayashi, S., McMahon, A., Rao, M., and Pevny, L. 2004. SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult. Dev. Neurosci. 26:148-165.
    Elms, P., Scurry, A., Davies, J., Willoughby, C., Hacker, T., Bogani, D., and Arkell, R. 2004. Overlapping and distinct expression domains of Zic2 and Zic3 during mouse gastrulation. Gene Expr. Patterns 4:505-511.
    Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154-156.
    Flecknell, P.A. 1993. Anesthesia and perioperative care. Methods Enzymol. 225:16-33.
    Gertow, K., Wolbank, S., Rozell, B., Sugars, R., Andang, M., Parish, C.L., Imreh, M.P., Wendel, M., and Ahrlund-Richter, L. 2004. Organized development from human embryonic stem cells after injection into immunodeficient mice. Stem Cells Dev. 13:421-435.
    Gertow, K., Cedervall, J., Bogdanovic, N., Szöke, K., Kärner, E., Imreh, M.P., and Ährlund-Richter, L. 2006. hESC in vivo xeno-grafting results in an initial growth of ectodermal origin, followed by processes akin to normal human development. Submitted.
    Grinnemo, K.H., Mansson-Broberg, A., Leblanc, K., Corbascio, M., Wardell, E., Siddiqui, A.J., Hao, X., Sylven, C., and Dellgren, G. 2006. Human mesenchymal stem cells do not differentiate into cardiomyocytes in a cardiac ischemic xenomodel. Ann. Med. 38:144-153.
    Hart, A.H., Hartley, L., Sourris, K., Staddler, E.S., Li, R., Stanley, E.G., Tam, P.P., Elefanty, A.G., and Rob, L. 2002. Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo. Development 129:3597-3608.
    Hatta, K. and Takeichi, M. 1986. Expression of N-cadherin adhesion molecules associated with early morphogenetic events in chick development. Nature 320:447-449.
    Hayman, M.W., Smith, K.H., Cameron, N.R., and Przyborski, S.A. 2004. Enhanced neurite outgrowth by human neurons grown on solid three-dimensional scaffolds. Biochem. Biophys. Res. Commun. 314:483-488.
    Heins, N., Englund, M.C., Sjoblom, C., Dahl, U., Tonning, A., Bergh, C., Lindahl, A., Hanson, C., and Semb, H. 2004. Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells 22:367-376.
    Hofman, F. 2002. Immunohistochemistry. Curr. Protoc. Immunol. 49:21.4.1-21.4.23.
    Hovatta, O., Mikkola, M., Gertow, K., Stromberg, A. M., Inzunza, J., Hreinsson, J., Rozell, B., Blennow, E., Andang, M., and Ahrlund-Richter, L. 2003. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18:1404-1409.
    Ito, M., Hiramatsu, H., Kobayashi, K., Suzue, K., Kawahata, M., Hioki, K., Ueyama, Y., Koyanagi, Y., Sugamura, K., Tsuji, K., Heike, T., and Nakahata, T. 2002. NOD/SCID/gamma(c)(null) mouse: An excellent recipient mouse model for engraftment of human cells. Blood 100:3175-3182.
    Keller, G. 2005. Embryonic stem cell differentiation: Emergence of a new era in biology and medicine. Genes Dev. 19:1129-1155.
    Kunath, T., Amaud, D., Uy, G.D., Okamota, I., Churaeu, C., Yamanaka, Y., Heard, E., Gardner, R.L., Avner, P., and Rossant, J. 2005. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development 132:1649-1661.
    Lanzendorf, S.E., Boyd, C.A., Wright, D.L., Muasher, S., Oehninger, S., and Hodgen, G.D. 2001. Use of human gametes obtained from anonymous donors for the production of human embryonic stem cell lines. Fertil. Steril. 76:132-137.
    Levenberg, S., Golub, J.S., Amit, M., Itskovitz-Eldor, J., and Langer, R. 2002. Endothelial cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 99:4391-4396.
    Levenberg, S., Huang, N.F., Lavik, E., Rogers, A.B., Itskovitz-Eldor, J., and Langer, R. 2003. Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proc. Natl. Acad. Sci. U.S.A. 100:12741-12746.
    Li, H., Liu, Y., Shin, S., Sun, Y., Loring J. F., Mattson, M.P., Rao, M.S., and Zhan, M. 2006. Transcriptome coexpression map of human embryonic stem cells. BMC Genomics 7:103.
    Li, L., Baroja, M.L., Majumdar, A., Chadwick, K., Rouleau, A., Gallacher, L., Ferber, I., Lebkowski, J., Martin, T., Madrenas, J., and Bhatia, M. 2004. Human embryonic stem cells possess immune-privileged properties. Stem Cells 22:448-456.
    Liu, Y., Shin, S., Zeng, X., Zhan, M., Gonzalez, R., Mueller, F.J., Schwartz, C.M., Xue, H., Li, H., Baker, S.C., Chudin, E., Barker, D.L., McDaniel, T.K., Oeser, F., Loring, J.F., Mattson, M.P., and Rao, M.S. 2006. Genome wide profiling of human embryonic stem cells (hESCs), their derivatives and embryonal carcinoma cells to develop base profiles of U.S. Federal government approved hESC lines. BMC Dev. Biol. 6:20.
    Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K., and Tochino, Y. 1980. Breeding of a non-obese, diabetic strain of mice. Jikken Dobutsu 29:1-13.
    Martin, G. R. 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. U.S.A. 78:7634-7638.
    McDaniel, T., Baker, S., Williams, R., Mueller, F-J., and Barker, D. 2007. Gene expression profiling of stem cells by microarray. In Human Stem Cell Manual: A Laboratory Guide (J.F. Loring, P.H. Schwartz, and R.L. Wesselschmidt, eds.) pp. 149-161. Academic Press Elsevier, New York.
    Mikkola, M., Olsson, C., Palgi, J., Ustinov, J., Palomaki, T., Horelli-Kuitunen, N., Knuutila, S., Lundin, K., Otonkoski, T., and Tuuri, T. 2006. Distinct differentiation characteristics of individual human embryonic stem cell lines. BMC Dev. Biol. 6:40.
    Mitalipova, M., Calhoun, J., Shin, S., Wininger, D., Schulz, T., Noggle, S., Venable, A., Lyons, I., Robins, A., and Stice, S. 2003. Human embryonic stem cell lines derived from discarded embryos. Stem Cells 21:521-526.
    Monaghan, A.P., Kaestner, K.H., Grau, E., Schultz, G. 1993. Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 alpha, beta, and gamma genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm. Development 119:567-578.
    Mosier, D.E., Stell, K.L., Gulizia, R.J., Torbett, B.E., and Gilmore, G.L. 1993. Homozygous scid/scid;beige/beige mice have low levels of spontaneous or neonatal T cell-induced B cell generation. J. Exp. Med. 177:191-194.
    Nagai, T., Aruga, J., Takada, S., Günther, T., Spörle, R., Schughart, K., and Mikoshiba, K. 1997. The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation. Dev. Biol. 182:299-313.
    Nieto, M.A., Bennett, M.F., Sargent, M.G., and Wilkinson, D.G. 1992. Cloning and developmental expression of Sna, a murine homologue of the Drosophila snail gene. Development 116:227-237.
    Park, J.H., Kim, S.J., Oh, E.J., Moon, S.Y., Roh, S.I., Kim, C.G., and Yoon, H.S. 2003. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol. Reprod. 69:2007-2014.
    Pearce, J.J. and Evans, M.J. 1999. Mml, a mouse Mix-like gene expressed in the primitive streak. Mech. Dev. 87:189-192.
    Pesce, M. and Scholer, H.R. 2001. Oct-4: Gatekeeper in the beginning of mammalian development. Stem Cells 19:271-278.
    Pevny, L.H., Sockanathan, S., Placzek, M., and Lovell-Badge, R. 1998. A role for SOX1 in neural determination. Development 125:1967-1978.
    Pickering, S.J., Minger, S.L., Patel, M., Taylor, H., Black, C., Burns, C.J., Ekonomou, A., and Braude, P.R. 2005. Generation of a human embryonic stem cell line encoding the cystic fibrosis mutation deltaF508, using preimplantation genetic diagnosis. Reprod. Biomed. Online 10:390-397.
    Plaia, T.W., Josephson, R., Liu, Y., Zeng, X., Ording, C., Toumadje, A., Brimble, S.N., Sherrer, E.S., Uhl, E.W., Freed, W.J., Schulz, T.C., Maitra, A., Rao, M.S., Auerbach, J.M., 2006. Characterization of a new NIH-registered variant human embryonic stem cell line, BG01V: A tool for embryonic stem cell research. Stem Cells 24:531-546.
    Przyborski, S.A. 2005. Differentiation of human embryonic stem cells after transplantation in immune-deficient mice. Stem Cells 23:1242-1250.
    Radice, G.L., Rayburn, G.L., Matsunami, H., Knudsen, K.A., Takeichi, M., and Hynes, R.O. 1997. Developmental defects in mouse embryos lacking N-cadherin. Dev. Biol. 181:64-78.
    Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A. 2000. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18:399-404.
    Richards, M., Fong, C.Y., Chan, W.K., Wong, P.C., and Bongso, A. 2002. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20:933-936.
    Roder, J. and Duwe, A. 1979. The beige mutation in the mouse selectively impairs natural killer cell function. Nature 278:451-453.
    Rosner, M.H., Vigano, M.A., Ozato, K., Timmons, P.M., Poirier, F., Rigby, P.W., and Staudt, L.M. 1990. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345:686-692.
    Sasaki, H. and Hogan, B.L. 1993. Differential expression of multiple fork head genes during gastrulation and axial pattern formation in the mouse embryo. Development 118:47-59.
    Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D.A., and Benvenisty, N. 2000. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 97:11307-11312.
    Schwartz, C.M., Spivak, C.E., Baker, S.C., McDaniel, T.K., Loring, J.F., Nguyen, C., Chrest, F.J., Wersto, R., Arenas, E., Zeng, X., Freed, W.J., and Rao, M.S. 2005. NTera2: A model system to study dopaminergic differentiation of human embryonic stem cells. Stem Cells Dev. 14:517-534.
    Shook, D. and Keller, R. 2003. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech. Dev. 120:1351-1383.
    Stemmler, M.P., Hecht, A., Kinzel, B., and Kemler, R. 2003. Analysis of regulatory elements of E-cadherin with reporter gene constructs in transgenic mouse embryos. Dev. Dyn. 227:238-245.
    Stevens, L.C. 1967. The biology of teratomas. Adv. Morphog. 6:1-31.
    Stojkovic, M., Lako, M., Stojkovic, P., Stewart, R., Przyborski, S., Armstrong, L., Evans, J., Herbert, M., Hyslop, L., Ahmad, S., Murdoch, A., and Strachan, T. 2004a. Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture. Stem Cells 22:790-797.
    Stojkovic, M., Lako, M., Strachan, T., and Murdoch, A. 2004b. Derivation, growth and applications of human embryonic stem cells. Reproduction 128:259-267.
    Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145-1147.
    Tzukerman, M., Rosenberg, T., Ravel, Y., Reiter, I., Coleman, R., and Skorecki, K. 2003. An experimental platform for studying growth and invasiveness of tumor cells within teratomas derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 100:13507-13512.
    Van Vliet, G. 2003. Development of the thyroid gland: Lessons from congenitally hypothyroid mice and men. Clin. Genet. 63:445-455.
    Varlet, I., Collignon, J., and Robertson, E.J. 1997. Nodal expression in the primitive endoderm is required for specification of the anterior axis during mouse gastrulation. Development 124:1033-1044.
    Waynforth, H.B. and Flecknell, P.A. 1999. Experimental and Surgical Techniques. Academic Press.
    Wilkinson, D.G., Bhatt, S., and Herrmann, B.G. 1990. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343:657-659.
    Winnier, G., Blessing, M., Labosky, P.A., and Hogan., B.L. 1995. Bone morpohogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9:2105-2116.
    Yao, S., Chen, S., Clark, J., Hao, E., Beattie, G.M., Hayek, A., and Ding, S. 2006. Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions. Proc. Natl. Acad. Sci. U.S.A. 103:6907-6912.
    Zhou, X., Sasaki, H., Lowe, L., Hogan, B.L., and Kuehn, M.R. 1993. Nodal is a novel TGF-beta-like gene expressed in the mouse node during gastrulation. Nature 361:543-547.
 Key References
    Hofman, F. 2002. See above.

Detailed knowledge on histology and collections of protocols used in histology.

    Van Zutphen, L.F.M., Baumans, V., Beynes, A.C. 2001. Principles of Laboratory Animal Science; Revised edition. Amsterdam, Netherlands.

This book covers the main theoretical aspects of laboratory animal science.

    Waynforth and Flecknell, 1999. See above.

This book covers standard surgical procedures

 Internet Resources
    http://iacuc.cwru.edu/policy/nihpolicies/surguide.htm

To learn more about animal experimentation, particularly rodent surgery, the authors recommend NIH Guidelines for Rodent Surgery.

    http://home.primus.com.au/royellis/histo.html

Detailed knowledge on histology and collections of protocols used in histology.

    http://www.ncbi.nlm.nih.gov/geo

Published expression patterns of adult tissues.

    http://www.stemcellcommunity.org

Provides expression profiles of the same genes in the HESCs.

    http://www-stat.stanford.edu/~tibs/SAM/

Further reading on Significance Analysis for Microarrays (SAM) used to obtain lists of genes that are up or down regulated within a given microarray dataset.

     
 
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