AATEX 14, Special Issue, 601-603 Proc. 6th World Congress on Alternatives & Animal Use in the Life Sciences August 21-25, 2007, Tokyo, Japan
Development of defined medium for mouse, monkey and human ES cell culture
Miho Kusuda Furue1,2, Jie Na3, Jamie P. Jackson3, Takamichi Miyazaki2, Kei Takada2, Hirofumi Suemori2, Ryu-Ichiro Hata1, Norio Nakatsuji2, Tetsuji Okamoto4, J. Denry Sato5 and Peter W. Andrews3 1
Institute for Frontier Oral Science, Kanagawa Dental College, 2 Institute for Frontier Medical Sciences, Kyoto University, 3 The Centre for Stem Cell Biology, The University of Sheffield, Western Bank, 4 Graduate Science School of Biomedical Sciences, Hiroshima University, 5 Mount Desert Island Biological Laboratory Corresponding author: Miho Kusuda Furue Division of Bioresources, National Institute of Biomedical Innovation 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan
[email protected]
Abstract Embryonic stem (ES) cells have great potential for tissue engineering application. However, major limitations in the use of the human ES cells for a variety of biotechnological applications are the current requirement for the use of feeder cells, and relatively poor understanding of their responses to growth factors to manipulate cell differentiation. Studies using mouse or monkey ES cells are useful for understanding of ES cell characteristic. Generally, mouse ES cells are maintained on mouse embryonic fibroblast (MEF) cells in a medium supplemented with serum, and human ES cells are also maintained on MEF in a medium supplemented with knock-out serum-replacement or on matrigel with an MEF-conditioned medium (CM). These culture conditions hamper the cell biological analysis of ES cells due to the presence of various unknown products. We have established a defined serum-free ESF7 medium for mouse ES cells without feeders or BMP4. In this medium, mouse ES cells express Oct-3/4, Nanog and Sox2. Building on this experience, we have developed a defined serum-free medium for the culture of human ES cells. We are now testing this culture medium for monkey ES cells. These culture conditions may be useful for understanding of ES cell responses to differentiation factors. Keywords: embryonic stem cells, defined medium, Oct-3/4, LIF, feeder-free
Introduction Embryonic stem (ES) cells derived from the inner cell mass of preimplantation embryos are pluripotent, retaining the ability to differentiate into all three germ layers, and they have the potential to generate all differentiated cell types in vitro (Martin and Evans 1975; Evans and Kaufman 1981). Thus, ES cells represent a model of mammalian development that is amenable to biochemical and molecular analyses (Smith 2001). In recent years, human ES cells since their first derivation have a lot of attention as a source for regeneration cell therapy (Thomson, ItskovitzEldor et al. 1998). Murine ES (mES) cells are commonly maintained on primary mouse embryonic fibroblast feeder cells in culture medium supplemented with bovine serum and leukaemia inhibitory factor (LIF) (Pease and Williams 1990; Smith 2001). In the absence of LIF, mES cells differentiate spontaneously in serum-
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containing culture medium. Culture protocols have been recently developed that permit the derivation of some types of cells from undifferentiated ES cells. However, these procedures require the cultivation o f E S c e l l a g g r e g a t e s ( Wi l e s a n d J o h a n s s o n 1999), conditioned medium containing undefined components (Lake, Rathjen et al. 2000), or feeder cells (Kawasaki, Mizuseki et al. 2000), and produce variable and heterogeneous results. Serum contains variable amounts of soluble factors including activins and fibroblast growth factors and feeder cells secrete also secrete variable factors. Differentiation protocols based upon knowledge of ES cell responses to specific growth factors are required for various types of mouse cells in defined culture condition. Johansson and Wiles (Johansson and Wiles 1995), and Ying, et al. (Ying, Sravridis et al. 2003) reported that populations with a non-ES cell morphology arose when ES cells were maintained for more than three
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passages in the absence of feeder cells in chemically defined serum-free media containing LIF (10 ng/ml). In contrast to their report, we developed a chemically defined serum-free culture medium containing LIF for mouse ES cells, designated ESF7 (Furue, Okamoto et al. 2005). In this simple serum-free adherent monolayer culture condition in the absence of feeder cells, LIF promotes ES cell proliferation and selfrenewal, and withdrawal of LIF from this medium resulted in ES cell apoptosis. Craniofacial tissues are mainly constituted of the neural crest. The neural crest is a migratory population of multipotent cells derived from the lateral edges of the neural plates. These cells differentiate into various cell types, including neurons and glia of the peripheral nervous system, craniofacial skeletal elements, smooth muscle cells and melanocytes (Dupin, Real et al. 2001). Our previous studies in Xenopus have shown that the developmental fate of undifferentiated presumptive ectodermal cells can be decided by concentration-sensitive inducing factors, such as activin A, FGF-2, and BMP4 (Furue and Asashima 2004). In this study, we tried to induce neural crest cells from mouse, monkey and human ES cells in adherent monolayer defined culture condition using growth factors. Materials and methods Cell culture The murine D3 embryonic stem cell (mES-D3) line (ATCC) was cultured in 75cm 2 plastic flasks (Corning, NY, U.S.A.) coated with type I collagen (Nitta gelatine, Tokyo, Japan) in a humidified atmosphere of 5% CO2 at 37°C in ESF7 medium Cell Science & Technology Institute, Inc., Tokyo, Japan). ESF7 consisted of ESF basal medium (Cell Science & Technology Institute) supplemented with insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, sodium selenite, oleic acid conjugated with fatty acid-free bovine serum albumin (FAF-BSA; Sigma, St. Louis, Missouri), and 10 ng/ml LIF (Chemicon, Billerica, Massachusetts), as described previously (Furue, Okamoto et al. 2005). Monkey and human ES cells were cultured in 25cm2 plastic flasks coated with type I collagen in modified ESF7 medium containing fibroblast growth factor-2 (submitted). ES differentiation in vitro. mES-D3 cells were inoculated on 5 μ g/cm 2 laminin- (Sigma, St. Louis, MO, U.S.A.) coated cell disks (Sumitomo Bakelite Co., Tokyo, Japan) in ESF5 medium supplemented with 10 ng/ml FGF-2 (Upstate Biotechnology, Inc. Lake Placid, NY. U.S.A.). ESF5 consisted of ESF basal medium supplemented with insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, and sodium selenite, supplemented with. Cells cultured with the growth factors and
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100 ng/ml heparin (Sigma) After 2 days-culture, the cells were then cultured without the growth factors and heparin for more 4 days or more. Cultures were scored for cells with differentiated morphologies on day 6. Immunohistochemical analysis Detailed protocol was described previously (Furue, Okamoto et al. 2005). Briefly, for the cell surface differentiation antigen A2B5, unfixed cells were incubated with mouse anti-A2B5 antibody (Chemicon, Temecula, CA, U.S.A.) and then incubated with FITC-conjugated goat anti-mouse IgG (Sigma). For intracellular differentiation antigens, fixed and permeabilized cells were immunostained with antinestin antibody (BD Biosciences Immunocytometry Systems, Rockville, MD, U.S.A.), mouse antiβ - t u b u l i n I I I ( C h e m i c o n ) , r a b b i t a n t i - G FA P (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.). Primary antibodies were detected with FITC-conjugated goat anti-mouse IgG (Sigma) or FITC-conjugated goat anti-rabbit IgG (Sigma). 4,6-diamidino-2-phenylindole (DAPI) (Sigma) was used as a counterstain. Results and discussion Ying reported that BMP4 was required for maintaining undifferentiated mES cells even in the presence of LIF in defined medium and withdrawal of LIF promoted ES cell differentiation into neuronal and glial cells (Ying, Nichols et al. 2003; Ying, Sravridis et al. 2003). In contrast, in ESF7 medium containing LIF, undifferentiated mES-D3 cells can grow without BMP4 for more than 2 years (Fig. 1A), and withdrawal of LIF resulted in apoptosis of the cells (Furue, Okamoto et al. 2005). Addition of FGF-2 stimulated ES-D3 cell differentiation into neuronal cells when inoculated on laminin-coated dishes in ESF7 medium deleted of LIF and oleic acid (Furue, Okamoto et al. 2005). The induced populations of cells (Fig. 1B) were immunoreactive with antibodies to nestin, β-tubulin III, A2B5 antigen, and GFAP (Fig. 2). These results indicated that addition of FGF-2 induced ES-D3 cell differentiation into neuronal
Fig. 1: Phase-contrast photomicrograph of ES-D3 cells. ES D3 cells cultured in ESF7 medium on a collagen-coated plate (A). ES-D3 cells differentiating into neuron-like cells after the withdrawal of FGF-2 (B). This figure is cited from (Furue and Hata 2005.)
Fig. 2: Neuronal and glial differentiation of ES-D3 cells by FGF-2. The cells were immunoreactive with antibodies to nestin (A), β-tubulin III (B), A2B5 (C), and GFAP (D). This figure is cited from (Furue and Hata 2005.)
and glial cells. We determined the expression of a primitive ectoderm gene, FGF-5, a neural crest marker gene, AP-2, and anterior marker gene, Otx-2 in these cells, using real-time RT-PCR method (data not shown). These results suggested that ES cells differentiated into neuronal and glial cells through primitive ectoderm cells and neural crest cells. Next, we have developed a defined culture condition for human ES cells (submitted) and tried to differentiate neuronal cells. We are now trying to culture monkey ES cells in the defined condition. Further analysis of this protocol will lead to understand the mechanisms of neural crest cell differentiation. Acknowledgements A part of this work was done in collaboration with Mr. Yohei Hayahsi, Dr. Makoto Asashima (The University of Tokyo). This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Culture, Sports, Science and Technology of Japan. References
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