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Development of cystic embryoid bodies with visceral yolk-sac-like structures from mouse embryonic stem cells using low-adherence 96-well plate
Journal of Bioscience and Bioengineering VOL. 107 No. 4, 442 – 446, 2009 www.elsevier.com/locate/jbiosc
Development of cystic embryoid bodies with visceral yolk-sac-like structures from mouse embryonic stem cells using low-adherence 96-well plate Emiko Yasuda,1 Yuji Seki,1 Takatoshi Higuchi,1 Fumio Nakashima,2 Tomozumi Noda,2 and Hiroshi Kurosawa1,⁎ Division of Medicine and Engineering Science, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan 1 and Tsukuba Research Laboratory Section II, Life Science Division, NOF Corporation, 5-10 Tokodai, Tsukuba, Ibaraki 300-2635, Japan 2 Received 17 October 2008; accepted 9 December 2008
Cystic embryoid bodies with visceral yolk-sac-like structure (cystic EB-Vs) are used as a model for the study of early extraembryonic tissue formation containing visceral endoderm-like derivatives. In this study, we optimized the cell density of embryonic stem (ES) cells for developing cystic EB-Vs in a low-adherence 96-well plate. When ES cells were seeded at a density of 4000 cells/well, the cystic EB-Vs were most efficiently developed from ES cells via forming multicellular spherical aggregates called embryoid bodies (EBs). The suspension culture in the low-adherence plate was preferable for developing EBs into cystic EB-Vs rather than the attachment culture in the plate coated with 0.1% gelatin. The seeding cell density of 4000 cells/well was always superior to 1000 cells/well in the efficiency of cystic EB-V development. Because the high-cell density culture generally raises the limitation of oxygen and nutrient supplies, we investigated the effects of low-oxygen and low-nutrient conditions on the development of cystic EB-Vs. It was found that low oxygen tension was not a factor for promoting the development of cystic EB-Vs. It was suggested that a low-nutrient medium is preferred for developing cystic EB-Vs rather than a sufficient-nutrient medium. In conclusion, the suspension culture in the low-adherence 96-well plate seeded with 4000 ES cells/well was optimum for developing cystic EB-Vs. The low-nutrient condition may be one of the factors for promoting the development of cystic EB-Vs. © 2008, The Society for Biotechnology, Japan. All rights reserved. [Key words: ES cells; Embryoid body; Cystic EBs; MPC; Visceral yolk sac]
Embryonic stem (ES) cells are self-renewing and theoretically immortal cells derived from the inner cell mass of developing blastocysts (1–3). A principal method of inducing the in vitro differentiation of ES cells is to form multicellular spherical aggregates of ES cells called embryoid bodies (EBs) (4,5). They recapitulate many aspects of cell differentiation during early embryogenesis and differentiate into derivatives of all three germ layers (6,7). EB formation has been employed as an in vitro differentiation system for not only ES cells (4,8) but also induced pluripotent stem (iPS) cells (9,10). Therefore, the quality (i.e., differentiation status) of EBs affects the generation of derivatives from the EBs in a subsequent differentiation culture. The term “EBs” has been widely used by many researchers to indicate embryoid bodies in the wide differentiation stage, even though their characteristics are varied embryologically and morphologically. Strictly speaking, the EBs are classified as simple EBs and cystic EBs according to the differentiation stage (11–13). Simple EBs represent the spherical ES cell aggregates with morula-like structures, which have no central cavity. In some cases, cystic EBs represent the ⁎ Corresponding author. Fax: +81 55 220 8536. E-mail address: [email protected] (H. Kurosawa).
cavitating EBs of a regular form with blastula-like structures. In other cases, cystic EBs represent the expanding EBs with visceral yolk-saclike structure. In this study, cystic EBs with visceral yolk-sac-like structures displaying inconsistent morphology are referred to as cystic EB-Vs. In a previous report, we have described that using a low-adherence 96-well plate is appropriate for the formation of EBs with high uniformity from mouse ES cells, and the density of ES cells seeded in a well affects the differentiation status of EBs (14). When the seeding cell density was increased from 1000 to 4000 cells/well, the EBs that resulted from 4000 cells/well showed markedly high expression levels of marker genes for endoderm of transthyretin (TTR) and αfetoprotein (AFP) compared with the EBs that resulted from 1000 cells/well. Cystic EB-Vs have been expected as a model for the study of early extraembryonic tissue formation containing visceral endoderm-like derivatives (15). Therefore, the reproducible development of ES cells into cystic EB-Vs is important to establish an efficient experimental system and produce reliable data. In general, the formation of cystic EB-Vs is achieved following a relatively long period of suspension culture of ES cells. The low-adherence 96-well plate is considered to be appropriate for suspension culture for developing cystic EB-Vs. In
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FIG. 1. Schematic of the culture procedures for cystic EB-V development.
this study, first, we optimized the density of ES cells for developing cystic EB-Vs in the low-adherence 96-well plate. Second, we investigated the effects of low oxygen tension and low nutrient supply on the development of cystic EB-Vs. MATERIALS AND METHODS ES cell culture Mouse ES cells of the cell line 129/SvEv (Thromb-X; N.V. Stem Cell Technologies, Leuven, Belgium) were maintained in Dulbecco's modified Eagle's medium (DMEM [high glucose], Invitrogen, Carlsbad, CA, USA) supplemented with 20% knockout serum replacement (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma, St. Louis, MO, USA), 50 U/ml penicillin, 50 μg/ml streptomycin (Invitrogen), and 1000 U/ ml murine leukemia inhibitory factor (mLIF; Chemicon, Temecula, CA, USA) on 0.1% gelatin-coated dish with feeder layer cells at 37 °C in humidified air with 5% CO2. As feeder layer cells, STO fibroblasts treated with 10 μg/ml mitomycin C (Sigma) were used. For these experiments, ES cells were grown for a maximum of 22 passages in the presence of feeder layer cells and mLIF. EB formation in low-adherence 96-well plate ES cells were dissociated with 0.1% trypsin-EDTA (Invitrogen) and suspended in Iscove's modified Dulbecco's medium (IMDM, Invitrogen) supplemented with 20% fetal bovine serum (Invitrogen), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 50 U/ ml penicillin, and 50 μg/ml streptomycin (EB medium). An ES cell suspension was poured into round-bottomed wells of a low-adherence 96-well polystyrene plate coated with 2-methacryloyloxethyl phosphorylcoline (MPC) (Lipidure coat®, NOF Co., Tokyo). Each well contained ES cells at a density of 1000 or 4000 cells per 200 μl of medium. The low-adherence 96-well plates were statically incubated to form EBs at 37 °C in humidified air with 5% CO2 for 5 days. The EBs that resulted from 1000 and 4000 cells by culture day 5 are referred to as EB5-1000 and EB5-4000, respectively. Direct development of cystic EB-Vs from ES cells in 96-well MPC plates The procedures for cystic EB-V development are schematically shown in Fig. 1. Cystic EB-Vs were developed from ES cells by one continuous operation using the low-adherence 96-well plates. To investigate the effect of cell density on cystic EB-V development, ES cells were seeded at the densities of 500, 1000, 4000, 8000, 10,000, and 20,000 cells/ well in 200 μl of EB medium and cultured for 20 days in the incubator with 20% O2 atmosphere. To investigate the effect of low oxygen tension, the ES cells were seeded at the densities of 1000 and 4000 cells/well in 200 μl of EB medium and cultured for 20 days under oxygen tension of 5% O2. To investigate the effect of low nutrient supply, the ES cells were seeded at the densities of 1000 cells/well and cultured for 14 days in 200 μl of EB medium based on DMEM. The EB medium based on DMEM was prepared by just replacing IMDM of the EB medium with DMEM. From culture day 5, half of the medium was changed daily. Periodically, microscopy was performed to detect the development of cystic EB-Vs in the wells. When the size of EBs increased drastically and cystic sphere with clear boundary appeared, we decided that cystic EB-Vs were developed. Three independent experiments were conducted for each cell density. Development of cystic EB-Vs from 5-d EBs in suspension and attachment cultures As illustrated in Fig. 1, the above-mentioned 5-d-old EBs (EB5-1000 and EB5-4000) were transferred to two types of culture system, which are suspension culture and attachment culture, to induce the organization of visceral yolk-sac-like structures in the EBs. In the suspension culture system, one EB was transferred into each well of a 24-well plate coated with MPC (low-adherence 24-well plate) containing 1 ml of EB medium. In the attachment culture system, one EB was placed on each well of a 24-well plate coated with 0.1% gelatin containing 1 ml of EB medium. Cultivations were performed for 15 days and half of the medium was changed every 3 days. Periodically, microscopy was performed to detect the development of cystic EB-Vs in the wells. Three independent experiments were conducted for each culture system.
Quantitative real-time PCR analysis The expression levels of several specific marker genes in EBs were quantitatively evaluated by real-time PCR analysis using a DNA Engine Opticon™ system (MJ Research Inc, Waltham, MA, USA). The GATA transcription factors GATA-4 (16, 17) was regarded as marker that is expressed in endoderm- and mesoderm-derived lineages in EBs. Transthyretin (TTR) and α-fetoprotein (AFP) were used as the markers specifically expressed in the visceral endoderm of yolk sac (18, 19). The gene of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an internal reference. Total RNA was extracted from EBs using the Micro-to-Midi Total RNA Purification system (Invitrogen, Carlsbad, CA, USA). To prepare a sample for respective control, total RNA was extracted from the dissociated ES cells in the same manner, just before transferring the cells to the EB formation culture. The quality of each RNA sample was analyzed using a UV–Vis spectrophotometer for RNA quality (A260/A280 N1.8). RNA integrity was verified electrophoretically by ethidium bromide staining. Complementary DNA (cDNA) was synthesized from 5 μg of total RNA using the SuperScript First-Strand Synthesis system for RT-PCR (Invitrogen) according to the manufacturer's instructions. Two microliters of cDNA template was added to 12.5 μl of the 2× SYBR Premix Ex Taq (Takara Bio, Otsu) and 10 pmol of each specific primer. Reactions were carried out for 45 cycles. The cycling parameters were as follows: denaturation at 95 °C for 5 s, and annealing and elongation at 60 °C for 20 s. The primer sequences were as follows: G3PDH, 5′-ACCACAAGTCCATGCCATCAC-3′ sense and 5′TCCACCACCCTGTTGCTGTA-3′ antisense; GATA-4, 5′-GAAAACGGAAGCCCAAGAACC-3′ sense and 5′-TGCTGTGCCCATAGTGAGATGAC-3′ antisense; TTR, 5′-CTCACCACAGATGAGAAG-3′ sense and 5′-GGCTGAGTCTCTCAATTC-3′ antisense; AFP, 5′-TCGTATTCCAACAGGAGG-3′ sense and 5′-AGGCTTTTGCTTCACCAG-3′ antisense. Fluorescence intensity was measured at the end of the annealing-extension phase of each cycle. The threshold value for the fluorescence intensity of all the samples was set manually. The reaction cycle at which PCR products exceeded this fluorescence intensity threshold was identified as the threshold cycle (CT) in the exponential phase of the PCR amplification. Relative quantification The expression levels of these target genes were quantified against that of the internal reference gene (G3PDH). Relative quantification is based on the comparison of the CT at a constant fluorescence intensity (20, 21). The amount of transcript present is inversely related to the observed CT, and for every twofold
FIG. 2. Effect of cell density on cystic EB-V development. The percentage of cystic EB-V development is the ratio of the wells in which cystic EB-Vs were observed on culture day 20 to the wells seeded with ES cells. Forty-eight wells were seeded with 200 μl of ES cell suspension of each cell density. Bar: standard error of the mean (n = 3).
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FIG. 3. Cumulative changes in the percentage of cystic EB-V development in the suspension and attachment cultures. The percentage of cystic EB-V development is the ratio of the wells in which cystic EB-Vs were observed to the 24 wells seeded with 5-d-old EBs. (A) 5-d-old EBs that resulted from 1000 ES cells (EB5-1000) were seeded; (B) 5-d-old EBs that resulted from 4000 ES cells (EB5-4000) were seeded. Symbols: open circles, suspension culture of EB5-1000; closed circles, attachment culture of EB5-1000; open squares, suspension culture of EB5-4000; closed squares, attachment culture of EB5-4000. The arrow indicates sample collection for quantitative RT-PCR analysis. Bar: standard error of the mean (n = 3). dilution in transcript, the CT is expected to increase by 1. The relative expression (R) is calculated using the equation R = 2− [ΔCT sample − ΔCT control]. That is, to determine a normalized arbitrary value for each gene, every data point was normalized to the reference gene (G3PDH), as well as to their respective control. Data (mean± SEM) are from triplicate runs of one sample set representative of three independent experiments.
RESULTS Effect of cell density on cystic EB-V development The optimum density of ES cells for developing cystic EB-Vs was investigated. Fig. 2 shows the ratio of the wells in which cystic EB-Vs were observed on culture day 20 to the wells seeded with ES cells (48 wells). As can be seen in Fig. 2, cystic EB-Vs were observed with more than 80% efficiency when the ES cells were seeded at the densities of 1000 and 4000 cells/well. Increasing the density to over 4000 cells/ well resulted in decreased efficiency and increased deviation. Developments of cystic EB-V from 5-d-old EBs in suspension and attachment cultures Two types of 5-d-old EB (EB5-1000 and EB5-4000) were transferred to the culture systems of suspension and attachment. As shown in Fig. 3, the development of cystic EB-Vs was more efficiently caused in suspension culture regardless of the type of 5-d-old EB. On day 6, the suspended EB5-1000 developed into cystic EB-Vs with approximately 60% efficiency, but the attached EB5-1000 rarely developed. Regarding cell density, the EB5-4000 was superior
to the EB5-1000 in terms of the efficiency of cystic EB-Vs development in any culture system. The suspended EB5-4000 most rapidly developed into cystic EB-Vs. The efficiency above 80% was achieved by day 6. Therefore, the suspension culture of EB5-4000 is most preferable for developing 5-d-old EBs into cystic EB-Vs. Fig. 4 shows the typical morphology change of the EB5-4000 in the suspension culture. Quantitative PCR analysis of endodermal genes To clarify the effect of culture system and cell density on the differentiation of EBs, the expression levels of visceral endoderm-related genes (GATA4, TTR, and AFP) were quantitatively analyzed on culture day 6 as shown in Fig. 3. In each of EB5-1000 and EB5-4000, as shown in Fig. 5, the expression levels of all of the genes were higher in the suspension culture than in the attachment culture. This tendency was notable in the EB5-1000. The increase in the expression levels of TTR and AFP indicates that the EBs are differentiating toward visceral endoderm (18, 19). Therefore, the results of quantitative PCR analysis are consistent with the results shown in Fig. 3. Development of cystic EB-Vs under low oxygen tension and low-nutrient condition The development of cystic EB-Vs was performed under low oxygen tension (5% O2) and compared with that under normal oxygen tension (20% O2). As shown in Fig. 6, the efficiency of cystic EB-V development was better in 20% O2 than that in 5% O2 regardless of cell density (1000 or 4000 cells/well). The density
FIG. 4. Typical morphology change of the EB5-4000 in the suspension culture using the low-adherence 24-well plate. The scale bar indicates 200 μm. (A) Day 0, just after seeding, (B) day 3, and (C) day 6.
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FIG. 5. Effect of culture system on the relative expression levels of marker genes related to visceral endoderm development. Samples for quantitative RT-PCR analysis were collected on day 6 in the cultures shown in Fig. 3. Abbreviations: Att., attachment culture; Sus., suspension culture. Bar: standard error of the mean (n = 3).
of 4000 cells/well in 20% O2 showed the highest efficiency of cystic EB-V development. It was shown that low oxygen tension did not promote cystic EB-V development. The effect of nutrient condition on the development of cystic EB-Vs was investigated in the low-adherence 96-well plates. The medium based on DMEM is relatively low in nutrients when compared with the medium based on IMDM. Fig. 7 shows that cystic EB-V development was caused more efficiently in the EB medium based on DMEM. It was shown that the low-nutrient condition promoted the development of cystic EB-Vs. DISCUSSION First, we investigated the optimum cell density required to form cystic EB-Vs in the low-adherence 96-well plate (Fig. 2). When the ES cells were seeded at the density of 4000 cells/well, the ES cells most efficiently developed into cystic EB-Vs via forming EBs. This result is consistent with the findings reported by Koike et al. (14). When the ES cells were seeded at the densities of 8000–20,000 cells/well, spherical smooth-faced EBs were not formed before the beginning of cystic EB-V development, and the efficiency of cystic EB-V development was decreased. The decrease in the efficiency of cystic EB-V development may be due to the incompleteness of EB formation because the EB
FIG. 6. Cumulative changes in the percentage of cystic EB-V development under low oxygen tension (5% O2) and normal oxygen tension (20% O2). Symbols: open circles, 1000 cells/well in 20% O2; closed circles, 1000 cells/well in 5% O2; open squares, 4000 cells/well in 20% O2; closed squares, 4000 cells/well in 5% O2. Bar: standard error of the mean (n = 3).
formation has a stimulating effect on the differentiation of ES cells in vitro. As for the culture system, the suspension culture of EBs in the low-adherence plate was suitable for developing cystic EB-Vs (Figs. 3 and 5). The EBs developed into cystic EB-Vs while maintaining their three-dimensional structure in the suspension culture, while they flattened into a two-dimensional structure in the attachment culture. In either culture system, however, the EBs formed from 4000 cells/ well were advantageous over those from 1000 cells/well for developing cystic EB-Vs. Therefore, it was concluded that the suspension culture at 4000 cells/well as seeding cell density was optimum for developing cystic EB-Vs. Next, we determined why 4000 cells/well was superior to 1000 cells/well as the seeding cell density in the development of cystic EB-Vs. Generally, high-density, or three-dimensional culture of cells is conducive to cell–cell contact, but unfavorable to oxygen and nutrient supplies. In this study, therefore, we investigated the effects of low-oxygen and low-nutrient conditions on the development of cystic EB-Vs. As the low-oxygen environment, we chose the oxygen tension of 5% O2 because it was similar to the in vivo environment in which embryogenesis is taking place (22). As shown in Fig. 6, the low oxygen
FIG. 7. Cumulative changes in the percentage of cystic EB-V development in the EB medium based on DMEM (low-nutrient medium) and in the EB medium based on IMDM (standard medium). Symbols: open circles, 1000 cells/well in the EB medium based on IMDM; closed circles, 1000 cells/well in the EB medium based on DMEM. ⁎P b 0.05 by Student's t-test, compared with the standard medium. Bar: standard error of the mean (n = 3).
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tension retarded the development of EB-Vs. The expression levels of visceral endoderm-related genes, AFP and TTR, were not increased in the culture samples under low oxygen tensions of 5% O2 and 10% O2 (data not shown). Therefore, it was found that low oxygen tension was not a factor for promoting the development of cystic EB-Vs. As a low-nutrient condition, we chose DMEM as the basal medium for cystic EB-V development instead of IMDM. Either DMEM or IMDM is widely used as the basal medium for EB formation, but IMDM is generally considered as a high-nutrient medium for high-cell-density culture (23). The IMDM is an enriched modification of DMEM containing additional amino acids (L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, and proline), vitamins (biotin and vitamin B12), and inorganic salts (KNO3 and Na2SeO3). The EB medium based on IMDM contains 34 mg/l L-alanine, 38 mg/l L-asparagine, 43 mg/l L-aspartic acid, 90 mg/l L-glutamic acid, and 52 mg/l proline. On the other hand, the EB medium based on DMEM contains 9 mg/l L-alanine, 13 mg/l L-asparagine, 13 mg/l L-aspartic acid, 15 mg/l Lglutamic acid, and 11.5 mg/l proline. As shown in Fig. 7, it can be seen that cystic EB-Vs were more efficiently formed in the EB medium based on DMEM. The gene expression levels of AFP and TTR in the culture samples on day 5 were quantitatively analyzed. The gene expression levels of AFP and TTR in the samples from the EB medium based on DMEM were higher than those from the EB medium based on IMDM. However, there were no statistically significant differences in the gene expression levels of AFP and TTR between the EB medium based on DMEM and the EB medium based on IMDM (data not shown). The lownutrient condition may be one of the factors for promoting the development of cystic EB-Vs, but the effect of nutrient supply on the cell differentiation in the EBs may be more complicated. In conclusion, this study showed that the suspension culture system using the low-adherence 96-well plate seeded with 4000 ES cells/well was optimum for developing cystic EB-Vs. In the differentiation system involving EB formation, however, cell differentiation must be a multicausal event. The cell–cell contact in the EBs would also be an important factor. Further investigation to clarify the factors that affect the differentiation status of EBs in the EB formation culture is necessary. References 1. Evans, M. J. and Kaufman, M. H.: Establishment in culture of pluripotential cells from mouse embryos, Nature, 292, 154–156 (1981). 2. Martin, G.: Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells, Proc. Natl. Acad. Sci. USA, 78, 7634–7638 (1981). 3. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., and Jones, J. M.: Embryonic stem cell lines derived from human blastocysts, Science, 282, 1145–1147 (1998). 4. Keller, G. M.: In vitro differentiation of embryonic stem cells, Curr. Opin. Cell Biol., 7, 862–869 (1995).
J. BIOSCI. BIOENG., 5. Kurosawa, H.: Methods for the formation of embryoid bodies: in vitro differentiation system of embryonic stem cells, J. Biosci. Bioeng., 103, 389–398 (2007). 6. Desbaillets, I., Ziegler, U., Groscurth, P., and Gassmann, M.: Embryoid bodies: an in vitro model of mouse embryogenesis, Exp. Physiol., 85, 645–651 (2000). 7. Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., and Benvenisty, N.: Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers, Mol. Med., 6, 88–95 (2000). 8. Höpfl, G., Gassmann, M., and Desbaillets, I.: Differentiating embryonic stem cells into embryoid bodies, Methods Mol. Biol., 254, 79–98 (2004). 9. Takahashi, K. and Yamanaka, S.: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell, 126, 663–676 (2006). 10. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichikawa, T., Tomoda, K., and Yamanaka, S.: Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell, 131, 1–12 (2007). 11. Conley, B. J., Young, J. C., Trounson, A. O., and Mollard, R.: Derivation, propagation and differentiation of human embryonic stem cells, Int. J. Biochem. Cell Biol., 36, 555–567 (2004). 12. Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R.: The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium, J. Embryol. Exp. Morph., 87, 27–45 (1985). 13. Magyar, J. P., Nemir, M., Ehler, E., Suter, N., Perriard, J., and Eppenberger, H. M.: Mass production of embryoid bodies in microbeads, Ann. N.Y. Acad. Sci., 944, 135–143 (2001). 14. Koike, M., Sakaki, S., Amano, Y., and Kurosawa, H.: Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies, J. Biosci. Bioeng., 104, 294–299 (2007). 15. Conley, B. J., Trounson, A. O., and Mollard, R.: Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives, Fetal Diagn. Ther., 19, 218–223 (2004). 16. Arceci, R. J., King, A. A., Simon, M. C., Orkin, S. H., and Wilson, D. B.: Mouse GATA-4: a retinoic acid-inducible GATA-binding transcription factor expressed in endodermally derived tissues and heart, Mol. Cell Biol., 13, 2235–2246 (1993). 17. Siltanen, S., Anttonen, M., Heikkila, P., Narita, N., Laitinen, M., Ritvos, O., Wilson, D. B., and Heikinheimo, M.: Transcription factor GATA-4 is expressed in pediatric yolk sac tumors, Am. J. Pathol., 155, 1823–1829 (1999). 18. Costa, R. H., Van Dyke, T. A., Yan, C., Kuo, F., and Darnell, J. E.: Similarities in transthyretin gene expression and differences in transcription factors: liver and yolk sac compared to choroid plexus, Proc. Natl. Acad. Sci. USA, 87, 6589–6593 (1990). 19. Lazarevich, N. L.: Molecular mechanisms of alpha-fetoprotein gene expression, Biochemistry (Mosc), 65, 117–133 (2000). 20. Zhang, H., Guo, D., Wang, L., Zhao, Y., Cheng, Y., and Qiao, Z.: Effect of nicotine on Oct-4 and Rex-1 expression of mouse embryonic stem cells, Reprod. Toxicol., 19, 473–478 (2005). 21. Yoon, B. S., Yoo, S. J., Lee, J. E., You, S., Lee, H. T., and Yoon, H. S.: Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment, Differentiation, 74, 149–159 (2006). 22. Ezashi, T., Das, P., and Robert, R. M.: Low O2 tensions and the prevention of differentiation of hES cells, Proc. Natl. Acad. Sci. USA, 102, 4783–4788 (2005). 23. Iscove, N. N. and Melchers, F.: Complete replacement of serum by albumin, transferring, and soybean lipid in cultures of lipopolysaccharide-reactive B lymphocytes, J. Exp. Med., 147, 923–933 (1978).
Development of cystic embryoid bodies with visceral yolk-sac-like structures from mouse embryonic stem cells using low-adherence 96-well plate Emiko Yasuda,1 Yuji Seki,1 Takatoshi Higuchi,1 Fumio Nakashima,2 Tomozumi Noda,2 and Hiroshi Kurosawa1,⁎ Division of Medicine and Engineering Science, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan 1 and Tsukuba Research Laboratory Section II, Life Science Division, NOF Corporation, 5-10 Tokodai, Tsukuba, Ibaraki 300-2635, Japan 2 Received 17 October 2008; accepted 9 December 2008
Cystic embryoid bodies with visceral yolk-sac-like structure (cystic EB-Vs) are used as a model for the study of early extraembryonic tissue formation containing visceral endoderm-like derivatives. In this study, we optimized the cell density of embryonic stem (ES) cells for developing cystic EB-Vs in a low-adherence 96-well plate. When ES cells were seeded at a density of 4000 cells/well, the cystic EB-Vs were most efficiently developed from ES cells via forming multicellular spherical aggregates called embryoid bodies (EBs). The suspension culture in the low-adherence plate was preferable for developing EBs into cystic EB-Vs rather than the attachment culture in the plate coated with 0.1% gelatin. The seeding cell density of 4000 cells/well was always superior to 1000 cells/well in the efficiency of cystic EB-V development. Because the high-cell density culture generally raises the limitation of oxygen and nutrient supplies, we investigated the effects of low-oxygen and low-nutrient conditions on the development of cystic EB-Vs. It was found that low oxygen tension was not a factor for promoting the development of cystic EB-Vs. It was suggested that a low-nutrient medium is preferred for developing cystic EB-Vs rather than a sufficient-nutrient medium. In conclusion, the suspension culture in the low-adherence 96-well plate seeded with 4000 ES cells/well was optimum for developing cystic EB-Vs. The low-nutrient condition may be one of the factors for promoting the development of cystic EB-Vs. © 2008, The Society for Biotechnology, Japan. All rights reserved. [Key words: ES cells; Embryoid body; Cystic EBs; MPC; Visceral yolk sac]
Embryonic stem (ES) cells are self-renewing and theoretically immortal cells derived from the inner cell mass of developing blastocysts (1–3). A principal method of inducing the in vitro differentiation of ES cells is to form multicellular spherical aggregates of ES cells called embryoid bodies (EBs) (4,5). They recapitulate many aspects of cell differentiation during early embryogenesis and differentiate into derivatives of all three germ layers (6,7). EB formation has been employed as an in vitro differentiation system for not only ES cells (4,8) but also induced pluripotent stem (iPS) cells (9,10). Therefore, the quality (i.e., differentiation status) of EBs affects the generation of derivatives from the EBs in a subsequent differentiation culture. The term “EBs” has been widely used by many researchers to indicate embryoid bodies in the wide differentiation stage, even though their characteristics are varied embryologically and morphologically. Strictly speaking, the EBs are classified as simple EBs and cystic EBs according to the differentiation stage (11–13). Simple EBs represent the spherical ES cell aggregates with morula-like structures, which have no central cavity. In some cases, cystic EBs represent the ⁎ Corresponding author. Fax: +81 55 220 8536. E-mail address: [email protected] (H. Kurosawa).
cavitating EBs of a regular form with blastula-like structures. In other cases, cystic EBs represent the expanding EBs with visceral yolk-saclike structure. In this study, cystic EBs with visceral yolk-sac-like structures displaying inconsistent morphology are referred to as cystic EB-Vs. In a previous report, we have described that using a low-adherence 96-well plate is appropriate for the formation of EBs with high uniformity from mouse ES cells, and the density of ES cells seeded in a well affects the differentiation status of EBs (14). When the seeding cell density was increased from 1000 to 4000 cells/well, the EBs that resulted from 4000 cells/well showed markedly high expression levels of marker genes for endoderm of transthyretin (TTR) and αfetoprotein (AFP) compared with the EBs that resulted from 1000 cells/well. Cystic EB-Vs have been expected as a model for the study of early extraembryonic tissue formation containing visceral endoderm-like derivatives (15). Therefore, the reproducible development of ES cells into cystic EB-Vs is important to establish an efficient experimental system and produce reliable data. In general, the formation of cystic EB-Vs is achieved following a relatively long period of suspension culture of ES cells. The low-adherence 96-well plate is considered to be appropriate for suspension culture for developing cystic EB-Vs. In
1389-1723/$ - see front matter © 2008, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2008.12.004
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FIG. 1. Schematic of the culture procedures for cystic EB-V development.
this study, first, we optimized the density of ES cells for developing cystic EB-Vs in the low-adherence 96-well plate. Second, we investigated the effects of low oxygen tension and low nutrient supply on the development of cystic EB-Vs. MATERIALS AND METHODS ES cell culture Mouse ES cells of the cell line 129/SvEv (Thromb-X; N.V. Stem Cell Technologies, Leuven, Belgium) were maintained in Dulbecco's modified Eagle's medium (DMEM [high glucose], Invitrogen, Carlsbad, CA, USA) supplemented with 20% knockout serum replacement (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma, St. Louis, MO, USA), 50 U/ml penicillin, 50 μg/ml streptomycin (Invitrogen), and 1000 U/ ml murine leukemia inhibitory factor (mLIF; Chemicon, Temecula, CA, USA) on 0.1% gelatin-coated dish with feeder layer cells at 37 °C in humidified air with 5% CO2. As feeder layer cells, STO fibroblasts treated with 10 μg/ml mitomycin C (Sigma) were used. For these experiments, ES cells were grown for a maximum of 22 passages in the presence of feeder layer cells and mLIF. EB formation in low-adherence 96-well plate ES cells were dissociated with 0.1% trypsin-EDTA (Invitrogen) and suspended in Iscove's modified Dulbecco's medium (IMDM, Invitrogen) supplemented with 20% fetal bovine serum (Invitrogen), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 50 U/ ml penicillin, and 50 μg/ml streptomycin (EB medium). An ES cell suspension was poured into round-bottomed wells of a low-adherence 96-well polystyrene plate coated with 2-methacryloyloxethyl phosphorylcoline (MPC) (Lipidure coat®, NOF Co., Tokyo). Each well contained ES cells at a density of 1000 or 4000 cells per 200 μl of medium. The low-adherence 96-well plates were statically incubated to form EBs at 37 °C in humidified air with 5% CO2 for 5 days. The EBs that resulted from 1000 and 4000 cells by culture day 5 are referred to as EB5-1000 and EB5-4000, respectively. Direct development of cystic EB-Vs from ES cells in 96-well MPC plates The procedures for cystic EB-V development are schematically shown in Fig. 1. Cystic EB-Vs were developed from ES cells by one continuous operation using the low-adherence 96-well plates. To investigate the effect of cell density on cystic EB-V development, ES cells were seeded at the densities of 500, 1000, 4000, 8000, 10,000, and 20,000 cells/ well in 200 μl of EB medium and cultured for 20 days in the incubator with 20% O2 atmosphere. To investigate the effect of low oxygen tension, the ES cells were seeded at the densities of 1000 and 4000 cells/well in 200 μl of EB medium and cultured for 20 days under oxygen tension of 5% O2. To investigate the effect of low nutrient supply, the ES cells were seeded at the densities of 1000 cells/well and cultured for 14 days in 200 μl of EB medium based on DMEM. The EB medium based on DMEM was prepared by just replacing IMDM of the EB medium with DMEM. From culture day 5, half of the medium was changed daily. Periodically, microscopy was performed to detect the development of cystic EB-Vs in the wells. When the size of EBs increased drastically and cystic sphere with clear boundary appeared, we decided that cystic EB-Vs were developed. Three independent experiments were conducted for each cell density. Development of cystic EB-Vs from 5-d EBs in suspension and attachment cultures As illustrated in Fig. 1, the above-mentioned 5-d-old EBs (EB5-1000 and EB5-4000) were transferred to two types of culture system, which are suspension culture and attachment culture, to induce the organization of visceral yolk-sac-like structures in the EBs. In the suspension culture system, one EB was transferred into each well of a 24-well plate coated with MPC (low-adherence 24-well plate) containing 1 ml of EB medium. In the attachment culture system, one EB was placed on each well of a 24-well plate coated with 0.1% gelatin containing 1 ml of EB medium. Cultivations were performed for 15 days and half of the medium was changed every 3 days. Periodically, microscopy was performed to detect the development of cystic EB-Vs in the wells. Three independent experiments were conducted for each culture system.
Quantitative real-time PCR analysis The expression levels of several specific marker genes in EBs were quantitatively evaluated by real-time PCR analysis using a DNA Engine Opticon™ system (MJ Research Inc, Waltham, MA, USA). The GATA transcription factors GATA-4 (16, 17) was regarded as marker that is expressed in endoderm- and mesoderm-derived lineages in EBs. Transthyretin (TTR) and α-fetoprotein (AFP) were used as the markers specifically expressed in the visceral endoderm of yolk sac (18, 19). The gene of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an internal reference. Total RNA was extracted from EBs using the Micro-to-Midi Total RNA Purification system (Invitrogen, Carlsbad, CA, USA). To prepare a sample for respective control, total RNA was extracted from the dissociated ES cells in the same manner, just before transferring the cells to the EB formation culture. The quality of each RNA sample was analyzed using a UV–Vis spectrophotometer for RNA quality (A260/A280 N1.8). RNA integrity was verified electrophoretically by ethidium bromide staining. Complementary DNA (cDNA) was synthesized from 5 μg of total RNA using the SuperScript First-Strand Synthesis system for RT-PCR (Invitrogen) according to the manufacturer's instructions. Two microliters of cDNA template was added to 12.5 μl of the 2× SYBR Premix Ex Taq (Takara Bio, Otsu) and 10 pmol of each specific primer. Reactions were carried out for 45 cycles. The cycling parameters were as follows: denaturation at 95 °C for 5 s, and annealing and elongation at 60 °C for 20 s. The primer sequences were as follows: G3PDH, 5′-ACCACAAGTCCATGCCATCAC-3′ sense and 5′TCCACCACCCTGTTGCTGTA-3′ antisense; GATA-4, 5′-GAAAACGGAAGCCCAAGAACC-3′ sense and 5′-TGCTGTGCCCATAGTGAGATGAC-3′ antisense; TTR, 5′-CTCACCACAGATGAGAAG-3′ sense and 5′-GGCTGAGTCTCTCAATTC-3′ antisense; AFP, 5′-TCGTATTCCAACAGGAGG-3′ sense and 5′-AGGCTTTTGCTTCACCAG-3′ antisense. Fluorescence intensity was measured at the end of the annealing-extension phase of each cycle. The threshold value for the fluorescence intensity of all the samples was set manually. The reaction cycle at which PCR products exceeded this fluorescence intensity threshold was identified as the threshold cycle (CT) in the exponential phase of the PCR amplification. Relative quantification The expression levels of these target genes were quantified against that of the internal reference gene (G3PDH). Relative quantification is based on the comparison of the CT at a constant fluorescence intensity (20, 21). The amount of transcript present is inversely related to the observed CT, and for every twofold
FIG. 2. Effect of cell density on cystic EB-V development. The percentage of cystic EB-V development is the ratio of the wells in which cystic EB-Vs were observed on culture day 20 to the wells seeded with ES cells. Forty-eight wells were seeded with 200 μl of ES cell suspension of each cell density. Bar: standard error of the mean (n = 3).
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FIG. 3. Cumulative changes in the percentage of cystic EB-V development in the suspension and attachment cultures. The percentage of cystic EB-V development is the ratio of the wells in which cystic EB-Vs were observed to the 24 wells seeded with 5-d-old EBs. (A) 5-d-old EBs that resulted from 1000 ES cells (EB5-1000) were seeded; (B) 5-d-old EBs that resulted from 4000 ES cells (EB5-4000) were seeded. Symbols: open circles, suspension culture of EB5-1000; closed circles, attachment culture of EB5-1000; open squares, suspension culture of EB5-4000; closed squares, attachment culture of EB5-4000. The arrow indicates sample collection for quantitative RT-PCR analysis. Bar: standard error of the mean (n = 3). dilution in transcript, the CT is expected to increase by 1. The relative expression (R) is calculated using the equation R = 2− [ΔCT sample − ΔCT control]. That is, to determine a normalized arbitrary value for each gene, every data point was normalized to the reference gene (G3PDH), as well as to their respective control. Data (mean± SEM) are from triplicate runs of one sample set representative of three independent experiments.
RESULTS Effect of cell density on cystic EB-V development The optimum density of ES cells for developing cystic EB-Vs was investigated. Fig. 2 shows the ratio of the wells in which cystic EB-Vs were observed on culture day 20 to the wells seeded with ES cells (48 wells). As can be seen in Fig. 2, cystic EB-Vs were observed with more than 80% efficiency when the ES cells were seeded at the densities of 1000 and 4000 cells/well. Increasing the density to over 4000 cells/ well resulted in decreased efficiency and increased deviation. Developments of cystic EB-V from 5-d-old EBs in suspension and attachment cultures Two types of 5-d-old EB (EB5-1000 and EB5-4000) were transferred to the culture systems of suspension and attachment. As shown in Fig. 3, the development of cystic EB-Vs was more efficiently caused in suspension culture regardless of the type of 5-d-old EB. On day 6, the suspended EB5-1000 developed into cystic EB-Vs with approximately 60% efficiency, but the attached EB5-1000 rarely developed. Regarding cell density, the EB5-4000 was superior
to the EB5-1000 in terms of the efficiency of cystic EB-Vs development in any culture system. The suspended EB5-4000 most rapidly developed into cystic EB-Vs. The efficiency above 80% was achieved by day 6. Therefore, the suspension culture of EB5-4000 is most preferable for developing 5-d-old EBs into cystic EB-Vs. Fig. 4 shows the typical morphology change of the EB5-4000 in the suspension culture. Quantitative PCR analysis of endodermal genes To clarify the effect of culture system and cell density on the differentiation of EBs, the expression levels of visceral endoderm-related genes (GATA4, TTR, and AFP) were quantitatively analyzed on culture day 6 as shown in Fig. 3. In each of EB5-1000 and EB5-4000, as shown in Fig. 5, the expression levels of all of the genes were higher in the suspension culture than in the attachment culture. This tendency was notable in the EB5-1000. The increase in the expression levels of TTR and AFP indicates that the EBs are differentiating toward visceral endoderm (18, 19). Therefore, the results of quantitative PCR analysis are consistent with the results shown in Fig. 3. Development of cystic EB-Vs under low oxygen tension and low-nutrient condition The development of cystic EB-Vs was performed under low oxygen tension (5% O2) and compared with that under normal oxygen tension (20% O2). As shown in Fig. 6, the efficiency of cystic EB-V development was better in 20% O2 than that in 5% O2 regardless of cell density (1000 or 4000 cells/well). The density
FIG. 4. Typical morphology change of the EB5-4000 in the suspension culture using the low-adherence 24-well plate. The scale bar indicates 200 μm. (A) Day 0, just after seeding, (B) day 3, and (C) day 6.
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FIG. 5. Effect of culture system on the relative expression levels of marker genes related to visceral endoderm development. Samples for quantitative RT-PCR analysis were collected on day 6 in the cultures shown in Fig. 3. Abbreviations: Att., attachment culture; Sus., suspension culture. Bar: standard error of the mean (n = 3).
of 4000 cells/well in 20% O2 showed the highest efficiency of cystic EB-V development. It was shown that low oxygen tension did not promote cystic EB-V development. The effect of nutrient condition on the development of cystic EB-Vs was investigated in the low-adherence 96-well plates. The medium based on DMEM is relatively low in nutrients when compared with the medium based on IMDM. Fig. 7 shows that cystic EB-V development was caused more efficiently in the EB medium based on DMEM. It was shown that the low-nutrient condition promoted the development of cystic EB-Vs. DISCUSSION First, we investigated the optimum cell density required to form cystic EB-Vs in the low-adherence 96-well plate (Fig. 2). When the ES cells were seeded at the density of 4000 cells/well, the ES cells most efficiently developed into cystic EB-Vs via forming EBs. This result is consistent with the findings reported by Koike et al. (14). When the ES cells were seeded at the densities of 8000–20,000 cells/well, spherical smooth-faced EBs were not formed before the beginning of cystic EB-V development, and the efficiency of cystic EB-V development was decreased. The decrease in the efficiency of cystic EB-V development may be due to the incompleteness of EB formation because the EB
FIG. 6. Cumulative changes in the percentage of cystic EB-V development under low oxygen tension (5% O2) and normal oxygen tension (20% O2). Symbols: open circles, 1000 cells/well in 20% O2; closed circles, 1000 cells/well in 5% O2; open squares, 4000 cells/well in 20% O2; closed squares, 4000 cells/well in 5% O2. Bar: standard error of the mean (n = 3).
formation has a stimulating effect on the differentiation of ES cells in vitro. As for the culture system, the suspension culture of EBs in the low-adherence plate was suitable for developing cystic EB-Vs (Figs. 3 and 5). The EBs developed into cystic EB-Vs while maintaining their three-dimensional structure in the suspension culture, while they flattened into a two-dimensional structure in the attachment culture. In either culture system, however, the EBs formed from 4000 cells/ well were advantageous over those from 1000 cells/well for developing cystic EB-Vs. Therefore, it was concluded that the suspension culture at 4000 cells/well as seeding cell density was optimum for developing cystic EB-Vs. Next, we determined why 4000 cells/well was superior to 1000 cells/well as the seeding cell density in the development of cystic EB-Vs. Generally, high-density, or three-dimensional culture of cells is conducive to cell–cell contact, but unfavorable to oxygen and nutrient supplies. In this study, therefore, we investigated the effects of low-oxygen and low-nutrient conditions on the development of cystic EB-Vs. As the low-oxygen environment, we chose the oxygen tension of 5% O2 because it was similar to the in vivo environment in which embryogenesis is taking place (22). As shown in Fig. 6, the low oxygen
FIG. 7. Cumulative changes in the percentage of cystic EB-V development in the EB medium based on DMEM (low-nutrient medium) and in the EB medium based on IMDM (standard medium). Symbols: open circles, 1000 cells/well in the EB medium based on IMDM; closed circles, 1000 cells/well in the EB medium based on DMEM. ⁎P b 0.05 by Student's t-test, compared with the standard medium. Bar: standard error of the mean (n = 3).
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tension retarded the development of EB-Vs. The expression levels of visceral endoderm-related genes, AFP and TTR, were not increased in the culture samples under low oxygen tensions of 5% O2 and 10% O2 (data not shown). Therefore, it was found that low oxygen tension was not a factor for promoting the development of cystic EB-Vs. As a low-nutrient condition, we chose DMEM as the basal medium for cystic EB-V development instead of IMDM. Either DMEM or IMDM is widely used as the basal medium for EB formation, but IMDM is generally considered as a high-nutrient medium for high-cell-density culture (23). The IMDM is an enriched modification of DMEM containing additional amino acids (L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, and proline), vitamins (biotin and vitamin B12), and inorganic salts (KNO3 and Na2SeO3). The EB medium based on IMDM contains 34 mg/l L-alanine, 38 mg/l L-asparagine, 43 mg/l L-aspartic acid, 90 mg/l L-glutamic acid, and 52 mg/l proline. On the other hand, the EB medium based on DMEM contains 9 mg/l L-alanine, 13 mg/l L-asparagine, 13 mg/l L-aspartic acid, 15 mg/l Lglutamic acid, and 11.5 mg/l proline. As shown in Fig. 7, it can be seen that cystic EB-Vs were more efficiently formed in the EB medium based on DMEM. The gene expression levels of AFP and TTR in the culture samples on day 5 were quantitatively analyzed. The gene expression levels of AFP and TTR in the samples from the EB medium based on DMEM were higher than those from the EB medium based on IMDM. However, there were no statistically significant differences in the gene expression levels of AFP and TTR between the EB medium based on DMEM and the EB medium based on IMDM (data not shown). The lownutrient condition may be one of the factors for promoting the development of cystic EB-Vs, but the effect of nutrient supply on the cell differentiation in the EBs may be more complicated. In conclusion, this study showed that the suspension culture system using the low-adherence 96-well plate seeded with 4000 ES cells/well was optimum for developing cystic EB-Vs. In the differentiation system involving EB formation, however, cell differentiation must be a multicausal event. The cell–cell contact in the EBs would also be an important factor. Further investigation to clarify the factors that affect the differentiation status of EBs in the EB formation culture is necessary. References 1. Evans, M. J. and Kaufman, M. H.: Establishment in culture of pluripotential cells from mouse embryos, Nature, 292, 154–156 (1981). 2. Martin, G.: Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells, Proc. Natl. Acad. Sci. USA, 78, 7634–7638 (1981). 3. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., and Jones, J. M.: Embryonic stem cell lines derived from human blastocysts, Science, 282, 1145–1147 (1998). 4. Keller, G. M.: In vitro differentiation of embryonic stem cells, Curr. Opin. Cell Biol., 7, 862–869 (1995).
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