August 2006


Biol. Pharm. Bull. 29(8) 1747—1750 (2006)


Conditioned Medium from Feeder STO Cells Increases the Attachment of Mouse Embryonic Stem Cells
Kenichiro AMANO, Tadahide FURUNO,1) and Mamoru NAKANISHI*,1)
Graduate School of Pharmaceutical Sciences, Nagoya City University; Tanabe-dori, Mizuho-ku, Nagoya 467–8603, Japan. Received January 24, 2006; accepted May 30, 2006; published online June 2, 2006 Mouse embryonic stem (ES) cell lines, which were established by culturing on feeder cells, have usually been cultured without feeder cells in the presence of leukemia inhibitory factor. However, proliferating rate of ES cells in the condition is often lower than that with feeder cells. Here, we found that conditioned medium (CM) from feeder cells (STO cells) increased the number of undifferentiated cells in a culture dish by promoting attachment of ES cells. The attached cells were increased in 4 h after replating ES cells in the presence of CM from STO cells, and they formed flat colonies composed of undifferentiated cells. This culture system with CM from feeder cells is useful in preparing a large number of well-defined ES cells. In addition, from present experiments we found that the dish double-coated with gelatin and CM was extremely useful for culturing ES cells.
Key words embryonic stem cell; feeder STO cell; conditioned medium

Pluripotent stem cell lines, embryonic stem (ES) cells are undifferentiated cell lines that are capable of forming virtually any cell type in the body.2) Mouse ES cells have been useful and versatile cells to study developmental biology, and are indispensable for creation of knockout mice to analyze function of specific protein.3) ES cell lines derived from not only mouse but also human have been established and their wide developmental potential and unlimited life span in culture make them extremely interesting and important for basic and applied research.4) They can undergo differentiation in vivo and in vitro, allowing them to be used for the study basic processes in developmental biology, and be used as a renewable cell source for cell engineering, transplantation and regenerative medicine. Maintenance of ES cells in the undifferentiated state was performed by the culture with feeder cells. There, isolated inner cell mass-derived cells were seeded to gelatin-coated dishes containing a confluent layer of mitomycin C-inactivated STO fibroblasts.5,6) Because STO cells are easily cultured for a long period, they have been then often used as feeder cells to culture mouse ES cells. This indicated that feeder cells such as STO fibroblasts and mouse embryo fibroblasts (MEF) provided some factors to promote self-renewal and suppress differentiation. Thereafter, the cytokine leukemia inhibitory factor (LIF) was found to be one of important factors for maintaining the pluripotency of mouse ES cells.7,8) LIF activates signal transducer and activator of transcription 3 (STAT3) which has been shown to play an essential role in maintaining self-renewal, while it also induces activation of a number of signaling protein including extracellular signal-regulated kinase (ERKs) which appears to promote differentiation.9—12) Therefore, it is considered that the balance between STAT3 and ERKs signaling is critical for determining the fate of undifferentiated ES cells.13) In contrast, maintenance of human ES cells still required a monolayer of feeder cells, since LIF has no effect on preventing differentiation of human ES cells. Mouse ES cells have recently been cultured in the presence of LIF and fetal bovine serum (FBS) or serum replacement (SR) on gelatin-coated dishes in feeder cell-free system. However, FBS is put under suspicion for its unknown factor to promote differentiation
∗ To whom correspondence should be addressed.

and SR often decreases the proliferation. Under those situations, we thought that the re-examination of feeder cell function for maintaining undifferentiated ES cells was necessary for establishment of the proper ES cell culture system. We found here that conditioned medium (CM) derived from STO cells enhanced proliferation of undifferentiated ES cells through the promotion of ES cell attachment on gelatincoated dishes. The result gave useful information for the role of feeder cells in the maintenance of undifferentiated ES cells. MATERIALS AND METHODS Cell Cultures Mouse 129/sv ES cell lines (passage 15, Dainippon Pharmaceutical Corporation, Osaka, Japan) were routinely cultured on tissue culture plates (Falcon) coated with 0.1% (v/v) gelatin (Dainippon Pharmaceutical Corporation) in KnockoutTM Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Scottland) in the presence of 15% (v/v) KnockoutTM serum replacement (Invitrogen), 0.1 mM b mercaptoethanol, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 1000 units/ml LIF (Chemicon) in 5% CO2 incubator at 37 °C. Cells were trypsinized and replated every 3 d. Mouse fibroblast STO cells (ATCC, Rockville, MD, U.S.A.) were cultured in DMEM in the presence of 15% fetal bovine serum (FBS) (Trace), 5 mg/ml D-glucose, 1.5 mg/ml sodium bicarbonate, 100 units/ml penicillin, 0.1 mg/ml streptomycin) in 5% CO2 incubator at 37 °C. Cells were treated with 10 m g/ml mitomycin C (Wako, Osaka) for 4 h, trypsinized, and replated on tissue culture dishes coated with 0.1% (v/v) gelatin to utilize as feeder cells. CM from STO Cells STO cells were maintained in culture flasks. The medium was changed to a culture medium for ES cells (KnockoutTM DMEM in the presence of 15% (v/v) KnockoutTM SR, 0.1 mM b -mercaptoethanol, 2 mM Lglutamine, 0.1 mM non-essential amino acids, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 1000 units/ml LIF). STO cells were cultured for 3 d, and the cultured medium was centrifuged at 1000 rpm for 5 min. The supernatant was used as CM after the filtration by a 0.45 m m mesh filter (Mil© 2006 Pharmaceutical Society of Japan


5)).25% Coomassie Brilliant Blue R250 (Wako). medium were much lower than those in FBS. we scratched the bottom of dishes by rubber policeman in lysis buffer (25 mM Tris–HCl.and FBS-containing media. CM-coated and gelatin plus CM-coated ones. which were lost on removal of LIF when the ES cells differentiated into a variety of cell types.14) We checked that the ES cell in such colonies expressed the stem cell markers alkaline phosphatase.M. SDS-PAGE To extract proteins existing in gelatin. To detect CM-containing factors which induced the promotion of ES cell attachment.01 compared with the medium in the absence of CM from STO cells. 2. which were gelatin-coated. 4 m g/ml leupeptin (pH 7. We used the 30 % CM-containing medium in the following experiments. After removing supernatants. In addition.05 was accepted as a level of statistically significant difference. ∗∗ pϽ0. the ES cell colonies in CM-containing medium were normal shape (Fig. and centrifuged them at 23500ϫg for 5 min. 3a. 0. the resulting precipitations were solubilized by treatment with Laemmli buffer at 100 °C for 5 min. CM-. On the contrary. 2. data were analyzed by unpaired Student’s t-test. Two significant bands of proteins at ca. ES cells in FBS-containing medium also showed some thick colonies as like as the case of LIF withdraw after 3-d culture as shown in Fig. Data Presentation Data were presented as meanϮ S. Removal of LIF for 3 d induced the differentiation of ES cells and the morphological changes of colonies to be thick (Fig. we measured the number of ES cells after culturing in the medium for ES cells containing CM from STO cells on tissue culture dishes coated with gelatin in the presence of LIF. Effect of CM from STO Cells on ES Cell Proliferation The numbers of ES cells attached to gelatin-coated dishes were counted at 3 d after replating 1. After culturing in SR-. we analyzed the proteins on the bottom of dishes coated with gelatin and gelatin plus CM. Here. When ES cells were cultured for a long-time (2 or 3 d) in FBS-containing medium. and then used for the experiments.5% Nonidet P-40. 2-tailed. Triplicate cell counts were performed in each experiment. The attached cells formed colonies whose morphology was characteristic of undifferentiated ES cell colonies. The number of ES cells cultured in CM-containing medium for 24 h was apparently higher than those in SR. kept them at Ϫ20 °C for 1 h.0ϫ106 cells in the medium containing CM from STO cells. 7 kDa and ca. the number of attachment of ES cells increased in the dishes pre-coated with CM. 4a.01 compared with the SR.05 and ∗∗ pϽ0. Further. the boundary of cells was apparently obscure in flat colonies. The number of attached ES cells to gelatin-coated dishes for 2-h culture was much higher in CM-containing medium (grey column in left) than in SR-containing medium (white column in left) in Fig. as shown . 1. and then attached cells were collected by trypsinization. 1mM sodium vanadate.15) The number of undifferentiated ES cells attached to gelatin-coated dishes did not increase in the medium containing 10% CM but increased in the medium containing more than 20% CM as shown in Fig. We made this experiment using three kinds of culture dishes. 5 mM MgCl2. 0. the medium containing 30% CM was added to non-coated or gelatin-coated dishes. we compared the number of ES cells attached to gelatin-coated dishes among the culture media containing 15% SR. the number in the dishes pre-coated with both gelatin and CM also more increased in comparison with that in the CMcoated dishes.0ϫ106 cells/dish). Triplicate cell counts were performed in each experiment. the number of the cells increased to the values in CM-containing medium. we studied the effects of dishes pre-coated with gelation and/or CM on the attachment of ES cells. The values in SR-containing Fig. we counted the number of ES cells attached to dishes. In statistical analysis. 3d). 15% FBS. They were washed PBS.and FBS-containing media as shown in Fig. 3c. Then.1748 Vol. more than half of ES cells (ca. 60%) attached on gelatincoated dishes. and CM (30%)-containing medium (᭹) were counted. CM-. and the experiment was repeated three times. as shown in Fig. and 30% CM from STO cells. or FBS-containing medium for appropriate period. the medium were removed. that is. As described above. In coating culture dishes with CM. No. FBS (15%)-containing medium (ᮀ). This suggested that some soluble factors in CM accelerated the proliferation of ES cells. After they were incubated overnight. separated by electrophoresis in 10% SDS-polyacrylamide gel and stained with 0. RESULTS To study the effects of CM from STO cells on proliferation of ES cells. 3b). and the experiment was repeated three times. we added acetone. 10% glycerol. 8 lipore). the attached ES cells formed flat colonies where the boundary of cells was apparently obscure in SR-containing medium in the presence of LIF. pϽ0. They were counted with Burker-Turk cell-counting plate.0ϫ106 cells/dish and were cultured in the SR (15%)-containing medium (᭝). 1 mM dithiothreitol.15 M NaCl. however. 1. 29. or gelatin plus CM-coated dishes (1. Cells were washed with PBS to remove floating cells. Next. but not in gelatin-coated ones. whether the medium contained SR or CM.or CM-containing medium (data not shown). 34 kDa were observed by SDS-PAGE in gelatin plus CM-coated dishes. 1 mM EDTA.and gelatin plus CM-coated dishes. 1 mM phenylmethylsulfonyl fluoride. Some floating ES cells differentiated. ∗ pϽ0. Effect of CM from STO Cells on Attachment of ES Cells The numbers of ES cells attached to gelatin-coated dishes which were replated at 1.E. Fig. Figure 2 shows the time-courses of attached ES cells in three different media. Cell Counts ES cells were replated on gelatin-.

the presence of LIF without feeder cells in the medium containing FBS or SR. S. Triplicate cell counts were performed in each experiment.. (c) ES cell colonies cultured in FBS (15%)-containing medium in the presence of LIF for 3 d. 7634—7638 (1981)... Science. Jones J. The data presented here show that the ES cell culture system utilizing CM from feeder cells enables us to prepare a large number of well-defined ES cells in the feeder cell-free and serum-free condition.. (d) ES cell colonies cultured in CM (30%)-containing medium in the presence of LIF for 3 d.. The procedure of ES cell culture using CM from feeder cells described in the present paper has two major advantages: first. a strong band was observed at ca. ES cells in this culture system are well-defined because there is no anxiety about the direct contamination of feeder cells. All of these results indicated that the CM from STO cells seems to be useful in preparing a large number of well-defined ES cells. Proc. H. but the growth rate was not so high. Kuehn M. 635—678 (2005). The adhesion to extracellular matrix of ES cells is necessary for the maintenance of undifferentiated state. The CM from STO cells showed the promotion of attachment to culture dishes in ES cell. we can obtain more undifferentiated ES cells in routine culture using CM from STO cells which was able to stock for at least 6 months at Ϫ20 °C. Promotion of Attachment of ES Cells to Culture Dishes Coated with CM from STO Cells (a) The numbers of ES cells attached to culture dishes coated with gelatin.19) The present results clearly indicated that the dish double-coated with gelatin and CM was extremely useful for culturing ES cells. 2) Wobus A.17) SR is completely devoid of any undefined growth factors or differentiation-promoting factors. In this experiment. M.S. 445—448 (1986). 323. U. 3. Kaufman M. We focused here on the role of feeder cells in the proliferation and maintenance of undifferentiated ES cells. In fact. Serum contains uncharacterized growth and differentiation factors. ES cells formed flat colonies. 282... A.August 2006 1749 Fig. # pϽ0. and the experiment was repeated three times. Waknitz M.. Sci. suggesting that this protein might be entactin from the previous paper analyzing CM from MEF and STO cells by two-dimensional electrophoresis mass spectrometry. J. A. R. REFERENCES AND NOTES 1) Present address: School of Pharmacy. or gelatin plus CM were counted at 2 h after replating 1.0ϫ106 cells in the SR (15%)-containing medium (white bars) and CM (30%)-containing medium (grey bars) were counted. Rev. 34 kDa. Bars ϭ100 m m. J. Equal amounts of protein (10 m g/lane) were electrophoresed. We detected two kinds of proteins from the gelatin plus CMcoated dishes.18. After LIF was identified as an important factor for the activity. R. Evans M. they are prepared at low passage number because a large number of ES cells adhere to culture dishes. and succeeded to find an important role of feeder cells to mouse ES cells. Boheler K.. floating ES cells are known to be led to apoptotic cell death or differentiation through the formation of embryonic body. mouse ES cells have been usually maintained in gelatin-coated dishes in .. Swiergiel J. Acad. 1145—1147 (1998). Actually. (b) ES cell colonies cultured in the SR (15%)-containing medium in the absence of LIF for 3 d. but the present results suggested that some components of soluble proteins derived from STO cells seem to play a significant role in the attachment of ES cells. ∗ ∗ pϽ0. and proliferated in undifferentiated states in the medium containing SR. DISCUSSION A monolayer of inactivated feeder cells was required for establishment of mouse ES cell lines. Itskovitz-Eldor J.. S. there are a number of commercially available chemically defined synthetic serum substitutes such as KnockoutTM SR. 292. Nature (London). Kusumoto-cho. in Fig. Chikusa-ku Nagoya 464–8650.20) The identification of this protein is needed by other experiments. Fig. because feeder cells are considered to produce some crucial factors either to promote self-renewal or suppress differentiation. 3) Robertson G. the supplement of FBS should be completely eliminated from the in vitro culture of ES cells because the composition of serum is not well defined. Japan. but they formed thick colonies which were likely to be differentiating.. Natl. Physiol. Nature (London). and has variations among their lots. Differential Interference Contrast Images of ES Cell Colonies (a) ES cell colonies cultured in the SR (15%)-containing medium in the presence of LIF. (b) Proteins extracted from gelatin-coated dishes (lane 1) and the gelatin plus CM-coated dish (lane 2) were analyzed by SDS-PAGE.. CM. Bradley A. Marshall V .16) However. The growth rate of ES cells was actually high in the medium containing FBS. Aichi-Gakuin University. 78. M. 4. R.01 compared with the SR-containing medium in the gelatin-coated dish. 5) Evans M. and second. 85. 4b.. 6) Martin G. 4) Thompson J. Especially. Shapiro S.05 compared with the CM-containing medium in the gelatincoated dish. At present.A..... 154—156 (1981).

1750 7) Williams R.... L. Donaldson D. Grail D. Savatier P. 12) Furuno T.. Nature (London)... Nakao K. 2.. R. Med. 12. 205—230. Sim E. Chap.. Gage F. Nichols J.. Price P. Onizawa S.... Katsuki M. Immunol.... 10) Niwa H. Nicola N... Chambers I. D.. 336. Tilkins M. J... P... Cell Tissue Res.. Wang D. Morphol. Gough N..... Nakanishi M. K. 11) Burdon T... 336. Haider H. 29. . J. K. Cao T.. Pease S.. 1187—1203 (2002). EMBO J. Schmidt W. Burdon T. Rogers D. F. Proteomics. 8—12 (1998). Braghetta P. Gottlieb D. pp. Morrison J. 291—303 (2004). Nature (London). R. 2048—2060 (1998). Gearing D.. Focus. G. 13) Burdon T.. 17) Goldsborough M. Gardner R. 87. Willson T. Wong G. ed..... M. 15) Pease S. 2001.. L.. Bodnar A.. L. 210... Trends Cell Biol. Smith A.... Muotri A.. C. Biol. Metcalf D.. 10. by Marshak D.. Gearing D. 20) Lim J. Smith A. D.. Z.. No. 141. 228—232 (2005). Stewart C.. Stahl M. 12. 18) Martin M. Stevens M. 4261—4269 (1999). 344—352 (1990). Cold Spring Harbor Laboratory Press. Nakamura T.. Hirashima N. A.. 19) Doetschman T. 8 14) Smith A. Wagner E.. Moreau J.. Katz M.. C. J.. 315. Ng S. K. Sagiya N. New York. Kemler R. L.... 20. L... Embryol. “Embryonic Stem Cells. 11. A.. Yokota T. 27—45 (1985).. Varki A.” Section II. Hilton D. 684—687 (1988). J... 8) Smith A. 18. Heath J. 30—43 (1999). Exp.. 16) Heng B. Dev.. E. Chambers I.. Nichols J. Vol. Eistetter H... 4416—4421 (2001)... J. 432—438 (2002). Genes Dev. Dev. Biol.... 688—690 (1988).. G. C. Stracey C.. Arai T. Heike T. 166. 9) Matsuda T. W. Nat.. Lobo-Alfonso J.. Williams R... Smith A.