{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T11:47:53Z","timestamp":1791805673818,"version":"4.3.4"},"reference-count":55,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5396","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1998,12,11]]},"abstract":"<jats:p>The trophoblast cell lineage is essential for the survival of the mammalian embryo in utero. This lineage is specified before implantation into the uterus and is restricted to form the fetal portion of the placenta. A culture of mouse blastocysts or early postimplantation trophoblasts in the presence of fibroblast growth factor 4 (FGF4) permitted the isolation of permanent trophoblast stem cell lines. These cell lines differentiated to other trophoblast subtypes in vitro in the absence of FGF4 and exclusively contributed to the trophoblast lineage in vivo in chimeras.<\/jats:p>","DOI":"10.1126\/science.282.5396.2072","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:44:47Z","timestamp":1027763087000},"page":"2072-2075","source":"Crossref","is-referenced-by-count":1266,"title":["Promotion of Trophoblast Stem Cell Proliferation by FGF4"],"prefix":"10.1126","volume":"282","author":[{"given":"Satoshi","family":"Tanaka","sequence":"first","affiliation":[{"name":"S. Tanaka and A.-K. Hadjantonakis, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. T. Kunath, A. Nagy, J. Rossant, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tilo","family":"Kunath","sequence":"additional","affiliation":[{"name":"S. Tanaka and A.-K. Hadjantonakis, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. T. Kunath, A. Nagy, J. Rossant, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna-Katerina","family":"Hadjantonakis","sequence":"additional","affiliation":[{"name":"S. Tanaka and A.-K. Hadjantonakis, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. T. Kunath, A. Nagy, J. Rossant, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andras","family":"Nagy","sequence":"additional","affiliation":[{"name":"S. Tanaka and A.-K. Hadjantonakis, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. T. Kunath, A. Nagy, J. Rossant, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Janet","family":"Rossant","sequence":"additional","affiliation":[{"name":"S. Tanaka and A.-K. Hadjantonakis, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. T. Kunath, A. Nagy, J. Rossant, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"#cr-split#-e_1_3_1_2_2.1","doi-asserted-by":"crossref","unstructured":"J. Rossant in Experimental Approaches to Mammalian Embryonic Development J. Rossant and R. A. Pederson Eds. (Cambridge Univ. Press London 1986) pp. 97-120","DOI":"10.1017\/CBO9780511665493.005"},{"key":"#cr-split#-e_1_3_1_2_2.2","unstructured":"Semin. Dev. Biol. 6 237 (1995)."},{"key":"e_1_3_1_3_2","first-page":"239","volume":"48","author":"Rossant J.","year":"1978","unstructured":"Rossant J., Gardner R. L., Alexandre H. L., J. Embryol. Exp. Morphol. 48, 239 (1978);","journal-title":"J. Embryol. Exp. Morphol."},{"key":"e_1_3_1_3_3","first-page":"103","volume":"61","author":"Johnson M. H.","year":"1981","unstructured":"Johnson M. H., Rossant J., ibid. 61, 103 (1981).","journal-title":"ibid."},{"key":"#cr-split#-e_1_3_1_4_2.1","doi-asserted-by":"crossref","unstructured":"R. L. Gardner and M. H. Johnson ibid. 28 279 (1972)","DOI":"10.1242\/dev.28.2.279"},{"key":"#cr-split#-e_1_3_1_4_2.2","doi-asserted-by":"crossref","unstructured":"J. Rossant and W. Tamura-Lis ibid. 62 217 (1981);","DOI":"10.1242\/dev.62.1.217"},{"key":"e_1_3_1_4_3","doi-asserted-by":"publisher","DOI":"10.1016\/S0143-4004(83)80010-1"},{"key":"e_1_3_1_5_2","first-page":"183","volume":"39","author":"Rossant J.","year":"1977","unstructured":"Rossant J., Ofer L., J. Embryol. Exp. Morphol. 39, 183 (1977);","journal-title":"J. Embryol. Exp. Morphol."},{"key":"e_1_3_1_5_3","first-page":"183","volume":"62","author":"Ilgren E. B.","year":"1981","unstructured":"Ilgren E. B., ibid. 62, 183 (1981).","journal-title":"ibid."},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1242\/dev.114.3.755"},{"key":"e_1_3_1_6_3","first-page":"2259","volume":"120","author":"Rappolee D. A.","year":"1994","unstructured":"Rappolee D. A., Basilico C., Patel Y., Werb Z., ibid. 120, 2259 (1994).","journal-title":"ibid."},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1006\/dbio.1993.1205"},{"key":"e_1_3_1_7_3","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.9.5082"},{"key":"e_1_3_1_8_2","unstructured":"J. Partanen and J. Rossant unpublished data."},{"key":"e_1_3_1_9_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.7809630"},{"key":"e_1_3_1_10_2","doi-asserted-by":"publisher","DOI":"10.1006\/dbio.1997.8858"},{"key":"e_1_3_1_11_2","unstructured":"The TS cell medium was RPMI 1640 supplemented with 20% fetal bovine serum (Hyclone Logan UT) sodium pyruvate (1 mM) (Gibco-BRL) \u03b2-mercaptoethanol (100 \u03bcM) (Sigma) l -glutamine (2 mM) (Gibco-BRL) and penicillin and streptomycin (50 \u03bcg\/ml each). Human recombinant FGF4 (25 ng\/ml) (Sigma) and heparin (1 \u03bcg\/ml) were added to aliquots of TS cell medium and used immediately."},{"key":"#cr-split#-e_1_3_1_12_2.1","unstructured":"B. Hogan R. Beddington F. Costantini E. Lacy Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press Cold Spring Harbor NY ed. 2 1994) pp. 265-272"},{"key":"#cr-split#-e_1_3_1_12_2.2","unstructured":"E. J. Robertson in Teratocarcinomas and Embryonic Stem Cells E. J. Robertson Ed. (IRL Oxford 1987) pp. 71-112."},{"key":"e_1_3_1_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/0143-4004(95)90033-0"},{"key":"e_1_3_1_14_2","unstructured":"TS 3.5 cell lines were obtained with techniques similar to those used for ES cell line derivation (11). Blastocysts (3.5 dpc) were individually plated into four-well plates on EMFI cells and cultured in TS medium with FGF4 and heparin (10). The medium was changed after day 2 and the blastocyst outgrowth was trypsinized on day 3. On day 5 or 6 flat colonies [referred to as \u201cepithelial-like cells\u201d in (11)] were picked and passaged. Once established the cell lines were grown without EMFI cells but in the presence of EMFI-CM (14). Under these culture conditions ES cell colonies were not observed."},{"key":"e_1_3_1_15_2","unstructured":"EMFI-CM was prepared by incubating TS medium (10) without FGF4 or heparin on confluent plates of mitomycin-treated EMFI cells for 72 hours. The conditioned medium was filtered (0.45 \u03bcm) and stored at \u201320\u00b0C. Established TS cell lines were routinely cultured in 70% EMFI-CM 30% TS medium FGF4 (25 ng\/ml) and heparin (1 \u03bcg\/ml) on gelatinized plates. The medium was changed every 2 days and 1 part in 25 of the cells was passaged every 4 days or at 80 to 90% confluency."},{"key":"e_1_3_1_16_2","unstructured":"S. Tanaka T. Kunath J. Rossant data not shown."},{"key":"e_1_3_1_17_2","unstructured":"TS cells were grown in the absence of EMFI cells (14) and were collected by cell scraping at 0 2 4 and 6 days after the removal of FGF4 heparin and EMFI-CM. The cells were fixed and stained with propidium iodide (PI) (Molecular Probes Eugene OR) as described [Z. Darzynkiewicz and G. Juan in Current Protocols in Cytometry J. P. Robinson Ed. (Wiley New York 1997) pp. 7.5.2\u20137.5.3]. Cell fluorescence was measured by flow cytometry with an argon ion laser (488 nm). The data were analyzed with Coulter EXPO Cytometer software [version 2.0 (1998)] by Applied Cytometry Systems (Sheffield UK)."},{"key":"e_1_3_1_18_2","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.9.4.795"},{"key":"e_1_3_1_19_2","unstructured":"Total RNA was prepared from cells and embryos with TRIzol (Gibco BRL) according to the manufacturer's instructions. Northern (RNA) blotting was performed by a standard protocol. Antisense RNA probes for Err\u03b2 (19) eomesodermin (21) Cdx2 ["},{"key":"e_1_3_1_19_3","first-page":"7340","volume":"14","author":"Suh E.","year":"1994","unstructured":"Suh E., Chen L., Taylor J., Traber P. G., Mol. Cell. Biol. 14, 7340 (1994);","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_19_4","unstructured":"] Fgfr2 (7) 4311 (22) Mash2 (23) Pl-1 ["},{"key":"e_1_3_1_19_5","doi-asserted-by":"publisher","DOI":"10.1210\/mend-1-11-767"},{"key":"e_1_3_1_19_6","unstructured":"] Hand1 (25) Oct-3\/4 (26) Brachyury (26) and GAPDH ["},{"key":"e_1_3_1_19_7","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/13.5.1431"},{"key":"e_1_3_1_19_8","unstructured":"] were labeled with either [\u03b1- 32 P]uridine 5\u2032-triphosphate (UTP) or digoxigenin (DIG)-11-UTP (Boehringer Mannheim) by using a Strip-EZ RNA kit (Ambion Austin TX). Blots were hybridized overnight at 65\u00b0C in NorthernMax prehybridization\/hybridization buffer (Ambion) and were washed in 0.1\u00d7 standard saline citrate\/0.1% SDS at 65\u00b0C. DIG-labeled probes were detected with the DIG luminescent detection kit (Boehringer Mannheim). The removal of hybridized RNA probes was performed with the Strip-EZ RNA kit (Ambion) according to the manufacturer's recommendations. To assess the expression of Hnf4 in the TS cell lines we synthesized first-strand cDNA from total RNA (0.5 \u03bcg) of TS cells and 7.5-dpc embryos with random hexamers were subjected to 35 cycles of polymerase chain reaction (PCR) (62\u00b0C annealing temperature) with Hnf4 -specific primers [5\u2032-CACGTCCCCATCTGAAGGTG-3\u2032 and 5\u2032-CTTCCTTCTTCATGCCAGCCC-3\u2032 (0.2 \u03bcM each)] and with \u03b2-actin\u2013specific primers [5\u2032-GACAACGGCTCCGGCATGTGCAAAG-3\u2032 and 5\u2032-TTCACGGTTGGCCTTAGGGTTCAG-3\u2032 (0.1 \u03bcM each)]. The primer sequences were adapted from"},{"key":"e_1_3_1_19_9","doi-asserted-by":"publisher","DOI":"10.1242\/dev.125.8.1529"},{"key":"e_1_3_1_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/0925-4773(95)00479-3"},{"key":"e_1_3_1_20_3","doi-asserted-by":"publisher","DOI":"10.1038\/42022"},{"key":"e_1_3_1_21_2","doi-asserted-by":"publisher","DOI":"10.1002\/aja.1002040302"},{"key":"e_1_3_1_22_2","unstructured":"B. Ciruna and J. Rossant unpublished data."},{"key":"e_1_3_1_23_2","doi-asserted-by":"publisher","DOI":"10.1101\/gad.2.12a.1639"},{"key":"e_1_3_1_24_2","doi-asserted-by":"publisher","DOI":"10.1038\/371333a0"},{"key":"e_1_3_1_24_3","doi-asserted-by":"publisher","DOI":"10.1006\/dbio.1997.8685"},{"key":"e_1_3_1_25_2","doi-asserted-by":"publisher","DOI":"10.1095\/biolreprod44.2.327"},{"key":"e_1_3_1_26_2","doi-asserted-by":"publisher","DOI":"10.1242\/dev.121.8.2513"},{"key":"e_1_3_1_26_3","doi-asserted-by":"publisher","DOI":"10.1038\/ng0398-271"},{"key":"e_1_3_1_27_2","doi-asserted-by":"publisher","DOI":"10.1006\/dbio.1994.1312"},{"key":"e_1_3_1_27_3","doi-asserted-by":"publisher","DOI":"10.1038\/343657a0"},{"key":"e_1_3_1_27_4","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.91.16.7598"},{"key":"e_1_3_1_28_2","unstructured":"A. Nagy and J. Rossant in Gene Targeting A. L. Joyner Ed. (IRL Oxford 1993) pp. 147\u2013180;"},{"key":"e_1_3_1_28_3","doi-asserted-by":"publisher","DOI":"10.1038\/365087a0"},{"key":"e_1_3_1_29_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0925-4773(98)00093-8"},{"key":"e_1_3_1_30_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81769-9"},{"key":"e_1_3_1_31_2","doi-asserted-by":"publisher","DOI":"10.1242\/dev.125.17.3379"},{"key":"e_1_3_1_31_3","first-page":"2983","volume":"121","author":"Griffin K.","year":"1995","unstructured":"Griffin K., Patient R., Holder N., ibid. 121, 2983 (1995).","journal-title":"ibid."},{"key":"e_1_3_1_32_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(98)70257-X"},{"key":"e_1_3_1_32_3","doi-asserted-by":"publisher","DOI":"10.1093\/emboj\/17.12.3413"},{"key":"e_1_3_1_33_2","unstructured":"We thank J. Cross for generously providing cDNAs for Hand1 Err\u03b2 and GAPDH and also for critically reviewing the manuscript; C. Smith for FACS analysis; D. Dufort M. Gertsenstein L. Schwartz and S. McMaster for helping in the production of chimeric embryos; and A. Spence and S. Egan for critically reviewing the manuscript. S.T. thanks K. Shiota for kindly allowing him to continue investigating TS cells in his laboratory. T.K. is the recipient of a Medical Research Council (MRC) studentship. A.N. is a MRC\/Bristol-Myers Squibb scientist. J.R. is a MRC distinguished scientist and a Howard Hughes international scholar. Supported by the MRC of Canada."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.282.5396.2072","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T05:29:07Z","timestamp":1705123747000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.282.5396.2072"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1998,12,11]]},"references-count":55,"journal-issue":{"issue":"5396","published-print":{"date-parts":[[1998,12,11]]}},"alternative-id":["10.1126\/science.282.5396.2072"],"URL":"https:\/\/doi.org\/10.1126\/science.282.5396.2072","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1998,12,11]]}}}