Bone morphogenetic protein-4 affects both trophoblast and non-trophoblast lineage-associated gene expression in human embryonic stem cells


Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation; however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.

Share and Cite:

L. Shirley, M. , Venable, A. , R. Rao, R. , L. Boyd, N. , L. Stice, S. , Puett, D. and Narayan, P. (2012) Bone morphogenetic protein-4 affects both trophoblast and non-trophoblast lineage-associated gene expression in human embryonic stem cells. Stem Cell Discovery, 2, 163-175. doi: 10.4236/scd.2012.24021.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Rama, S. and Rao, A.J. (2003) Regulation of growth and function of the human placenta. Molecular and Cellular Biochemistry, 253, 263-268. doi:10.1023/A:1026076219126
[2] Sullivan, M.H. (2004) Endocrine cell lines from the placenta. Molecular and Cellular Endocrinology, 228, 103-119. doi:10.1016/j.mce.2003.03.001
[3] Udayashankar, R., Baker, D., Tuckerman, E., Laird, S., Li, T.C. and Moore, H.D. (2011) Characterization of invasive trophoblasts generated from human embryonic stem cells. Human Reproduction, 26, 398-406. doi:10.1093/humrep/deq350
[4] Jameson, J.L. and Hollenberg, A.N. (1993) Regulation of chorionic gonadotropin gene expression. Endocrine Reviews, 14, 203-221.
[5] Mesiano, S. (2009) The endocrinology of human pregnancy and fetoplacental neuroendocrine development. In: Strauss, J.F. and Barbieri, R.L. Eds., Yen and Jaffe’s Reproductive Endocrinology, 6th Edition, Physiology, Pathophysiology and Clinical Management, Philadelphia, 249-281.
[6] Thomson, J.A., Itskovitz-Eldor. J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S. and Jones, J.M. (1998) Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145-1147. doi:10.1126/science.282.5391.1145
[7] Mitalipova, M., Calhoun, J., Shin, S., Wininger, D., Schulz, T., Noggle, S., Venable, A., Lyons, I., Robins, A. and Stice, S. (2003) Human embryonic stem cell lines derived from discarded embryos. Stem Cells, 21, 521-526. doi:10.1634/stemcells.21-5-521
[8] Gerami-Naini, B., Dovzhenko, O.V., Durning, M., Wegner, F.H., Thomson, J.A. and Golos, T.G. (2004) Trophoblast differentiation in embryoid bodies derived from human embryonic stem cells. Endocrinology, 145, 1517-1524. doi:10.1210/en.2003-1241
[9] Giakoumopoulos, M., Siegfried, L.M., Dambaeva, S.V., Garthwaite, M.A., Glennon, M.C. and Golos, T.G. (2010) Placental-derived mesenchyme influences chorionic gonadotropin and progesterone secretion of human embryonic stem cell-derived trophoblasts. Reproductive Science, 17, 798-808. doi:10.1177/1933719110371853
[10] Golos, T.G., Pollastrini, L.M. and Gerami-Naini, B. (2006) Human embryonic stem cells as a model for trophoblast differentiation. Semin Reproductive Medicine, 24, 314-321. doi:10.1055/s-2006-952154
[11] Harun, R., Ruban, L., Matin, M., Draper, J., Jenkins, N.M., Liew, G.C., Andrews, P.W., Li, T.C., Laird, S.M. and Moore, H.D. (2006) Cytotrophoblast stem cell lines derived from human embryonic stem cells and their capacity to mimic invasive implantation events. Human Reproduction, 21, 1349-1358. doi:10.1093/humrep/del017
[12] Peiffer, I., Belhomme, D., Barbet, R., Haydont, V., Zhou, Y.P., Fortunel, N.O., Li, M., Hatzfeld, A., Fabiani, J.N. and Hatzfeld, J.A. (2007) Simultaneous differentiation of endothelial and trophoblastic cells derived from human embryonic stem cells. Stem Cells and Development, 16, 393-402. doi:10.1089/scd.2006.0013
[13] Boyd, N.L., Dhara, S.K., Rekaya, R., Godbey, E.A., Hasneen, K., Rao, R.R., West, F.D., Gerwe, B.A. and Stice, S.L. (2007) BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies. Experimental Biology and Medicine, 232, 833-843.
[14] Chen, G., Ye, Z., Yu, X., Zou, J., Mali, P., Brodsky, R.A. and Cheng, L. (2008) Trophoblast differentiation defect in human embryonic stem cells lacking PIG-A and GPI-anchored cell-surface proteins. Cell Stem Cell, 2, 345-355. doi:10.1016/j.stem.2008.02.004
[15] Kee, K., Gonsalves, J.M., Clark, A.T. and Pera, R.A. (2006) Bone morphogenetic proteins induce germ cell differentiation from human embryonic stem cells. Stem Cells and Development, 15, 831-837. doi:10.1089/scd.2006.15.831
[16] Liu, Y.P., Dovzhenko, O.V., Garthwaite, M.A., Dambaeva, S.V., Durning, M., Pollastrini, L.M. and Golos, T.G. (2004) Maintenance of pluripotency in human embryonic stem cells stably over-expressing enhanced green fluorescent protein. Stem Cells and Development, 13, 636-645. doi:10.1089/scd.2004.13.636
[17] Marchand, M., Horcajadas, J.A., Esteban, F.J., McElroy, S.L., Fisher, S.J. and Giudice, L.C. (2011) Transcriptomic signature of trophoblast differentiation in a human embryonic stem cell model. Biology of Reproduction, 84, 1258-1271. doi:10.1095/biolreprod.110.086413
[18] Pera, M.F., Andrade, J., Houssami, S., Reubinoff, B., Trounson, A., Stanley, E.G., Ward-van Oostwaard, D. and Mummery, C. (2004) Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. Journal of Cell Science, 117, 1269-1280. doi:10.1242/jcs.00970
[19] Schulz, L.C., Ezashi, T., Das, P., Westfall, S.D., Livingston, K.A. and Roberts, R.M. (2008) Human embryonic stem cells as models for trophoblast differentiation. Placenta, 29, S10-S16.
[20] Xu R.H. (2006) In vitro induction of trophoblast from human embryonic stem cells. Methods in Molecular Medicine, 121, 189-202.
[21] Xu, R.H., Chen, X., Li, D.S., Li, R., Addicks, G.C., Glennon, C., Zwaka, T.P. and Thomson, J.A. (2002) BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nature Biotechnology, 20, 1261-1264. doi:10.1038/nbt761
[22] Yu, P., Pan, G., Yu, J. and Thomson, J.A. (2011) FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation. Cell Stem Cell, 8, 326-334. doi:10.1016/j.stem.2011.01.001
[23] Yu, X., Zou, J., Ye, Z., Hammond, H., Chen, G., Tokunaga, A., Mali, P., Li, Y.M., Civin, C., Gaiano, N. and Cheng, L. (2008) Notch signaling activation in human embryonic stem cells is required for embryonic, but not trophoblastic, lineage commitment. Cell Stem Cell, 2, 461-471. doi:10.1016/j.stem.2008.03.001
[24] Zhang, P., Li, J., Tan, Z., Wang, C., Liu, T., Chen, L., Yong, J., Jiang, W., Sun, X., Du, L., Ding, M. and Deng, H. (2008) Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells. Blood, 111, 1933-1941. doi:10.1182/blood-2007-02-074120
[25] Douglas, G.C., Vande-Voort, C.A., Kumar, P., Chang, T.C. and Golos, T.G. (2009) Trophoblast stem cells: models for investigating trophectoderm differentiation and placental development. Endocrine Reviews, 30, 228-240. doi:10.1210/er.2009-0001
[26] Golos, T.G., Giakoumopoulos, M. and Garthwaite, M.A. (2010) Embryonic stem cells as models of trophoblast differentiation: progress, opportunities, and limitations. Reproduction, 140, 3-9. doi:10.1530/REP-09-0544
[27] Pera M.F.and Trounson A.O. (2004) Human embryonic stem cells: prospects for development. Development, 131, 5515-5525. doi:10.1242/dev.01451
[28] Roberts, R.M., Ezashi, T. and Das, P. (2004) Trophoblast gene expression: transcription factors in the specification of early trophoblast. Reproductive Biology and Endocrinology, 2, 47. doi:10.1186/1477-7827-2-47
[29] Hay, D.C., Sutherland, L., Clark, J. and Burdon, T. (2004) Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells, 22, 225-235. doi:10.1634/stemcells.22-2-225
[30] Matin, M.M., Walsh, J.R., Gokhale, P.J., Draper, J.S., Bahrami, A.R., Morton, I., Moore, H.D. and Andrews, P.W. (2004) Specific knockdown of Oct4 and beta 2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells. Stem Cells, 22, 659-668. doi:10.1634/stemcells.22-5-659
[31] Bragdon, B., Moseychuk, O., Saldanha, S., King, D., Julian, J. and Nohe, A. (2011) Bone morphogenetic proteins: a critical review. Cell Signal, 23, 609-620. doi:10.1016/j.cellsig.2010.10.003
[32] Rider, C.C. and Mulloy, B. (2010) Bone morphogenetic protein and growth differentiation factor cytokine families and their protein antagonists. The Biochemical Journal, 429, 1-12. doi:10.1042/BJ20100305
[33] Walsh, D.W., Godson, C., Brazil, D.P. and Martin, F. (2010) Extracellular BMP-antagonist regulation in development and disease: tied up in knots. Trends in Cell Biology, 20, 244-256. doi:10.1016/j.tcb.2010.01.008
[34] Zeng, S., Chen, J. and Shen, H. (2010) Controlling of bone morphogenetic protein signaling. Cell Signal, 22, 888-893. doi:10.1016/j.cellsig.2009.12.007
[35] Malassine, A., Frendo, J.L. and Evain-Brion, D. (2003) A comparison of placental development and endocrine functions between the human and mouse model. Hum Reproduction, 9, 531-539. doi:10.1093/humupd/dmg043
[36] Rossant, J. (2001) Stem cells from the mammalian blastocyst. Stem Cells, 19, 477-482. doi:10.1634/stemcells.19-6-477
[37] Smith, A.G. (2001) Embryo-derived stem cells of mice and men. Annual Reviews of Cell and Developmental Biology, 17, 435-462. doi:10.1146/annurev.cellbio.17.1.435
[38] Erb, T.M., Schneider, C., Mucko, S.E., Sanfilippo, J.S., Lowry, N.C., Desai, M.N., Mangoubi, R.S., Leuba, S.H. and Sammak, P.J. (2011) Paracrine and epigenetic control of trophectoderm differentiation from human embryonic stem cells: the role of bone morphogenic protein 4 and histone deacetylases. Stem Cells Development, 20, 1601-1614. doi:10.1089/scd.2010.0281
[39] Bernardo, A.S., Faial, T., Gardner, L., Niakan, K.K., Ortmann, D., Senner, C.E., Callery, E.M., Trotter, M.W., Hemberger, M., Smith, J.C., Bardwell, L., Moffett, A. and Pedersen, R.A. (2011) BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extra embryonic lineages. Cell Stem Cell, 9, 144-155. doi:10.1016/j.stem.2011.06.015
[40] Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods, 25, 402-408.
[41] Pfaffl, M.W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 29, e45. doi:10.1093/nar/29.9.e45
[42] Aghajanova, L., Shen, S., Rojas, A.M., Fisher, S.J., Irwin, J.C. and Giudice, L.C. (2012) Comparative transcriptome analysis of human trophectoderm and embryonic stem cell-derived trophoblasts reveal key participants in early implantation. Biology and Reproduction, 86, 1-21. doi:10.1095/biolreprod.111.092775
[43] Wu, Z., Zhang, W., Chen, G., Cheng, L., Liao, J., Jia, N., Gao, Y., Dai, H., Yuan, J. and Xiao, L. (2008) Combinatorial signals of activin/nodal and bone morphogenic protein regulate the early lineage segregation of human embryonic stem cells. The Journal of Biological Chemistry, 283, 24991-25002. doi:10.1074/jbc.M803893200
[44] Das, P., Ezashi, T., Schulz, L.C., Westfall, S.D., Livingston, K.A. and Roberts, R.M. (2007) Effects of fgf 2 and oxygen in the bmp4-driven differentiation of trophoblast from human embryonic stem cells. Stem Cell Research, 1, 61-74. doi:10.1016/j.scr.2007.09.004
[45] West, F.D., Roche-Rios, M.I., Abraham, S., Rao, R.R., Natrajan, M.S., Bacanamwo, M. and Stice, S.L. (2010) KIT ligand and bone morphogenetic protein signaling enhances human embryonic stem cell to germ-like cell differentiation. Human Reproduction, 25, 168-178. doi:10.1093/humrep/dep338
[46] Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H. and Benvenisty, N. (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Molecular Medicine, 6, 88-95.

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.