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Both BMP4 and serum have significant roles in differentiation of embryonic stem cells to primitive and definitive endoderm

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Abstract

Differentiation of embryonic stem (ES) cells is a heterogeneous process which is influenced by different parameters, including growth and differentiation factors. The aim of the present study was to investigate the effect of bone morphogenetic protein-4 (BMP4) signaling on differentiation of mouse ES cells to endodermal lineages. For this purpose, differentiation of the ES cells was induced by embryoid body (EB) formation through hanging drop method. During the suspension stage, EBs were treated with BMP4 in a medium containing either fetal bovine serum (FBS) or knockout serum replacement (KoSR). After plating, EBs showed differentiation to a heterogeneous population of specialized cell types. Two weeks after plating, all the experimental groups expressed three germ layer markers and some primitive and definitive endoderm-specific genes. Quantitative real-time PCR analysis showed higher expression levels of Sox17, Pdx1, Cdx2 and Villin mRNAs in the KoSR plus BMP4 condition and higher Gata4 and Afp expression levels in the FBS plus BMP4 condition. Formation of visceral endoderm and derivatives of definitive endoderm was detected in the BMP4 treated EBs. In conclusion, we demonstrated that both BMP4 signaling and serum composition have significant roles in differentiation of mouse ES cells towards endodermal lineages.

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References

  • Aberdam D, Gambaro K, Rostagno P, Aberdam E, de la Forest Divonne S, Rouleau M (2007) Key role of p63 in BMP-4-induced epidermal commitment of embryonic stem cells. Cell Cycle 6:291–294

    Article  CAS  Google Scholar 

  • Amita M, Adachi K, Alexenko AP, Sinha S, Schust DJ, Schulz LC, Roberts RM, Ezashi T (2013) Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4. Proc Natl Acad Sci USA 110:E1212–E1221. doi:10.1073/pnas.1303094110

    Article  CAS  Google Scholar 

  • Artus J, Douvaras P, Piliszek A, Isern J, Baron MH, Hadjantonakis AK (2012) BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity. Dev Biol 361:245–262. doi:10.1016/j.ydbio.2011.10.015

    Article  CAS  Google Scholar 

  • Beck F, Erler T, Russell A, James R (1995) Expression of Cdx-2 in the mouse embryo and placenta: possible role in patterning of the extra-embryonic membranes. Dev Dyn 204:219–227. doi:10.1002/aja.1002040302

    Article  CAS  Google Scholar 

  • Conley BJ, Ellis S, Gulluyan L, Mollard R (2007) BMPs regulate differentiation of a putative visceral endoderm layer within human embryonic stem-cell-derived embryoid bodies. Biochem Cell Biol 85:121–132. doi:10.1139/o06-145

    Article  CAS  Google Scholar 

  • Coucouvanis E, Martin GR (1995) Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83:279–287

    Article  CAS  Google Scholar 

  • Coucouvanis E, Martin GR (1999) BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo. Development 126:535–546

    CAS  Google Scholar 

  • Davis S, Miura S, Hill C, Mishina Y, Klingensmith J (2004) BMP receptor IA is required in the mammalian embryo for endodermal morphogenesis and ectodermal patterning. Dev Biol 270:47–63. doi:10.1016/j.ydbio.2004.01.048

    Article  CAS  Google Scholar 

  • Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 87:27–45

    CAS  Google Scholar 

  • Dziadek M, Adamson E (1978) Localization and synthesis of alphafoetoprotein in post-implantation mouse embryos. J Embryol Exp Morphol 43:289–313

    CAS  Google Scholar 

  • Faure S, de Santa Barbara P, Roberts DJ, Whitman M (2002) Endogenous patterns of BMP signaling during early chick development. Dev Biol 244:44–65. doi:10.1006/dbio.2002.0579

    Article  CAS  Google Scholar 

  • Gardner RL (1983) Origin and differentiation of extraembryonic tissues in the mouse. Int Rev Exp Pathol 24:63–133

    CAS  Google Scholar 

  • Goldsborough MD, Tilkins ML, Price PJ, Lobo-Alfonso J, Morrison J, Stevens ME, Meneses J, Pederson R, Koller B, Latour A (1998) Serum-free culture of murine embryonic stem (ES) cells. Focus 20:8–12

    Google Scholar 

  • Gouon-Evans V, Boussemart L, Gadue P, Nierhoff D, Koehler CI, Kubo A, Shafritz DA, Keller G (2006) BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat Biotechnol 24:1402–1411. doi:10.1038/nbt1258

    Article  CAS  Google Scholar 

  • Hamazaki T, Oka M, Yamanaka S, Terada N (2004) Aggregation of embryonic stem cells induces Nanog repression and primitive endoderm differentiation. J Cell Sci 117:5681–5686. doi:10.1242/jcs.01489

    Article  CAS  Google Scholar 

  • Hansson M, Olesen DR, Peterslund JM, Engberg N, Kahn M, Winzi M, Klein T, Maddox-Hyttel P, Serup P (2009) A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells. Dev Biol 330:286–304. doi:10.1016/j.ydbio.2009.03.026

    Article  CAS  Google Scholar 

  • Hayashi Y, Furue MK, Tanaka S, Hirose M, Wakisaka N, Danno H, Ohnuma K, Oeda S, Aihara Y, Shiota K, Ogura A, Ishiura S, Asashima M (2010) BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin. In Vitro Cell Dev Biol Anim 46:416–430. doi:10.1007/s11626-009-9266-6

    Article  CAS  Google Scholar 

  • Hogan BL (1996) Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev 10:1580–1594

    Article  CAS  Google Scholar 

  • Howe JR, Bair JL, Sayed MG, Anderson ME, Mitros FA, Petersen GM, Velculescu VE, Traverso G, Vogelstein B (2001) Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis. Nat Genet 28:184–187. doi:10.1038/88919

    Article  CAS  Google Scholar 

  • Kanai-Azuma M, Kanai Y, Gad JM, Tajima Y, Taya C, Kurohmaru M, Sanai Y, Yonekawa H, Yazaki K, Tam PP, Hayashi Y (2002) Depletion of definitive gut endoderm in Sox17-null mutant mice. Development 129:2367–2379

    CAS  Google Scholar 

  • Kennedy M, D’Souza SL, Lynch-Kattman M, Schwantz S, Keller G (2007) Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109:2679–2687. doi:10.1182/blood-2006-09-047704

    CAS  Google Scholar 

  • Kuo CT, Morrisey EE, Anandappa R, Sigrist K, Lu MM, Parmacek MS, Soudais C, Leiden JM (1997) GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev 11:1048–1060

    Article  CAS  Google Scholar 

  • Lee SJ, Talamantes F, Wilder E, Linzer DI, Nathans D (1988) Trophoblastic giant cells of the mouse placenta as the site of proliferin synthesis. Endocrinology 122:1761–1768. doi:10.1210/endo-122-5-1761

    Article  CAS  Google Scholar 

  • Liu H, Collins SF, Suggs LJ (2006) Three-dimensional culture for expansion and differentiation of mouse embryonic stem cells. Biomaterials 27:6004–6014. doi:10.1016/j.biomaterials.2006.06.016

    Article  CAS  Google Scholar 

  • Martin GR, Wiley LM, Damjanov I (1977) The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells. Dev Biol 61:230–244

    Article  CAS  Google Scholar 

  • Mathew S, Jaramillo M, Zhang X, Zhang LA, Soto-Gutierrez A, Banerjee I (2012) Analysis of alternative signaling pathways of endoderm induction of human embryonic stem cells identifies context specific differences. BMC Syst Biol 6:154. doi:10.1186/1752-0509-6-154

    Article  CAS  Google Scholar 

  • Maunoury R, Robine S, Pringault E, Huet C, Guenet JL, Gaillard JA, Louvard D (1988) Villin expression in the visceral endoderm and in the gut anlage during early mouse embryogenesis. EMBO J 7:3321–3329

    CAS  Google Scholar 

  • Maunoury R, Robine S, Pringault E, Leonard N, Gaillard JA, Louvard D (1992) Developmental regulation of villin gene expression in the epithelial cell lineages of mouse digestive and urogenital tracts. Development 115:717–728

    CAS  Google Scholar 

  • Mfopou JK, Geeraerts M, Dejene R, Van Langenhoven S, Aberkane A, Van Grunsven LA, Bouwens L (2013) Efficient definitive endoderm induction from mouse embryonic stem cell adherent cultures: a rapid screening model for differentiation studies. Stem cell Res 12:166–177. doi:10.1016/j.scr.2013.10.004

    Article  Google Scholar 

  • Molkentin JD, Lin Q, Duncan SA, Olson EN (1997) Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev 11:1061–1072

    Article  CAS  Google Scholar 

  • Narita T, Saitoh K, Kameda T, Kuroiwa A, Mizutani M, Koike C, Iba H, Yasugi S (2000) BMPs are necessary for stomach gland formation in the chicken embryo: a study using virally induced BMP-2 and Noggin expression. Development 127:981–988

    CAS  Google Scholar 

  • Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan BL, Wright CV (1996) PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122:983–995

    CAS  Google Scholar 

  • Pekkanen-Mattila M, Pelto-Huikko M, Kujala V, Suuronen R, Skottman H, Aalto-Setala K, Kerkela E (2010) Spatial and temporal expression pattern of germ layer markers during human embryonic stem cell differentiation in embryoid bodies. Histochem Cell Biol 133:595–606. doi:10.1007/s00418-010-0689-7

    Article  CAS  Google Scholar 

  • Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36

  • Poulain M, Furthauer M, Thisse B, Thisse C, Lepage T (2006) Zebrafish endoderm formation is regulated by combinatorial Nodal, FGF and BMP signalling. Development 133:2189–2200. doi:10.1242/dev.02387

    Article  CAS  Google Scholar 

  • Roberts DJ, Smith DM, Goff DJ, Tabin CJ (1998) Epithelial–mesenchymal signaling during the regionalization of the chick gut. Development 125:2791–2801

    CAS  Google Scholar 

  • Silberg DG, Swain GP, Suh ER, Traber PG (2000) Cdx1 and cdx2 expression during intestinal development. Gastroenterology 119:961–971

    Article  CAS  Google Scholar 

  • Smith DM, Nielsen C, Tabin CJ, Roberts DJ (2000) Roles of BMP signaling and Nkx2.5 in patterning at the chick midgut–foregut boundary. Development 127:3671–3681

    CAS  Google Scholar 

  • Sudheer S, Bhushan R, Fauler B, Lehrach H, Adjaye J (2012) FGF inhibition directs BMP4-mediated differentiation of human embryonic stem cells to syncytiotrophoblast. Stem Cells Dev 21:2987–3000. doi:10.1089/scd.2012.0099

    Article  CAS  Google Scholar 

  • Sumi T, Tsuneyoshi N, Nakatsuji N, Suemori H (2008) Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, activin/nodal and BMP signaling. Development 135:2969–2979. doi:10.1242/dev.021121

    Article  CAS  Google Scholar 

  • Tada S, Era T, Furusawa C, Sakurai H, Nishikawa S, Kinoshita M, Nakao K, Chiba T (2005) Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132:4363–4374. doi:10.1242/dev.02005

    Article  CAS  Google Scholar 

  • Taha MF, Valojerdi MR, Mowla SJ (2007) Effect of bone morphogenetic protein-4 (BMP-4) on cardiomyocyte differentiation from mouse embryonic stem cell. Int J Cardiol 120:92–101. doi:10.1016/j.ijcard.2006.08.118

    Article  Google Scholar 

  • Teo AK, Ali Y, Wong KY, Chipperfield H, Sadasivam A, Poobalan Y, Tan EK, Wang ST, Abraham S, Tsuneyoshi N, Stanton LW, Dunn NR (2012) Activin and BMP4 synergistically promote formation of definitive endoderm in human embryonic stem cells. Stem Cells 30:631–642. doi:10.1002/stem.1022

    Article  CAS  Google Scholar 

  • Wiles MV, Johansson BM (1999) Embryonic stem cell development in a chemically defined medium. Exp Cell Res 247:241–248. doi:10.1006/excr.1998.4353

    Article  CAS  Google Scholar 

  • Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, Zwaka TP, Thomson JA (2002) BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol 20:1261–1264. doi:10.1038/nbt761

    Article  CAS  Google Scholar 

  • Yamamoto M, Tase N, Okuno T, Kondo Y, Akiba S, Shimozawa N, Terao K (2007) Monitoring of gene expression in differentiation of embryoid bodies from cynomolgus monkey embryonic stem cells in the presence of bisphenol A. J Toxicol Sci 32:301–310

    Article  CAS  Google Scholar 

  • Zhang P, Li J, Tan Z, Wang C, Liu T, Chen L, Yong J, Jiang W, Sun X, Du L, Ding M, 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

    Article  CAS  Google Scholar 

  • Zorn AM, Wells JM (2009) Vertebrate endoderm development and organ formation. Annu Rev Cell Dev Biol 25:221–251. doi:10.1146/annurev.cellbio.042308.113344

    Article  CAS  Google Scholar 

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Acknowledgments

This study was supported by a research grant from National Institute of Genetic Engineering and Biotechnology (434). We would like to thank Mrs. Ilnaz Sadeghi Sadr and Mr. Shahram Pour Beiranvand for their technical advice.

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Authors and Affiliations

  1. Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Shahrak-e-Pajoohesh, 15th Km, Tehran-Karaj Highway, P.O. Box 14965-161, Tehran, Iran

    Masoumeh Fakhr Taha, Arash Javeri & Tayebeh Majidizadeh

  2. Department of Anatomy, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

    Mojtaba Rezazadeh Valojerdi

  3. Department of Embryology, Royan Institute, Tehran, Iran

    Mojtaba Rezazadeh Valojerdi

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  1. Masoumeh Fakhr Taha
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  2. Arash Javeri
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  4. Mojtaba Rezazadeh Valojerdi
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Correspondence to Masoumeh Fakhr Taha.

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Taha, M.F., Javeri, A., Majidizadeh, T. et al. Both BMP4 and serum have significant roles in differentiation of embryonic stem cells to primitive and definitive endoderm. Cytotechnology 68, 1315–1324 (2016). https://doi.org/10.1007/s10616-015-9891-8

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