Functional & Integrative Genomics

, Volume 5, Issue 2, pp 59–69 | Cite as

Transcriptional program of bone morphogenetic protein-2-induced epithelial and smooth muscle differentiation of pluripotent human embryonal carcinoma cells

  • Rajendrakumar S. V. Chadalavada
  • Jane Houldsworth
  • Adam B. Olshen
  • George J. Bosl
  • Lorenz Studer
  • R. S. K. Chaganti
Original Paper


Pluripotent human embryonal carcinoma NTera2/cloneD1 (NT2/D1) cells respond to multiple vertebrate patterning factors and offer a unique model system to investigate the signaling events associated with lineage determination and cell differentiation. Here, we define the temporal changes in global gene expression patterns in NT2/D1 cells upon treatment with bone morphogenetic protein-2 (BMP-2). Exposure to BMP-2 rapidly induced the expression of several transcription factors involved in establishing non-neural ectodermal fate followed by the appearance of epithelial-specific markers. Subsequent loss of stem cell markers was coupled to gene expression changes associated with decreased proliferative activity. Temporal clustering of gene expression patterns revealed a concurrent down-regulation of multiple transcripts involved in neurogenesis, neurite outgrowth, and axonal guidance, suggesting that the BMP-mediated differentiation process involves pro-epithelial as well as anti-neurogenic mechanisms. In addition, increased expression of smooth muscle markers both by gene expression and immunohistochemistry was detected. Several neural crest markers were induced preceding such a differentiation, compatible with a neural crest origin of NT2/D1-derived smooth muscle cells. Comparison of changes in transcript expression between BMP-2-induced epithelial versus all-trans-retinoic acid (ATRA)-induced neural differentiation revealed potential candidates for regulation of BMP-2 signaling and suppression of neural fate by BMP-2. This study suggests that BMP-2-induced differentiation of NT2/D1 cells provides a powerful assay to study early human epithelial and smooth muscle development.


Embryonal carcinoma BMP-2 Micro-array Differentiation 



We thank the MSKCC Genomics Core Facility for hybridization, washing, and scanning of the arrays. This work was supported by grants from the NIH and the Byrne Research Fund.

Supplementary material

Figures S1–S4

s10142_2005_132_fig_s1_4.pdf (52 kb)
(PDF 52 KB)

Table S1 Gene expression data from the time course analysis in triplicate after BMP-2 treatment of NT2/D1 cells

s10142_2005_132_table_s1.xls (20 mb)
(Excel 20 MB)

Table S2 Signal log ratios of time course analysis in triplicate after BMP-2 treatment of NT2/D1 cells. Designated clusters from Fig.1 and Fig. S1 are indicated in separate and adjacent columns

s10142_2005_132_table_s2.xls (304 kb)
(PDF 1 MB)
s10142_2005_132_table_s2.xls (304 kb)
(Excel 304 KB)

Table S3 Signal log ratios of time course analysis in triplicate after ATRA treatment of NT2/D1 cells. Changes in transcript expression during ATRA-induced differentiation was analyzed using the Micro Array Suite 5.0, using the 24 hour untreated control culture as the baseline

s10142_2005_132_table_s3.xls (207 kb)
(PDF 792 KB)
s10142_2005_132_table_s3.xls (207 kb)
(Excel 207 KB)


  1. Andrews PW (1984) Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev Biol 103:285–293CrossRefGoogle Scholar
  2. Andrews PW, Damjanov I, Berends J, Kumpf S, Zappavigna V, Mavilio F, Sampath K (1994) Inhibition of proliferation and induction of differentiation of pluripotent human embryonal carcinoma cells by osteogenic protein-1 (or bone morphogenetic protein-7). Lab Invest 71:243–251Google Scholar
  3. Bloom L, Horvitz HR (1997) The Caenorhabditis elegans gene unc-76 and its human homologs define a new gene family involved in axonal outgrowth and fasciculation. Proc Natl Acad Sci USA 94:3414–3419CrossRefGoogle Scholar
  4. Byk T, Dobransky T, Cifuentes-Diaz C, Sobel A (1996) Identification and molecular characterization of unc-33-like phosphoprotein (Ulip), a putative mammalian homolog of the axonal guidance-associated unc-33 gene product. J Neurosci 16:688–701Google Scholar
  5. Caricasole A, Oostward DV, Zeinstra L, Eijnden-van Raaij AVD, Raaij EV, Mummery C (2000) Bone morphogenetic proteins (BMPs) induce epithelial differentiation of NT2D1 human embryonal carcinoma cells. Int J Dev Biol 44:443–450Google Scholar
  6. Chaganti RSK, Houldsworth J (2000) Genetics and biology of adult human male germ cell tumors. Cancer Res 60:1475–1482Google Scholar
  7. Chang DF, Belaguli NS, Iyer D, Roberts WB, Wu SP, Dong XR, Marx JG, Moore MS, Beckerle MC, Majesky MW, Schwartz RJ (2003) Cysteine-rich LIM-only proteins CRP1 and CRP2 are potent smooth muscle differentiation cofactors. Dev Cell 4:107–118CrossRefGoogle Scholar
  8. Chu PG, Weiss LM (2002) Keratin expression in human tissues and neoplasms. Histopathology 40:403–439CrossRefPubMedGoogle Scholar
  9. Coller HA, Grandori C, Tamayo P, Colbert T, Lander ES, Eisenman RN, Golub TR (2000) Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc Natl Acad Sci USA 97:3260–3265CrossRefGoogle Scholar
  10. Coppe JP, Smith AP, Desprez PY (2003) Id proteins in epithelial cells. Exp Cell Res 15:131–145CrossRefGoogle Scholar
  11. Coraux C, Hilmi C, Rouleau M, Spadafora A, Hinnrasky J, Ortonne JP, Dani C, Aberdam D (2003) Reconstituted skin from murine embryonic stem cells. Curr Biol 13:849–853CrossRefGoogle Scholar
  12. Coulthard MG, Duffy S, Down M, Evans B, Power M, Smith F, Stylianou C, Kleikamp S, Oates A, Lackmann M, Burns GF, Boyd AW (2001) Characterization of the Epha1 receptor tyrosine kinase: expression in epithelial tissues. Growth Factors 18:303–317Google Scholar
  13. Curto EV, Lambert GW, Davis RL, Wilborn TW, Dooley TP (2002) Biomarkers of human skin cells identified using DermArray DNA arrays and new bioinformatics methods. Biochem Biophys Res Commun 291:1052–1064CrossRefPubMedGoogle Scholar
  14. Danziger N, Yokoyama M, Jay T, Cordier J, Glowinski J, Chneiweiss H (1995) Cellular expression, developmental regulation, and phylogenic conservation of PEA-15, the astrocytic major phosphoprotein and protein kinase C substrate. J Neurochem 64:1016–1025Google Scholar
  15. de Boor C (1978) A practical guide to splines. Applied mathematical sciences, vol 27. Springer-Verlag, New York Heidelberg BerlinGoogle Scholar
  16. de la Pompa JL, Wakeham A, Correia KM, Samper E, Brown S, Aguilera RJ, Nakano T, Honjo T, Mak TW, Rossant J, Conlon RA (1997) Conservation of the Notch signaling pathway in mammalian neurogenesis. Development 124:1139–1148PubMedGoogle Scholar
  17. Draper JS, Pigott C, Thomson JA, Andrews PW (2002) Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat 200:249–258CrossRefGoogle Scholar
  18. Esteve P, Trousse F, Rodriguez J, Bovolenta P (2003) SFRP1 modulates retina cell differentiation through a beta-catenin-independent mechanism. J Cell Sci 116:2471–2481CrossRefPubMedGoogle Scholar
  19. Favier J, Kempf H, Corvol P, Gasc JM (1999) Cloning and expression pattern of EPAS1 in the chicken embryo. Colocalization with tyrosine hydroxylase. FEBS Lett 462:19–24CrossRefGoogle Scholar
  20. Fuchs E, Raghavan S (2002) Getting under the skin of epidermal morphogenesis. Nat Rev Genet 3:199–209CrossRefGoogle Scholar
  21. Galbiati F, Volonte D, Gil O, Zanazzi G, Salzer JL, Sargiacomo M, Scherer PE, Engelman JA, Schlegel A, Parenti M, Okamoto T, Lisanti MP (1998) Expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion neurons: caveolin-2 is up-regulated in response to cell injury. Proc Natl Acad Sci USA 95:10257–10262CrossRefGoogle Scholar
  22. Gandarillas A, Watt FM (1995) Changes in expression of members of the fos and jun families and myc network during terminal differentiation of human keratinocytes. Oncogene 11:1403–1407Google Scholar
  23. Gitai Z, Yu TW, Lundquist EA, Tessier-Lavigne M, Bargmann CI (2003) The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and, in parallel, Rac and UNC-115/AbLIM. Neuron 37:53–65CrossRefGoogle Scholar
  24. Gokhale PJ, Giesberts AM, Andrews PW (2000) Brachyury is expressed by human teratocarcinoma cells in the absence of mesodermal differentiation. Cell Growth Differ 11:157–162Google Scholar
  25. Gripp KW, Wotton D, Edwards MC, Roessler E, Ades L, Meinecke P, Richieri-Costa A, Zackai EH., Massague J, Muenke M, Elledge SJ (2000) Mutations in TGIF cause holoprosencephaly and link NODAL signaling to human neural axis determination. Nat Genet 25:205–208CrossRefPubMedGoogle Scholar
  26. Halayko AJ, Solway J (2001) Molecular mechanisms of phenotypic plasticity in smooth muscle cells. J Appl Physiol 90:358–368Google Scholar
  27. Hemavathy K, Ashraf SI, Ip YT (2000) Snail/slug family of repressors: slowly going into the fast lane of development and cancer. Gene 257(1):1–12CrossRefGoogle Scholar
  28. Hollnagel A, Oehlmann V, Heymer J, Ruther U, Nordheim A (1999) Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. J Biol Chem 274:19838–19845CrossRefGoogle Scholar
  29. Hoodless PA, Hemmati-Brivanlou A (1997) Inhibitory control of neural differentiation in mammalian cells. Dev Genes Evol 207:19–28CrossRefGoogle Scholar
  30. Houldsworth J, Reuter VE, Bosl GJ, Chaganti RSK (2001) ID gene expression varies with lineage during differentiation of pluripotential male germ cell tumor cell lines. Cell Tissue Res 303:371–379CrossRefGoogle Scholar
  31. Houldsworth J, Heath SC, Bosl GJ, Studer L, Chaganti RSK (2002) Expression profiling of lineage differentiation in pluripotential human embryonal carcinoma cells. Cell Growth Differ 13:257–264Google Scholar
  32. Katz AB, Taichman LB (1999) A partial catalog of proteins secreted by epidermal keratinocytes in culture. J Invest Dermatol 112:818–821CrossRefPubMedGoogle Scholar
  33. Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, Nishikawa SI, Sasai Y (2000) Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28:31–40CrossRefPubMedGoogle Scholar
  34. Kim TA, Lim J, Ota S, Raja S, Rogers R, Rivnay B, Avraham H, Avraham S (1998) NRP/B, a novel nuclear matrix protein, associates with p110(RB) and is involved in neuronal differentiation. J Cell Biol 141:553–566CrossRefGoogle Scholar
  35. Kishi M, Mizuseki K, Sasai N, Yamazaki H, Shiota K, Nakanishi S, Sasai Y (2000) Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm. Development 127:791–800Google Scholar
  36. Knecht AK, Bronner-Fraser M (2002) Induction of the neural crest: a multigene process. Nat Rev Genet 3:453–461Google Scholar
  37. Layne MD, Endege WO, Jain MK, Yet SF, Hsieh CM, Chin MT, Perrella MA, Blanar MA, Haber E, Lee ME (1998) Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation. J Biol Chem 273:15654–15660CrossRefGoogle Scholar
  38. Lin Q, Lu J, Yanagisawa H, Webb R, Lyons GE, Richardson JA, Olson EN (1998) Requirement of the MADS-box transcription factor MEF2C for vascular development. Development 125:4565–4574PubMedGoogle Scholar
  39. Lin A, Lenert LA, Hlatky MA, McDonald KM, Olshen RA, Hornberger J (1999) Clustering and the design of preference-assessment surveys in healthcare. Health Serv Res 34:1033–1045Google Scholar
  40. Lin L, Jeanclos EM, Treuil M, Braunewell KH, Gundelfinger ED, Anand R (2002) The calcium sensor protein visinin-like protein-1 modulates the surface expression and agonist sensitivity of the alpha 4beta 2 nicotinic acetylcholine receptor. J Biol Chem 277:41872–41878CrossRefGoogle Scholar
  41. Lloyd SP (1982) Least square quantization in PCM. IEEE Trans Inf Theory 28:29–137CrossRefGoogle Scholar
  42. Luo T, Lee YH, Saint-Jeannet JP, Sargent TD (2003) Induction of neural crest in Xenopus by transcription factor AP2alpha. Proc Natl Acad Sci USA 100:532–537CrossRefGoogle Scholar
  43. Luther T, Flossel C, Mackman N, Bierhaus A, Kasper M, Albrecht S, Sage EH, Iruela-Arispe L, Grossmann H, Strohlein A, Zhang Y, Nawroth PP, Carmeliet P, Loskutoff DJ, Muller M (1996) Tissue factor expression during human and mouse development. Am J Pathol 149:101–113Google Scholar
  44. Masunaga T, Shimizu H, Ishiko A, Fujiwara T, Hashimoto T, Nishikawa T (1995) Desmoyokin/AHNAK protein localizes to the non-desmosomal keratinocyte cell surface of human epidermis. J Invest Dermatol 104:941–945CrossRefGoogle Scholar
  45. Matin MM, Walsh JR, Gokhale PJ, Draper JS, Bahrami AR, Morton I, Moore HD, Andrews PW (2004) Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells. Stem Cells 22:659–668CrossRefGoogle Scholar
  46. McKee KK, Tan CP, Palyha OC, Liu J, Feighner SD, Hreniuk DL, Smith RG, Howard AD, van der Ploeg LH (1997) Cloning and characterization of two human G protein-coupled receptor genes (GPR38 and GPR39) related to the growth hormone secretagogue and neurotensin receptors. Genomics 46:426–434CrossRefGoogle Scholar
  47. Melnikova IN, Bounpheng M, Schatteman GC, Gilliam D, Christy BA (1999) Differential biological activities of mammalian Id proteins in muscle cells. Exp Cell Res 247:94–104CrossRefGoogle Scholar
  48. Menssen A, Hermeking H (2002) Characterization of the c-MYC-regulated transcriptome by SAGE: identification and analysis of c-MYC target genes. Proc Natl Acad Sci USA 99:6274–6279CrossRefGoogle Scholar
  49. Mizuseki K, Sakamoto T, Watanabe K, Muguruma K, Ikeya M, Nishiyama A, Arakawa A, Suemori H, Nakatsuji N, Kawasaki H, Murakami F, Sasai Y (2003) Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc Natl Acad Sci USA 100:5828–5833CrossRefGoogle Scholar
  50. Munoz-Sanjuan I, Brivanlou AH (2002) Neural induction, the default model and embryonic stem cells. Nat Rev Neurosci 3:271–280CrossRefGoogle Scholar
  51. Murphy S, Krainock R, Tham M (2002) Neuregulin signaling via erbB receptor assemblies in the nervous system. Mol Neurobiol 25:67–77CrossRefGoogle Scholar
  52. Nakashima K, Takizawa T, Ochiai W, Yanagisawa M, Hisatsune T, Nakafuku M, Miyazono K, Kishimoto T, Kageyama R, Taga T (2001) BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis. Proc Natl Acad Sci USA 98:5868–5873CrossRefGoogle Scholar
  53. Pleasure SJ, Page C, Lee VM (1992) Pure, postmitotic, polarized human neurons derived from NTera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons. J Neurosci 12:1802–1815Google Scholar
  54. Rajan P, Panchision DM, Newell LF, McKay RD (2003) BMPs signal alternately through a SMAD or FRAP-STAT pathway to regulate fate choice in CNS stem cells. J Cell Biol 161:911–921CrossRefGoogle Scholar
  55. Ramaekers F, van Niekerk C, Poels L, Schaafsma E, Huijsmans A, Robben H, Schaart G, Vooijs P (1990) Use of monoclonal antibodies to keratin 7 in the differential diagnosis of adenocarcinomas. Am J Pathol 136:641–655Google Scholar
  56. Read EM, Rodaway AR, Neave B, Brandon N, Holder N, Patient RK, Walmsley ME (1998) Evidence for non-axial A/P patterning in the nonneural ectoderm of Xenopus and zebrafish pregastrula embryos. Int J Dev Biol 42:763–774Google Scholar
  57. Reimers D, Lopez-Toledano MA, Mason I, Cuevas P, Redondo C, Herranz AS, Lobo MV, Bazan E (2001) Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell progeny. Modulation of neuronal and glial lineages by basic FGF treatment. Neurol Res 23:612–621CrossRefGoogle Scholar
  58. Rigato F, Garwood J, Calco V, Heck N, Faivre-Sarrailh C, Faissner A (2002) Tenascin-C promotes neurite outgrowth of embryonic hippocampal neurons through the alternatively spliced fibronectin type III BD domains via activation of the cell adhesion molecule F3/contactin. J Neurosci 22:6596–6609Google Scholar
  59. Schweiger S, Schneider R (2003) The MID1/PP2A complex: a key to the pathogenesis of Opitz BBB/G syndrome. BioEssays 25:356–366CrossRefGoogle Scholar
  60. Shi Y, Massague J (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113:685–700CrossRefPubMedGoogle Scholar
  61. Sonneveld E, van den Brink CE, Tertoolen LG, van der Burg B, van der Saag PT (1999) Retinoic acid hydroxylase (CYP26) is a key enzyme in neuronal differentiation of embryonal carcinoma cells. Dev Biol 213:390–404CrossRefGoogle Scholar
  62. Soukas A, Cohen P, Socci ND, Friedman JM (2000) Leptin-specific patterns of gene expression in white adipose tissue. Genes Dev 14:963–980Google Scholar
  63. Soussi-Yanicostas N, de Castro F, Julliard AK, Perfettini I, Chedotal A, Petit C (2002) Anosmin-1, defective in the X-linked form of Kallmann syndrome, promotes axonal branch formation from olfactory bulb output neurons. Cell 109:217–228CrossRefPubMedGoogle Scholar
  64. Sperger JM, Chen X, Draper JS, Antosiewicz JE, Chon CH, Jones SB, Brooks JD, Andrews PW, Brown PO, Thomson JA (2003) Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci USA 100:13350–13355CrossRefGoogle Scholar
  65. Spin JM, Nallamshetty S, Tabibiazar R, Ashley EA, King JY, Chen M, Tsao PS, Quertermous T (2004) Transcriptional profiling of in vitro smooth muscle cell differentiation identifies specific patterns of gene and pathway activation. Physiol Genomics 19:292–302Google Scholar
  66. Stelnicki EJ, Komuves LG, Holmes D, Clavin W, Harrison MR, Adzick NS, Largman C (1997) The human homeobox genes MSX-1, MSX-2, and MOX-1 are differentially expressed in the dermis and epidermis in fetal and adult skin. Differentiation 62:33–41CrossRefGoogle Scholar
  67. Ulloa L, Tabibzadeh S (2001) Lefty inhibits receptor-regulated Smad phosphorylation induced by the activated transforming growth factor-beta receptor. J Biol Chem 276:21397–21404CrossRefGoogle Scholar
  68. Watanabe M, Layne MD, Hsieh CM, Maemura K, Gray S, Lee ME, Jain MK (2002) Regulation of smooth muscle cell differentiation by AT-rich interaction domain transcription factors Mrf2alpha and Mrf2beta. Circ Res 91:382–389CrossRefGoogle Scholar
  69. Wei Y, Harris T, Childs G (2002) Global gene expression patterns during neural differentiation of P19 embryonic carcinoma cells. Differentiation 70:204–219CrossRefGoogle Scholar
  70. Wilson SI, Graziano E, Harland R, Jessel TM, Edlund T (2000) An early requirement for FGF signaling in the acquisition of neural cell fate in the chick embryo. Curr Biol 10:421–429CrossRefGoogle Scholar
  71. 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 20:1261–1264Google Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Rajendrakumar S. V. Chadalavada
    • 1
  • Jane Houldsworth
    • 1
    • 2
  • Adam B. Olshen
    • 3
  • George J. Bosl
    • 2
  • Lorenz Studer
    • 4
    • 5
  • R. S. K. Chaganti
    • 1
    • 2
    • 6
  1. 1.Cell Biology ProgramMemorial Sloan-Kettering Cancer CenterNew YorkUSA
  2. 2.Department of MedicineMemorial Sloan-Kettering Cancer CenterNew YorkUSA
  3. 3.Department of Epidemiology and BiostatisticsMemorial Sloan-Kettering Cancer CenterNew YorkUSA
  4. 4. Developmental Biology ProgramMemorial Sloan-Kettering Cancer CenterNew YorkUSA
  5. 5.Division of NeurosurgeryMemorial Sloan-Kettering Cancer CenterNew YorkUSA
  6. 6.Memorial Sloan-Kettering Cancer CenterNew YorkUSA

Personalised recommendations