Journal of Cell Science, Vol 96, Issue 1 131-141, Copyright © 1990 by Company of Biologists

JOURNAL ARTICLES

Differentiation of human trophoblast cells in vitro as revealed by immunocytochemical staining of desmoplakin and nuclei

GC Douglas and BF King
Department of Human Anatomy, School of Medicine, University of California, Davis 95616.

The differentiation of isolated human cytotrophoblast cells has been studied by staining cells with anti-desmoplakin antibodies, to reveal cell boundaries, and with anti-nuclear antibodies, to reveal nuclei. During the first 24 h after plating in Ham's/Waymouth medium, mononucleated cytotrophoblast cells began to spread and aggregate, forming colonies. This was accompanied by progressive changes in the pattern of desmoplakin staining. In single cells, desmoplakin was dispersed throughout the cytoplasm. As cells aggregated, desmoplakin was redistributed and formed linear, punctate arrays at regions of cell-cell contact, consistent with desmosome formation. A pavement-like staining pattern was maintained even at 5 days. Double staining for desmoplakin and nuclei revealed that most cells within colonies were mononucleated. When plated in a growth medium originally formulated for keratinocytes, cytotrophoblast cells aggregated and formed desmosomes normally. However, after 48 h, cell diameters were increased and nuclei changed from being evenly distributed to forming clusters within large cells, consistent with syncytiotrophoblast formation. While cells grown in Ham's/Waymouth medium for 2 days could be induced to differentiate by switching to keratinocyte growth medium, cells cultured for 5 days before switching were resistant to the differentiation-inducing effects of the keratinocyte medium. Desmosome-type junctions within colonies of trophoblast cells were unstable and, even after 5 days in culture, could be disrupted by lowering the extracellular Ca2+ concentration. While syncytiotrophoblast formation in keratinocyte growth medium (which contains epidermal growth factor, insulin and hydrocortisone) was accompanied by a 15- to 20-fold increase in chorionic gonadotropin secretion, syncytiotrophoblast formation occurred to a similar extent in keratinocyte basal medium (which does not contain these factors) but with only a twofold increase in chorionic gonadotropin release. These results support the notion that biochemical and morphological differentiation of trophoblast are independent events.

This article has been cited by other articles:

				E. Rimon, B. Chen, A. L. Shanks, D. M. Nelson, and Y. Sadovsky Hypoxia in Human Trophoblasts Stimulates the Expression and Secretion of Connective Tissue Growth Factor Endocrinology, June 1, 2008; 149(6): 2952 - 2958. [Abstract] [Full Text] [PDF]

				R. B. Donker, J.-F. Mouillet, D.M. Nelson, and Y. Sadovsky The expression of Argonaute2 and related microRNA biogenesis proteins in normal and hypoxic trophoblasts Mol. Hum. Reprod., April 1, 2007; 13(4): 273 - 279. [Abstract] [Full Text] [PDF]

				K.E. Green, C. Thota, G.D.V. Hankins, C. Yallampalli, and Y.-L. Dong Calcitonin gene-related peptide stimulates human villous trophoblast cell differentiation in vitro Mol. Hum. Reprod., July 1, 2006; 12(7): 443 - 450. [Abstract] [Full Text] [PDF]

				B. Chen, D. M. Nelson, and Y. Sadovsky N-Myc Down-regulated Gene 1 Modulates the Response of Term Human Trophoblasts to Hypoxic Injury J. Biol. Chem., February 3, 2006; 281(5): 2764 - 2772. [Abstract] [Full Text] [PDF]

				L. M. Holets, J. S. Hunt, and M. G. Petroff Trophoblast CD274 (B7-H1) Is Differentially Expressed Across Gestation: Influence of Oxygen Concentration Biol Reprod, February 1, 2006; 74(2): 352 - 358. [Abstract] [Full Text] [PDF]

				H. Li, J. Dakour, L. J. Guilbert, B. Winkler-Lowen, F. Lyall, and D. W. Morrish PL74, a Novel Member of the Transforming Growth Factor-{beta} Superfamily, Is Overexpressed in Preeclampsia and Causes Apoptosis in Trophoblast Cells J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3045 - 3053. [Abstract] [Full Text] [PDF]

				E. A. Cowley, M. C. Sellers, and N. P. Illsley Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification Am J Physiol Cell Physiol, April 1, 2005; 288(4): C891 - C898. [Abstract] [Full Text] [PDF]

				M. Knofler, L. Saleh, S. Bauer, B. Galos, H. Rotheneder, P. Husslein, and H. Helmer Transcriptional Regulation of the Human Chorionic Gonadotropin {beta} Gene during Villous Trophoblast Differentiation Endocrinology, April 1, 2004; 145(4): 1685 - 1694. [Abstract] [Full Text] [PDF]

				J. Chen, T. L. Thirkill, P. N. Lohstroh, S. R. Bielmeier, M. G. Narotsky, D. S. Best, R. A. Harrison, K. Natarajan, R. A. Pegram, J. W. Overstreet, et al. Bromodichloromethane Inhibits Human Placental Trophoblast Differentiation Toxicol. Sci., March 1, 2004; 78(1): 166 - 174. [Abstract] [Full Text] [PDF]

				J. Chen, G. C. Douglas, T. L. Thirkill, P. N. Lohstroh, S. R. Bielmeier, M. G. Narotsky, D. S. Best, R. A. Harrison, K. Natarajan, R. A. Pegram, et al. Effect of Bromodichloromethane on Chorionic Gonadotrophin Secretion by Human Placental Trophoblast Cultures Toxicol. Sci., November 1, 2003; 76(1): 75 - 82. [Abstract] [Full Text] [PDF]

				L. Cronier, J.-L. Frendo, N. Defamie, G. Pidoux, G. Bertin, J. Guibourdenche, G. Pointis, and A. Malassine Requirement of Gap Junctional Intercellular Communication for Human Villous Trophoblast Differentiation Biol Reprod, November 1, 2003; 69(5): 1472 - 1480. [Abstract] [Full Text] [PDF]

				J.-L. Frendo, L. Cronier, G. Bertin, J. Guibourdenche, M. Vidaud, D. Evain-Brion, and A. Malassine Involvement of connexin 43 in human trophoblast cell fusion and differentiation J. Cell Sci., August 15, 2003; 116(16): 3413 - 3421. [Abstract] [Full Text] [PDF]

				J.-L. Frendo, D. Olivier, V. Cheynet, J.-L. Blond, O. Bouton, M. Vidaud, M. Rabreau, D. Evain-Brion, and F. Mallet Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation Mol. Cell. Biol., May 15, 2003; 23(10): 3566 - 3574. [Abstract] [Full Text] [PDF]

				D. M. Nelson, S. D. Smith, T. C. Furesz, Y. Sadovsky, V. Ganapathy, C. A. Parvin, and C. H. Smith Hypoxia reduces expression and function of system A amino acid transporters in cultured term human trophoblasts Am J Physiol Cell Physiol, February 1, 2003; 284(2): C310 - C315. [Abstract] [Full Text] [PDF]

				D. G. Hemmings and L. J. Guilbert Polarized Release of Human Cytomegalovirus from Placental Trophoblasts J. Virol., June 5, 2002; 76(13): 6710 - 6717. [Abstract] [Full Text] [PDF]

				M.O. Babawale, M.A. Mobberley, T.A. Ryder, M.G. Elder, and M.H.F. Sullivan Ultrastructure of the early human feto-maternal interface co-cultured in vitro Hum. Reprod., May 1, 2002; 17(5): 1351 - 1357. [Abstract] [Full Text] [PDF]

				I. P. Crocker, B. K. Strachan, G. E. Lash, S. Cooper, A. Y. Warren, and P. N. Baker Vascular Endothelial Growth Factor But Not Placental Growth Factor Promotes Trophoblast Syncytialization In Vitro Reproductive Sciences, December 1, 2001; 8(6): 341 - 346. [Abstract] [PDF]

				K. Leitner, R. Szlauer, I. Ellinger, A. Ellinger, K.-P. Zimmer, and R. Fuchs Placental Alkaline Phosphatase Expression at the Apical and Basal Plasma Membrane in Term Villous Trophoblasts J. Histochem. Cytochem., September 1, 2001; 49(9): 1155 - 1164. [Abstract] [Full Text] [PDF]

				W. T. Schaiff, M. G. Carlson, S. D. Smith, R. Levy, D. M. Nelson, and Y. Sadovsky Peroxisome Proliferator-Activated Receptor-{gamma} Modulates Differentiation of Human Trophoblast in a Ligand-Specific Manner J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3874 - 3881. [Abstract] [Full Text]

				M. Knofler, L. Saleh, S. Bauer, R. Vasicek, G. Griesinger, H. Strohmer, H. Helmer, and P. Husslein Promoter Elements and Transcription Factors Involved in Differentiation-Dependent Human Chorionic Gonadotrophin-{alpha} Messenger Ribonucleic Acid Expression of Term Villous Trophoblasts Endocrinology, October 1, 2000; 141(10): 3737 - 3748. [Abstract] [Full Text] [PDF]

				F. Debieve, S. Pampfer, and K. Thomas Inhibin and activin production and subunit expression in human placental cells cultured in vitro Mol. Hum. Reprod., August 1, 2000; 6(8): 743 - 749. [Abstract] [Full Text] [PDF]

				S. Khan, H. Katabuchi, M. Araki, R. Nishimura, and H. Okamura Human Villous Macrophage-Conditioned Media Enhance Human Trophoblast Growth and Differentiation In Vitro Biol Reprod, April 1, 2000; 62(4): 1075 - 1083. [Abstract] [Full Text]

				M. Knofler, L. Saleh, H. Strohmer, P. Husslein, and M. F. Wolschek Cyclic AMP- and differentiation-dependent regulation of the proximal {alpha}HCG gene promoter in term villous trophoblasts Mol. Hum. Reprod., June 1, 1999; 5(6): 573 - 580. [Abstract] [Full Text] [PDF]

				G. I. Gallicano, P. Kouklis, C. Bauer, M. Yin, V. Vasioukhin, L. Degenstein, and E. Fuchs Desmoplakin Is Required Early in Development for Assembly of Desmosomes and Cytoskeletal Linkage J. Cell Biol., December 28, 1998; 143(7): 2009 - 2022. [Abstract] [Full Text] [PDF]

				D. G. Hemmings, R. Kilani, C. Nykiforuk, J. Preiksaitis, and L. J. Guilbert Permissive Cytomegalovirus Infection of Primary Villous Term and First Trimester Trophoblasts J. Virol., June 1, 1998; 72(6): 4970 - 4979. [Abstract] [Full Text] [PDF]

				G Keryer, E Alsat, K Tasken, and D Evain-Brion Cyclic AMP-dependent protein kinases and human trophoblast cell differentiation in vitro J. Cell Sci., January 4, 1998; 111(7): 995 - 1004. [Abstract] [PDF]