human embryonic stem cells; feeder-free culture; matrigel; differentiation; epithelial–mesenchymal transition
Abstract :
[en] Feeder-free human embryonic stem cell (hESC) culture is associated with the presence of mesenchymal-like cells appearing at the periphery of the colonies. The aim of this study was to identify this early differentiation process. Long-term feeder-free hESC cultures using matrigel and conditioned medium from mouse and from human origin revealed that the appearance of mesenchymal-like cells was similar regardless of the conditioned medium used. Standard characterization confirmed the preservation of hESC properties, but the feeder-free cultures could not be maintained longer than 37 passages. The early differentiation process was characterized in the short term after switching hESCs cultured on feeders to feeder-free conditions. Transmission electron microscopy showed an epithelium-like structure inside the hESC colonies, whereas the peripheral cells revealed the acquisition of a rather mesenchymal-like phenotype. Immunochemistry analysis showed that cells at the periphery of the colonies had a negative E-cadherin expression and a positive Vimentin expression, suggesting an epithelial–mesenchymal transition (EMT). Nuclear staining of ß-catenin, positive N-cadherin and negative Connexin 43 expression were also found in the mesenchymal-like cell population. After RT–PCR analysis, Slug and Snail, both EMT-related transcription factors, were detected as up-regulated in the mesenchymal-like cell population. Taken together, our data suggest that culturing hESCs in feeder-free conditions enhances an early differentiation process identified as an EMT.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Ullmann, U.
In'T Veld, P.
Gilles, Christine ; Université de Liège - ULiège > Département des sciences cliniques > Labo de biologie des tumeurs et du développement
Sermon, K.
De Rycke, M.
Van de Velde, H.
Van Steirteghem, A.
Liebears, I.
Language :
English
Title :
Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions.
Alikani M (2005) Epithelial cadherin distribution in abnormal human pre-implantation embryos. Hum Reprod 20,3369-3375.
Alvi A, Clayton H, Joshi C, Enver T, Ashworth A, Vivanco M, Dale T and Smalley M (2003) Functional and molecular characterisation of mammary side population cells. Breast Cancer Res 5,R1-R8.
Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R and Itskovitz-Eldor J (2003) Human feeder layers for human embryonic stem cells. Biol Reprod 68,2150-2156.
Amit M, Shariki C, Margulets V and Itskovitz-Eldor J (2004) Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 70,837-845.
Barrallo-Gimeno A and Nieto MA (2005) The Snail genes as inducers of cell movement and survival: Implications in development and cancer. Development 132,3151-3161.
Behr R, Heneweer C, Viebahn C, Denker HW and Thie M (2005) Epithelial-mesenchymal transition in colonies of rhesus monkey embryonic stem cells: A model for processes involved in gastrulation. Stem Cells 23,805-816.
Bloor DJ, Metcalfe AD, Rutherford A, Brison DR and Kimber SJ (2002) Expression of cell adhesion molecules during human preimplantation embryo development. Mol Hum Reprod 8,237-245.
Cai J, Olson JM, Rao MS, Stanley M, Taylor E and Ni H (2005) Development of antibodies to human embryonic stem cell antigens. BMC Dev Biol 5,26.
Carpenter MK, Rosler ES, Fisk GJ, Brandenberger R, Ares X, Miura T, Lucero M and Rao MS (2004) Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 229,243-258.
Cauffman G, Liebaers I, Van Steirteghem A and Van de Velde H (in press) POU5F1 isoforms show different expression patterns in human embryonic stem cells and preimplantation embryos. Stem Cells.
Cheng L, Hammond H, Ye Z, Zhan X and Dravid G (2003) Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21,131-142.
Ciruna B and Rossant J (2001) FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak. Dev Cell 1,37-49.
D'Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E and Baetge EE (2005) Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23,1534-1541.
Gerrard L, Zhao D, Clark AJ and Cui W (2005) Stably transfected human embryonic stem cell clones express OCT4-specific green feeder fluorescent protein and maintain self-renewal and pluripotency. Stem Cells 23,124-133.
Gilles C, Polette M, Zahm JM, Tournier JM, Volders L, Foidart JM and Birembaut P (1999) Vimentin contributes to human mammary epithelial cell migration. J Cell Sci 112,4615-4625.
Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter MK, Itskovitz-Eldor J et al. (2004) Differences between human and mouse embryonic stem cells. Dev Biol 269,360-380.
Grundemann C, Bauer M, Schweier O, von Oppen N, Lassing U, Saudan P, Becker K, Karp K, Hanke T, Bachmann M et al. (2006) Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1. J Immunol 176,1311-1315.
Guarino M (1995) Epithelial-to-mesenchymal change of differentiation. From embryogenetic mechanism to pathological patterns. Histol Histopathol 10,171-184.
Gumbiner BM (2005) Regulation of cadherin-mediated adhesion in morphogenesis. Nat Rev Mol Cell Biol 6,622-634.
Hajra KM, Chen DY and Fearon ER (2002) The SLUG zinc-finger protein represses E-cadherin in breast cancer. Cancer Res 62,1613-1618.
Hay ED (2005) The mesenchymal cell, its role in the embryo, and the remarkable signalling mechanisms that create it. Dev Dyn 233,706-720.
Hyslop L, Stojkovic M, Armstrong L, Walter T, Stojkovic P, Przyborski S, Herbert M, Murdoch A, Strachan T and Lako M (2005) Downregulation of NANOG induces differentiation of embryonic stem cells to extraembryonic lineages. Stem Cells 23,1035-1043.
Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD and Lanza R (2005) Human embryonic stem cells derived without feeder cells. Lancet 365,1636-1641.
Lee JM, Dedhar S, Kallouri R and Thompson EW (2006) The epithelial-mesenchymal transition: New insights in signaling, development, and disease. J Cell Biol 172,973-981.
Ludwig TE, Levenstein ME, Jones JM, Berggen WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyck MS et al. (2006) Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 24,185-187.
Maeda M, Johnson KR and Wheelock MJ (2005) Cadherin switching: Essential for behavioural but not morphological changes during an epithelium-to-mesenchyme transition. J Cell Sci 118,873-887.
Mateizel I, De Temmerman N, Ullmann U, Cauffman G, Sermon K, Van de Velde H, De Rycke M, Degreef E, Devroey P, Liebaers I et al. (2006) Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum Reprod 21,503-511.
Matin MM, Walsh JR, Gokhale PJ, Draper JS, Bahrami AR, Morton I, Moore HD and 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 22,659-668.
Muller T, Bain G, Wang X and Papkoff J (2002) Regulation of epithelial cell migration and tumor formation by beta-catenin signalling. Exp Cell Res 280,119-133.
Nieto MA, Sargent MG, Wilkinson DG and Cooke J (1994) Control of cell behavior during vertebrate development by Slug, a zinc finger gene. Science 264,835-839.
Philp D, Chen SS, Fitzgerald W, Orenstein J, Margolis L and Kleinman HK (2005) Complex extracellular matrices promote tissue-specific stem cell differentiation. Stem Cells 23,288-296.
Richards M, Fong CY, Chan WK, Wong PC and Bongso A (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 20,933-936.
Rosler ES, Fisk GJ, Ares X, Irving J, Miura T, Rao MS and Carpenter MK (2004) Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 229,259-274.
Sathananthan H, Pera M and Trounson A (2002) The fine structure of human embryonic stem cells. Reprod Biomed Online 4,56-61.
Savagner P, Yamada KM and Thiery JP (1997) The zinc-finger protein slug causes desmosome dissociation, an initial and necessary step for growth factor-induced epithelial-mesenchymal transition. J Cell Biol 137,1403-1419.
Stojkovic P, Lako M, Stewart R, Przyborski S, Armstrong L, Evans J, Murdoch A, Strachan T and Stojkovic M (2005) An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23,306-314.
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS and Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282,1145-1147.
Wong RC, Pebay A, Nguyen LT, Koh KL and Pera MF (2004) Presence of functional gap junctions in human embryonic stem cells. Stem Cells 22,883-889.
Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD and Carpenter MK (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19,971-974.
Xu RH, Peck RM, Li DS, Feng X, Ludwig T and Thomson JA (2005) Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2,185-190.