Mouse embryonic stem cells (ESC) make cell fate decisions based on intrinsic and extrinsic factors. The decision of ESC to differentiate to multiple lineages in vitro occurs during the formation of embryoid bodies (EB) and is influenced by cell-environment interactions. However, molecular mechanisms underlying cell-environmental modulation of ESC fate decisions are incompletely understood. Since adhesion molecules (AM) influence proliferation and differentiation in developing and adult tissues, we hypothesized that specific AM interactions influence ESC commitment toward hematopoietic and endothelial lineages. Expression of AM in the adherens, tight and gap junction pathways in ESC subpopulations were quantified. E-cadherin (E-cad), Claudin-4 (Cldn4), Connexin-43 (Cx43), Zona Occludens-1 (ZO-1) and Zona Occludens-2 (ZO-2) transcript levels were differentially expressed during early stages of hematopoietic/endothelial commitment. Stable ESC lines were generated with reduced expression of E-cad, Cldn4, Cx43, ZO-1 and ZO-2 using shRNA technology. Functional and phenotypic consequences of modulating AM expression were assessed using hematopoietic colony forming assays, endothelial sprouting assays and surface protein expression. A decrease in E-cad, Cldn4, Cx43 and ZO-1 expression was associated with less commitment to the hematopoietic lineage and increased endothelial differentiation as evidenced by functional and phenotypic analysis. A reduction in ZO-2 expression did not influence endothelial differentiation, but decreased hematopoietic commitment two-fold. These data indicate that a subset of AM influence ESC decisions to commit to endothelial and hematopoietic lineages. Furthermore, differentially expressed AM may provide novel markers to delineate early stages of ESC commitment to hematopoietic/endothelial lineages.
References
[1]
Dzau VJ, Gnecchi M, Pachori AS, Morello F, Melo LG (2005) Therapeutic Potential of Endothelial Progenitor Cells in Cardiovascular Diseases. Hypertension 46: 7–18.
[2]
D.o.H.a.H.S (2001) Hematopoietic Stem Cells. Stem Cells: Scientific Progress and Future Research Directions.
[3]
Mimeault M, Hauke R, Batra SK (2007) Stem Cells: A Revolution in Therapeutics - Recent Advances in Stem Cell Biology and Their Therapeutic Applications in Regenerative Medicine and Cancer Therapies. Nature 82: 252–264.
[4]
Ling V, Neben S (1997) In vitro differentiation of embryonic stem cells: immunophenotypic analysis of cultured embryoid bodies. J Cell Physiol 171: 104–115.
[5]
Keller G (2005) Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev 19: 1129–1155.
[6]
Vittet D, Prandini MH, Berthier R, Schweitzer A, Martin-Sisteron H, et al. (1996) Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood 88: 3424–3431.
[7]
Dang SM, Kyba M, Perlingeiro R, Daley GQ, Zandstra PW (2002) Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems. Biotechnol Bioeng 78: 442–453.
[8]
Helgason CD (2003) In vitro hematopoietic differentiation of murine embryonic stem (ES) cells: Stem Cell Technologies.
[9]
Wong RC, Dottori M, Koh KL, Nguyen LT, Pera MF, et al. (2006) Gap junctions modulate apoptosis and colony growth of human embryonic stem cells maintained in a serum-free system. Biochem Biophys Res Commun 344: 181–188.
[10]
Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, et al. (2004) Differences between human and mouse embryonic stem cells. Dev Biol 269: 360–380.
Nagano K, Taoka M, Yamauchi Y, Itagaki C, Shinkawa T, et al. (2005) Large-scale identification of proteins expressed in mouse embryonic stem cells. Proteomics 5: 1346–1361.
[13]
Palmqvist L, Glover CH, Hsu L, Lu M, Bossen B, et al. (2005) Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency. Stem Cells 23: 663–680.
[14]
Schneeberger EE, Lynch RD (2004) The tight junction: a multifunctional complex. American Journal of Physiology Cell Physiology 286: C1213–1228.
[15]
Matter K, Aijaz S, Tsapara A, Balda MS (2005) Mammalian tight junctions in the regulation of epithelial differentiation and proliferation. Curr Opin Cell Biol 17: 453–458.
[16]
Giepmans BNG (2004) Gap junctions and connexin-interacting proteins. Cardiovascular Research 62: 233–245.
[17]
Riethmacher D, Brinkmann V, Birchmeier C (1995) A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development. Proceedings of the National Academy of Sciences of the United States of America 92: 855–859.
Xu J, Kausalya PJ, Phua DCY, Ali SM, Hossain Z, et al. (2008) Early Embryonic Lethality of Mice Lacking ZO-2, but Not ZO-3, Reveals Critical and Nonredundant Roles for Individual Zonula Occludens Proteins in Mammalian Development. Molecular and Cellular Biology 28: 1669–1678.
[20]
Moriwaki K, Tsukita S, Furuse M (2007) Tight junctions containing claudin 4 and 6 are essential for blastocyst formation in preimplantation mouse embryos. Developmental Biology 312: 509–522.
[21]
Willison K (1990) The mouse Brachyury gene and mesoderm formation. Trends in Genetics 6: 104–106.
[22]
Fehling HJ, Lacaud G, Kubo A, Kennedy M, Robertson S, et al. (2003) Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130: 4217–4227.
[23]
Eichmann A, Corbel C, Nataf V, Vaigot P, Breant C, et al. (1997) Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2. Proceedings of the National Academy of Sciences of the United States of America 94: 5141–5146.
[24]
Fujimoto T, Ogawa M, Minegishi N, Yoshida H, Yokomizo T, et al. (2001) Step-wise divergence of primitive and definitive haematopoietic and endothelial cell lineages during embryonic stem cell differentiation. Genes Cells 6: 1113–1127.
[25]
D'Souza SL, Elefanty AG, Keller G (2005) SCL/Tal-1 is essential for hematopoietic commitment of the hemangioblast but not for its development. Blood 105: 3862–3870.
[26]
Kabrun N, Buhring HJ, Choi K, Ullrich A, Risau W, et al. (1997) Flk-1 expression defines a population of early embryonic hematopoietic precursors. Development 124: 2039–2048.
[27]
Robertson SM, Kennedy M, Shannon JM, Keller G (2000) A transitional stage in the commitment of mesoderm to hematopoiesis requiring the transcription factor SCL/tal-1. Development 127: 2447–2459.
[28]
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, et al. (2002) Molecular Biology of the Cell. New York: Garland Science.
[29]
Cancelas JA, Koevoet WLM, de Koning AE, Mayen AEM, Rombouts EJC, et al. (2000) Connexin-43 gap junctions are involved in multiconnexin-expressing stromal support of hemopoietic progenitors and stem cells. Blood 96: 498–505.
[30]
Wang H, Ding T, Brown N, Yamamoto Y, Prince LS, et al. (2008) Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse. Developmental Biology 318: 112–125.
[31]
Lacaud G, Keller G, Kouskoff V (2004) Tracking Mesoderm Formation and Specification to the Hemangioblast in Vitro. Trends in Cardiovascular Medicine 14: 314–317.
[32]
Hermant B, Desroches-Castan A, Dubessay M, Prandini M, Huber P, et al. (2007) Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis. BMC Biotechnology 7: 20.
[33]
Krtolica A, Genbacev O, Escobedo C, Zdravkovic T, Nordstrom A, et al. (2007) Disruption of Apical-Basal Polarity of Human Embryonic Stem Cells Enhances Hematoendothelial Differentiation. Stem Cells 25: 2215–2223.
[34]
Patan S (1998) TIE1 and TIE2 Receptor Tyrosine Kinases Inversely Regulate Embryonic Angiogenesis by the Mechanism of Intussusceptive Microvascular Growth. Microvascular Research 56: 1–21.
[35]
Nowak G, Karrar A, Holmen C, Nava S, Uzunel M, et al. (2004) Expression of Vascular Endothelial Growth Factor Receptor-2 or Tie-2 on Peripheral Blood Cells Defines Functionally Competent Cell Populations Capable of Reendothelialization. Circulation 110: 3699–3707.
[36]
Yamashita J, Itoh H, Hirashima M, Ogawa M, Nishikawa S, et al. (2000) Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408: 92–96.
[37]
Ema M, Takahashi S, Rossant J (2006) Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors. Blood 107: 111–117.
[38]
Yang X, Cepko CL (1996) Flk-1, a Receptor for Vascular Endothelial Growth Factor (VEGF), Is Expressed by Retinal Progenitor Cells. Journal of Neuroscience 16: 6089–6099.
[39]
Rosenstein JM, Krum JM (2004) New roles for VEGF in nervous tissue–beyond blood vessels. Experimental Neurology 187: 246–253.
[40]
Itoh M, Morita K, Tsukita S (1999) Characterization of ZO-2 as a MAGUK Family Member Associated with Tight as well as Adherens Junctions with a Binding Affinity to Occludin and alpha Catenin. Journal of Biological Chemistry 274: 5981–5986.
[41]
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.
[42]
Elefanty AG, Begley CG, Metcalf D, Barnett L, Kontgen F, et al. (1998) Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ “knock-in” strategy. Proc Natl Acad Sci U S A 95: 11897–11902.
[43]
Kennedy M, Keller GM, PM Wassarman aGMK (2003) Hematopoietic Commitment of ES Cells in Culture. Methods in Enzymology. Academic Press. pp. 39–59.
[44]
Fraser IDC (2004) A vector set for plasmid-based RNAi studies in mammalian cells. AfCS Reports Version 1.0.
