Background Progenitor cell therapy is emerging as a novel treatment for heart failure. However the molecular mechanisms regulating the generation of cardiac progenitor cells is not fully understood. We hypothesized that cardiac progenitor cells are generated from cardiac explant via a process similar to epithelial to mesenchymal transition (EMT). Methods/Findings Explant-derived cells were generated from partially digested atrial tissue. After 21 days in culture, c-Kit+ cells were isolated from cell outgrowth. The majority of explant-originated c-Kit+ cells expressed the epicardial marker Wt1. Cardiac cell outgrowth exhibits a temporal up-regulation of EMT-markers. Notch stimulation augmented, while Notch inhibition suppressed, mesenchymal transition in both c-Kit+ and c-Kit- cells. In c-Kit+ cells, Notch stimulation reduced, while Notch inhibition up-regulated pluripotency marker expressions such as Nanog and Sox2. Notch induction was associated with degradation of β-catenin in c-Kit- cells. In contrast, Notch inhibition resulted in β-catenin accumulation, acquisition of epitheloid morphology, and up-regulation of Wnt target genes in c-Kit- cells. Conclusion Our study suggests that Notch-mediated reversible EMT process is a mechanism that regulates explant-derived c-Kit+ and c-Kit- cells.
References
[1]
Messina E, De Angelis L, Frati G, Morrone S, Chimenti S, et al. (2004) Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res 95: 911–921.
[2]
Zakharova L, Mastroeni D, Mutlu N, Molina M, Goldman S, et al. (2010) Transplantation of cardiac progenitor cell sheet onto infarcted heart promotes cardiogenesis and improves function. Cardiovasc Res 87: 40–49.
[3]
Davis DR, Zhang Y, Smith RR, Cheng K, Terrovitis J, et al. (2009) Validation of the cardiosphere method to culture cardiac progenitor cells from myocardial tissue. PLoS One 4: e7195.
[4]
Davis DR, Kizana E, Terrovitis J, Barth AS, Zhang Y, et al. (2010) Isolation and expansion of functionally-competent cardiac progenitor cells directly from heart biopsies. J Mol Cell Cardiol 49: 312–321.
[5]
Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139: 871–890.
[6]
Thiery JP (2002) Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2: 442–454.
[7]
Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman AL, McMullen JR, et al. (2007) Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med 13: 952–961.
[8]
Zhou B, Ma Q, Rajagopal S, Wu SM, Domian I, et al. (2008) Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 454: 109–113.
[9]
Arnoux V, Nassour M, L’Helgoualc’h A, Hipskind RA, Savagner P (2008) Erk5 controls Slug expression and keratinocyte activation during wound healing. Mol Biol Cell 19: 4738–4749.
[10]
Gittenberger-de Groot AC, Winter EM, Poelmann RE (2010) Epicardium-derived cells (EPDCs) in development, cardiac disease and repair of ischemia. J Cell Mol Med 14: 1056–1060.
[11]
Limana F, Bertolami C, Mangoni A, Di Carlo A, Avitabile D, et al. (2010) Myocardial infarction induces embryonic reprogramming of epicardial c-Kit(+) cells: role of the pericardial fluid. J Mol Cell Cardiol 48: 609–618.
[12]
Winter EM, Grauss RW, Hogers B, van Tuyn J, van der Geest R, et al. (2007) Preservation of left ventricular function and attenuation of remodeling after transplantation of human epicardium-derived cells into the infarcted mouse heart. Circulation 116: 917–927.
Yang J, Weinberg RA (2008) Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell 14: 818–829.
[15]
Koyanagi M, Bushoven P, Iwasaki M, Urbich C, Zeiher AM, et al. (2007) Notch signaling contributes to the expression of cardiac markers in human circulating progenitor cells. Circ Res 101: 1139–1145.
[16]
Boni A, Urbanek K, Nascimbene A, Hosoda T, Zheng H, et al. (2008) Notch1 regulates the fate of cardiac progenitor cells. Proc Natl Acad Sci U S A 105: 15529–15534.
[17]
Li H, Yu B, Zhang Y, Pan Z, Xu W (2006) Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes. Biochem Biophys Res Commun 341: 320–325.
[18]
Tezuka K, Yasuda M, Watanabe N, Morimura N, Kuroda K, et al. (2002) Stimulation of osteoblastic cell differentiation by Notch. J Bone Miner Res 17: 231–239.
[19]
Bax NA, van Oorschot AA, Maas S, Braun J, van Tuyn J, et al. (2011) In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGFbeta-signaling and WT1. Basic Res Cardiol 106: 829–847.
[20]
Iso T, Kedes L, Hamamori Y (2003) HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol 194: 237–255.
[21]
Nemir M, Pedrazzini T (2008) Functional role of Notch signaling in the developing and postnatal heart. J Mol Cell Cardiol 45: 495–504.
[22]
Grieskamp T, Rudat C, Ludtke TH, Norden J, Kispert A (2011) Notch signaling regulates smooth muscle differentiation of epicardium-derived cells. Circ Res 108: 813–823.
[23]
von Gise A, Zhou B, Honor LB, Ma Q, Petryk A, et al. (2011) WT1 regulates epicardial epithelial to mesenchymal transition through beta-catenin and retinoic acid signaling pathways. Dev Biol 356: 421–431.
[24]
Chen HC, Zhu YT, Chen SY, Tseng SC (2012) Wnt signaling induces epithelial-mesenchymal transition with proliferation in ARPE-19 cells upon loss of contact inhibition. Lab Invest 92: 676–8710.
[25]
Logan C, Nusse R (2004) The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20: 781–810.
[26]
Compton LA, Potash DA, Mundell NA, Barnett JV (2006) Transforming growth factor-beta induces loss of epithelial character and smooth muscle cell differentiation in epicardial cells. Dev Dyn 235: 82–93.
[27]
Austin AF, Compton LA, Love JD, Brown CB, Barnett JV (2008) Primary and immortalized mouse epicardial cells undergo differentiation in response to TGFbeta. Dev Dyn 237: 366–376.
[28]
Castaldo C, Di Meglio F, Nurzynska D, Romano G, Maiello C, et al. (2008) CD117-positive cells in adult human heart are localized in the subepicardium, and their activation is associated with laminin-1 and alpha6 integrin expression. Stem Cells 26: 1723–1731.
[29]
Bhowmick NA, Ghiassi M, Bakin A, Aakre M, Lundquist CA, et al. (2001) Transforming growth factor-beta1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism. Mol Biol Cell 12: 27–36.
[30]
Zhou B, Ma Q, Rajagopal S, Wu SM, Domian I, et al. (2008) Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 454: 109–113.
[31]
Di Meglio F, Castaldo C, Nurzynska D, Romano V, Miraglia R, et al. (2010) Epithelial-mesenchymal transition of epicardial mesothelium is a source of cardiac CD117-positive stem cells in adult human heart. J Mol Cell Cardiol 49: 719–727.
[32]
van Tuyn J, Atsma DE, Winter EM, van der Velde-van Dijke I, Pijnappels DA, et al. (2007) Epicardial cells of human adults can undergo an epithelial-to-mesenchymal transition and obtain characteristics of smooth muscle cells in vitro. Stem Cells 25: 271–278.
[33]
Martinez-Estrada OM, Lettice LA, Essafi A, Guadix JA, Slight J, et al. (2010) Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin. Nat Genet 42: 89–93.
[34]
Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, et al. (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133: 704–715.
[35]
Morel AP, Lievre M, Thomas C, Hinkal G, Ansieau S, et al. (2008) Generation of breast cancer stem cells through epithelial-mesenchymal transition. PLoS One 3: e2888.
[36]
Zhou H, Wu S, Joo JY, Zhu S, Han DW, et al. (2009) Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4: 381–384.
[37]
Martinez-Estrada OM, Lettice LA, Essafi A, Guadix JA, Slight J, et al. (2010) Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin. Nat Genet 42: 89–93.
[38]
Urbanek K, Torella D, Sheikh F, De Angelis A, Nurzynska D, et al. (2005) Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure. Proc Natl Acad Sci U S A 102: 8692–8697.
