Factors released from embryonic stem cells inhibit apoptosis of H9c2 cells

Abstract

Our recent study (Singla DK, Hacker TA, Ma L, Douglas PS, Sullivan R, Lyons GE, Kamp TJ, J Mol Cell Cardiol 40: 195-200, 2006) suggests that transplanted embryonic stem (ES) cells subsequent to myocardial infarction differentiate into the major cell types in the heart and improve cardiac function. However, the extent of regeneration is relatively meager compared with the observed functional improvement. The mechanisms underlying their improved function are completely unknown. In this report, we provide evidence using a cell culture model system for novel mechanisms that involve the release of cytoprotective, antiapoptotic factor(s) from ES cells and inhibit H 2O2-induced apoptosis in the rat cardiomyocyte-derived cell line H9c2. Conditioned medium (CM) from growing mouse ES cells treated with and without H2O2 was generated. Apoptosis was induced after exposure to H2O2 in H9c2 cells for 2 h followed by replacement with fresh cell culture or ES cell-CM. After 24 h, H9c2 cells treated with both ES cell-CMs demonstrated significantly decreased apoptosis, as determined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining, apoptotic ELISA, caspase-3 activity, and DNA ladder. Next, using Luminex technology, we examined the presence of antiapoptotic proteins cystatin c, osteopontin, and clusterin and anti-fibrotic, tissue inhibitor of metalloproteinase-1 (TIMP-1) in both ES cell-CMs. The levels of released factors were 2-to 170-fold higher than those in H9c2 cell-CM. Antiapoptotic effects of ES cell-CM were significantly inhibited with TIMP-1 antibody, suggesting that TIMP-1 is an important factor to inhibit apoptosis. Furthermore, we used CM from an TIMP-1-overexpressing cell line and demonstrated that H2O2-induced apoptosis in the H9c2 cells was significantly inhibited. These observations demonstrate that factors released from ES cells contain antiapoptotic factors and that the effects are mediated by TIMP-1. Moreover, these findings suggest that released factors might be useful for therapeutic applications in ischemic heart disease as well as for many other diseases. hydrogen peroxide; tissue inhibitor of metalloproteinase-1

Factors Released from Embryonic Stem Cells Stimulate c-kit-FLK-1+ve Progenitor Cells and Enhance Neovascularization

“…Mouse ES cells were maintained in Dulbecco's minimum essential medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with sodium pyruvate, leukemia inhibitory factor (LIF), glutamine, penicillin-streptomycin, b-mercaptoethanol, nonessential amino acids, and 15% FBS (Invitrogen) as we previously reported (30,31). ES-CM was prepared by growing 9000 ES cells/cm 2 in Petri dishes containing cell culture medium with LIF for 24 h and then replaced with fresh cell culture medium without LIF for 48 h (30,31).…”

“…Furthermore, we developed oxidative stress-induced apoptosis in H9c2 cells and demonstrated that factors released from ES cells inhibit apoptosis in the cell culture model (30,31). However, whether factors released from ES cells can inhibit apoptosis and enhance neovascularization following transplantation in the infarcted mouse heart remains unknown.…”

The birth of ‘regenerative pharmacology’: a clinical perspective

“…SDF-1 (stromal cell-derived factor-1) (Askari et al, 2003) Anti-apoptotic, cell migration, pro-angiogenic VEGF (Kamihata et al, 2001;Kinnaird et al, 2004) Pro-angiogenic, anti-apoptotic, cell proliferation and migration, contractility bFGF (basic fibroblast growth factor) (Kamihata et al, 2001, Kinnaird et al, 2004 Cell survival, pro-angiogenic, cell proliferation, contractility IGF-1 (insulin-like growth factor) (Xu et al, 2007) Cell survival, pro-angiogenic, cell proliferation and migration MCP-1 (monocyte chemoattractant protein) (Boomsma and Geenen, 2012) Cell migration, pro-angiogenic, differentiation, survival HGF (hepatocyte growth factor) (Kitta et al, 2003) Pro-angiogenic, cell survival, remodelling, differentiation IL-1b (Kobayashi et al, 2000) Pro-angiogenic IL-6 (Pricola et al, 2009) Cell proliferation, IL-10 (Chen et al, 2010) Remodelling, cell survival TGF-b (transforming growth factor-b) (Doyle et al, 2008) Cell hypertrophy, proliferation TNF-a (Corallini et al, 2010) Pro-angiogenic, migration MIP-1a (macrophage inflammatory protein-1a) (Boomsma and Geenen, 2012) Cell migration TIMP 1 and 2 (tissue inhibitor of metalloproteinase) (Singla and McDonald, 2007;Glass and Singla, 2012) Cell migration, remodelling SFRP-2 (secreted frizzled-related protein) (Alfaro et al, 2008) Cell survival…”

Adhesion Proteins, Stem Cells, and Arrhythmogenesis

“…The ESC-conditioned media has been shown to contain antiapoptotic and antifibrotic factors and to protect cardiomyocyte-like cells from oxidative stress in vitro [69]. Therefore, functional improvement of ischemic myocardium seen in ESC engraftment studies, can also be a result of reduced atrial natriuretic peptide signaling and/or collagen production [67].…”