Elsevier

Acta Histochemica

Volume 116, Issue 1, January 2014, Pages 117-125
Acta Histochemica

In vitro protection of adipose tissue-derived mesenchymal stem cells by erythropoietin

https://doi.org/10.1016/j.acthis.2013.06.007Get rights and content

Abstract

Mobilization of stem cells and their differentiation into cardiomyocytes are known to have protective effects after myocardial infarction. The integrity of transplanted mesenchymal stem cells for cardiac regeneration is dependent on cell–cell or cell–matrix interaction, which is adversely affected by reactive oxygen species in an ischemic environment. Treatment with erythropoietin was shown to protect human adipose tissue derived mesenchymal stem cells in an ischemic injury in vitro model. The analyses indicated that expression of erythropoietin receptors played a pivotal role in erythropoietin mediated cell survival. In this study, the anti-apoptotic effect of erythropoietin on stem cells was analyzed in apoptosis-induced human mesenchymal stem cells. Apoptosis was induced in cultured adult human adipose tissue derived mesenchymal stem cells by hydrogen peroxide. A group of cultured cells was also treated with recombinant human erythropoietin in a concentration of 50 ng mL−1. The degree of apoptosis was analyzed by flow-cytometry and immunohistochemical staining for Caspase 3. The average percentages of apoptotic cells were significantly higher in H2O2-induced stem cells than in cells co-cultured with erythropoietin (63.03 ± 4.96% vs 29 ± 3.41%, p < 0.01). We conclude that preconditioning with erythropoietin suppresses apoptosis of mesenchymal stem cells and enhances their survival.

Introduction

Myocardial infarction is a leading cause of morbidity and mortality worldwide. Attempts to repair infarcted myocardium using stem cells have been reported (Leri et al., 2005, Liao et al., 2007, Pasha et al., 2008, Jing et al., 2008, Robey et al., 2008). Mesenchymal stem cells (MSCs) represent a suitable sub-cell type for regeneration of infarcted myocardium. MSCs are self-renewing and clonal precursors of non-hematopoietic tissues (Uemura et al., 2006, Liao et al., 2007, Pasha et al., 2008, Hwangbo et al., 2010, Madonna and De Caterina, 2010). The promising therapeutic effect(s) of MSCs depend on their capacity to survive and to be incorporated in the target tissue. Cardiac tissue regeneration is carried out either by differentiation of transplanted cells toward cardiomyocytes or by their paracrine activity, which has the potential to replace myocardial mass, to form a functional vascular network, and to correct the ventricular geometry (Mazo et al., 2012). Although a large number of MSCs have been transplanted into infarcted hearts, it has been observed that only a limited number of cells MSCs survived for one week after injection. This situation has partly been attributed to poor viability and increased apoptosis of the transplanted MSCs in the ischemic environment (Rangappa et al., 2003, Leri et al., 2005, Xie et al., 2006, Pasha et al., 2008). Therefore, to develop strategies to improve the ability of MSCs to survive in this environment it is important for their proliferation and differentiation into cardiac phenotypes, which can lead to cardiac regeneration in the infarcted myocardium.

Previous studies have shown that systemic administration of erythropoietin (EPO) improves survival and cardiac function in the experimental myocardial infarction models. In addition to the stimulation of erythropoiesis by EPO it has been shown that EPO has anti-apoptotic, anti-oxidative, and anti-inflammatory properties in various organs. Also, EPO enhances new vessel formation in cardiac ischemic injury (Moon et al., 2003, Sterin-Borda et al., 2003, van der Meer et al., 2004). EPO inhibits apoptosis and limits infarct size during ischemia and reperfusion through activation of various intracellular signaling pathways (Parsa et al., 2003). EPO enhances cardiac contractility and relaxation independently of the change in hematocrit values (Parsa et al., 2003). Inhibition of apoptosis in ischemic/hypoxic tissue is the major cytoprotective effect of EPO. Similar antiapoptotic and cardioprotective effects of EPO, independent of the hematopoietic effects, have been shown with carbamylated EPO, a non-erythropoietic derivative of EPO, and with helix B-surface peptide, a peptide mimicking the three dimensional structure of EPO (Fiordaliso et al., 2005, Ueba et al., 2010). Consequently, EPO may play an important role in enhancing survival of MSCs in an ischemic environment because of its prominent anti-apoptotic and cytoprotective effects. Because human adipose tissue derived mesenchymal stem cells (hAT-MSCs) abundantly express erythropoietin receptor (EPOR), preconditioning with EPO may have a role in extending survival of hAT-MSCs. In this study, we investigated whether EPO decreases apoptosis induced by hydrogen peroxide (H2O2) in cultured adult hAT-MSCs. H2O2 causes cellular changes similar to ischemia or anoxia in various cell types, and is used in stem cells as an oxidant or stimulant of apoptosis (Fandrey et al., 1994, Kim et al., 2006, Jiang et al., 2009a, Jiang et al., 2009b, Peng et al., 2009, Karaoz et al., 2010).

Section snippets

Isolation of hAT-MSCs

Human adipose tissues were obtained from subcutaneous tissue samples excised from standard surgical abdominal incision in eight healthy mothers undergoing uncomplicated elective cesarean delivery, and the procedures were approved by the Ethics Committee, University of Kocaeli (KOU-IAEK:13/22). To remove blood and residues, tissue samples were washed several times with Hanks’ balanced salt solution (HBSS) (Invitrogen/Gibco, Paisley, UK) containing 5% penicillin/streptomycin (Invitrogen/Gibco,

Culture of hAT-MSCs

A few hAT-MSCs attached to culture flasks in primary cell cultures, and the majority of these cells displayed a fibroblast-like, spindle-shaped morphology during the early days of incubation. After 10–15 days of plating, these primary cells reached 80% confluency (Fig. 1A). At later passages, majority of these cells exhibited large, flattened or fibroblast-like morphology (Fig. 1B and C). The cells were stored for long-term care at −80 °C for 1 year. These cells showed high vitality, and were

Discussion

MSCs express specific adhesion molecules and antigens, and are known to contribute to the formation of the stromal microenvironment, and to maintain the stem cell survival and function. Some studies have suggested that MSCs are multipotent cells capable of differentiating into cardiomyocytes (Rangappa et al., 2003, Passier and Mummery, 2005, Van Laake et al., 2005, Xie et al., 2006, Liao et al., 2007). Human MSCs derived from adipose tissue can differentiate into cardiac muscle cells when

Acknowledgements

We thank C. Subaşi and A. Okcu for their excellent technical assistance in the experiments and G. Duruksu for his contributions in the writing of the manuscript.

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