Abstract
The discovery of endothelial colony forming cells (ECFCs) with robust self-renewal and de novo vessel formation potentials suggests that ECFCs can be an excellent cell source for cardiovascular diseases treatment through improving neovascularization in the ischemic tissues. However, their engraftment after transplantation resulted to be low. Previous studies showed mesenchymal stem/stromal cells (MSCs) could improve the survival and capillary formation capacity of ECFCs in co-culture systems. In this article, we describe a protocol for in vitro co-culture of MSCs and ECFCs to prime ECFCs for better engraftment.
Keywords: Endothelial colony forming cells, Endothelial progenitor cells, Mesenchymal stem/stromal cells, Vascularization, Placenta, Co-culture, in vitro
Background
Endothelial progenitor cells (EPC) are defined as a cell population capable of forming new blood vessels through a vasculogenesis process. In 2004, Ingram et al. identified a specific highly proliferative population of EPC in ex vivo culture termed ‘endothelial colony-forming cells (ECFC)’ from human umbilical cord blood (Ingram et al., 2004) and these cells have recently been declared to represent EPCs (Medina et al., 2017). A similar population can also be isolated from the human term placenta tissue with equivalent vascularization potential and at clinically relevant quantities (Patel et al., 2013; Shafiee et al., 2015). Therefore, ECFC transplantation has been proposed as a therapeutical approach for ischemic diseases such as myocardial infarction or critical leg ischemia. However, ECFCs engraftment and vasculogenic potential after transplantation are well documented to be low (Shafiee et al., 2017; Medina et al., 2017). Previous experiments have shown enhanced ECFC engraftment and function by co-transplantation of mesenchymal stem/stromal cells (MSC) with ECFC (Shafiee et al., 2017). In vitro and in the presence of MSC, ECFC showed enhanced survival in serum deprivation conditions. In normal/growth culture conditions, MSC co-culture resulted in reduced ECFC proliferation and altered appearance towards an elongated mesenchymal-like morphology. Further investigations suggested that direct contact with MSC was required for changes in ECFC morphology and proliferation rate (Shafiee et al., 2017). In addition, after being co-cultured with MSCs for 4 days, ‘primed ECFCs’ showed reduced colony forming potential but improved capacity to form tube-like structures on MatrigelTM in vitro (Shafiee et al., 2017). In this article, we describe a protocol for in vitro co-culturing of ECFCs and bone marrow-derived MSCs (BM-MSCs).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This protocol has been adapted and slightly modified from the previously published article (Shafiee et al., 2017). This study was supported by National Health and Medical Research Council Project Grant.
References
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