Characterizing Endothelial Cells Derived from the Murine Embryonic Stem Cell Line CCE

Abstract

Embryonic stem cells (ESC) are defined by two main properties of self-renewal and their multipotency to differentiate into virtually all cell types of the body, including endothelial cells. ESCs have been widely regarded as an unlimited source of cells in regeneration medicine and also an ideal in vitro model to investigate complex developmental processes. Here, we report a simple and efficient in vitro model to derive a nearly pure population of endothelial cells from a murine ESC line. CCE ES cells are exposed to alpha-MEM medium containing 10% FBS for 4 days and then cultured in endothelial basal-2 medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and 2% FBS for 42 days. The cells acquired a relatively uniform endothelial cell morphology and were able to propagate and expand in culture. When murine ES cell-derived endothelial cells (MESDECs) were cultured on Matrigel and incubated for 48 h, vessel-like tube structures consisting of CD31 (PECAM-1) or BS-1 immunoreactive cells were developed. Immunocytochemistry and RT-PCR analyses revealed that MESDECs express endothelial cell-specific marker proteins such as Flk-1, PECAM-1, Tie-1, and Tie-2, in which the expressions persist for long periods of time after differentiation. The cells were also capable of taking up acetylated low-density lipoprotein (LDL) in culture. Our data suggest that MESDECs could provide a suitable in vitro model to study molecular events involved in vascular development and open up a new therapeutic strategy in regeneration medicine of cardiovascular disorders.

Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture

“…Previous studies have shown that VEGF treatment of murine ES cells grown on type IV collagen (a component of Matrigel) coated plates [3] or on Matrigel [27] undergo vasculogenesis. In addition, EB treated with VEGF grown in the presence [28] or absence [13] of Matrigel undergo vasculogenesis.…”

“…Although previous studies have demonstrated that VEGF treated murine or human ES cells or EB are capable of vascular sprouting [25,27,45] the mechanism of lumen formation was not explored. Since we observed apoptotic cells inside the vascular tubes, it is possible that the sprouts begin as solid tubes and form lumina by apoptotic loss of central cells.…”

“…Our previous studies with mammogenesis [20,21] have convinced us that 2D environments are not as efficient and reproducible as 3D culture conditions in providing a physiological setting for cell differentiation. Although others have had some success with simple 2D culture conditions [4] or growth on Matrigel coated plates [25,27,45], one can see that the extent of endothelial cell sprouting is more extensive in the 3D setting. Second, we have followed the expression of Ceacam1 in the ES/EB system for the first time, thus demonstrating that the Ceacam1 gene has a role not only in angiogenesis, but also in vasculogenesis.…”

3D study of capillary network derived from human cord blood mesenchymal stem cells and differentiated into endothelial cell with VEGFR2 protein expression

“…The functionality of the cells was confirmed by their ability to form capillary network in presence of vascular endothelial growth factor (VEGF) (8). Differentiation of the ECs from different sources inducing the angioblast in the embryo (8), the endothelial progenitor cells (EPCs), mesoangioblasts and multipotent progenitor cells-(MAPCs) or side population in the adult bone marrow (2,9) and murine embryonic stem cell line (10) has been reported (11). Various surface markers such as CD34, CD146, vascular endothelial growth factor 2 (VEG FR2) which is sometimes referred as KDR, tie 2, endothelial cadherin and E-selection have been used to identify the stem cell contributing to angiogenesis (12)(13)(14).…”

Molecular and ultrastructural characterization of endothelial cells differentiated from human bone marrow mesenchymal stem cells

“…Differentiation of the endothelial cells (ECs) from different sources including the angioblast in the embryo (Mikkola and Orkin, 2002), the EPCs, mesoangioblast and multipotent adult progenitor cells (MAPCs) or side population in the adult bone marrow (Luttun et al, 2002;Reyes et al, 2002) and a murine embryonic stem cell line (Fathi et al, 2008) has been reported. According to Rehman et al (2003), EPCs contribute to vessel growth by releasing angiogenic growth factors.…”