Purified and Proliferating Endothelial Cells Derived and Expanded In Vitro from Embryonic Stem Cells

Abstract

Embryonic stem (ES) cells serve as an excellent in vitro system for studying differentiation events and for developing methods of generating various specialized cells for future regenerative therapeutic applications. Two obstacles associated with using embryonic stem cells include (a) isolating homogeneous populations of differentiated cells and (b) obtaining terminally differentiated cell populations that are capable of proliferating further. Here, the authors describe methods in which they have overcome these two obstacles by generating highly purified populations (>96%) of actively proliferating endothelial cells from mouse ES cells. Briefly, 60,000 ES cells progress through three different stages of cell induction/expansion and two cell isolation procedures, generating over 300 million endothelial cells. These ES-derived endothelial cells display characteristics similar to vascular endothelial cells in that they express several common endothelial markers, they form two-dimensional (2D) tubelike structures as well as complex microvessels in three-dimensional (3D) collagen type I gels, and they retain the ability to reorganize their cytoskeleton in response to mechanical forces. Our findings indicate that it is possible to obtain proliferating populations of homogeneous endothelial cells from mouse ES cells without genetically manipulating the ES cells or coculturing with feeder cells.

Fluid Shear Stress Pre-Conditioning Promotes Endothelial Morphogenesis of Embryonic Stem Cells Within Embryoid Bodies

“…22,32 Early studies have reported that treating EBs with basic fibroblast growth factor (bFGF) promotes EC differentiation and vasculogenesis. 37 Furthermore, studies that utilized a combinatorial approach, using bFGF and VEGF, demonstrated the potency of these biochemical cues to promote EB endothelial morphogenesis and vasculogenesis.…”

“…6 During pre-conditioning, ESCs were cultured in Minimal Essential Media Alpha (Mediatech) supplemented with 10% FBS, 100 U/mL penicillin streptomycin, and 0.1 mM b-mercaptoethanol, similar to media commonly used for ESC endothelial differentiation. 6,32,33 Quantitative reverse-transcriptase-polymerase chain reaction…”

Fibronectin-mediated upregulation of α5β1 integrin and cell adhesion during differentiation of mouse embryonic stem cells

“…These findings support the concept that VEGFR-2 in ES-D3 cells is constitutivley expressed, independent of the differentiation status of the cells. 8 The time course of gene expression levels of the pluripotency marker Oct 3/4 and of the meso-endodermal lineage markers FoxA2 and Sox17 in LIF-dperived ES-D3 cells grown on fibronectin was assessed by qRT-PCR and immunostaining. As seen in Figure 7A, during the eight-day culture period, mRNA expression of FoxA2 and Sox17 significantly increased over time by up to 20-and 45-fold, respectively.…”

“…Fibronectin-mediated upregulation of α5β1 integrin and cell adhesion during differentiation of mouse embryonic stem cells derivatives of all three germ layers, 5,7 such as mesodermal-derived myocardial, 4 endothelial, 8 chondrogenic cells, 9 ectodermalderived neurons, 10 endodermal-derived hepatocytes, 11 and pancreatic β cells. 12 In the field of tissue engineering, ES-D3 cells have been used to generate 3D tissue-like structures in vitro, such as beating cardiomyocytes in a scaffold of collagen I and matrigel, 13 and to generate endodermal cells on fibronectin-coated collagen I gels.…”

Endothelial Cells from Embryonic Stem Cells in a Chemically Defined Medium

“…Successful methods for the in vitro differentiation of ECs from ESCs [9][10][11][12][13][14][15][16] and adult stem cells [17][18][19] have been previously described. One common method used in the derivation of several cell types from ESCs, including ECs, involves the formation of a 3-dimensional aggregate called an embryoid body [9,14].…”