Specialized mouse embryonic stem cells for studying vascular development

Abstract

Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.

Multifactorial Optimizations for Directing Endothelial Fate from Stem Cells

“…The effect of providing VEGF as insoluble form, soluble form, or both to mouse R1 and A3 ESC lines was investigated. After 2 or 3 days for the R1-ESC [17] and A3-ESC [41], respectively, the cellular expression of Flk-1 and Flt-1 was quantified for the 4 experimental conditions (Fig 8). The data (N = 3) show that the number of Flk-1 + and Flt-1 + cells was not significantly altered across all VEGF presentation conditions of soluble and insoluble VEGF supplementation (Fig 8).…”

“…ESC were seeded at 10,000 cells/cm 2 and cultured in induction medium, previously optimized by our laboratory [17], consisting of alpha-MEM, 20% KSR, 1× ps, 1×NEAA, 2 mM l-glutamine, 0.05 mM 2-mercaptoethanol, 20 ng/mL of VEGF, and 5 ng/mL BMP-4. The expression of VEGR receptors Flk-1 and Flt-1 were examined at the time in which that ESC line generated greatest numbers of Flk-1 + VPC—2 and 3 days for the R1-ESC [17] and A3-ESC [41], respectively.…”

Metabolic shift in density-dependent stem cell differentiation

“…Mouse A3-ESCs were extracted, generated, and cultured at 3,000/cm 2 on inactivated mouse embryonic fibroblasts (MEFs; 20,000/cm 2 ) [ 22 ]. Prior to induction, the A3-ESCs are purified from MEFs by gravity separation followed by MEF adhesion to tissue culture dishes for 1–2 hours and passaged onto 0.5% gelatin-coated plates in ESC culture media containing: Knockout Dulbecco’s Modified Eagle Medium (KO-DMEM; Invitrogen), 15% Knockout Serum Replacer (KSR; Invitrogen), 1Χ penicillin-streptomycin (Invitrogen), 1Χ non-essential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 0.1 mM 2-mercaptoethanol (Calbiochem), 2000 units/mL of leukemia inhibitory factor (LIF-ESGRO; Chemicon), and 10 ng/mL of bone morphogenetic protein 4 (BMP4, GeneBank: 652) (R&D Systems).…”

“…Experiments were conducted in triplicate ( N = 3) allowing for analysis of variance. The assessment of stage 1 VPCs, which are not contact-inhibited, was quantified by the percentage of FLK1 + cells over time, previously shown to correlate with down-regulation of the pluripotent stem cell marker POU class 5 homeobox 1 (POU5F1 = OCT3/4, GeneBank: 5460) over the same time period [ 22 ].…”

“…1a ), we examined a number of combinatorial variables (induction time, VEGF treatment, matrix signaling, and cell seeding density) for directing the generation of VPCs (stage 1). The results indicated that cell seeding density was a significant factor in the first stages of induction of ESCs into VPCs, especially in the A3-ESC cell line [ 22 ] generated by our own laboratory. Therefore, we set out to further examine the underlying mechanisms related to density-dependent differentiation in this ESC line.…”