Abstract
BackgroundVascular progenitor cells (VPCs) derived from embryonic stem cells (ESCs) are a valuable source for cell- and tissue-based therapeutic strategies. During the optimization of endothelial cell (EC) inductions from mouse ESCs using our staged and chemically-defined induction methods, we found that cell seeding density but not VEGF treatment between 10 ng/mL and 40 ng/mL was a significant variable directing ESCs into FLK1+ VPCs during stage 1 induction. Here, we examine potential contributions from cell-to-cell signaling or cellular metabolism in the production of VPCs from ESCs seeded at different cell densities.MethodsUsing 1D 1H-NMR spectroscopy, transcriptomic arrays, and flow cytometry, we observed that the density-dependent differentiation of ESCs into FLK1+ VPCs positively correlated with a shift in metabolism and cellular growth.ResultsSpecifically, cell differentiation correlated with an earlier plateauing of exhaustive glycolysis, decreased lactate production, lower metabolite consumption, decreased cellular proliferation and an increase in cell size. In contrast, cells seeded at a lower density of 1,000 cells/cm2 exhibited increased rates of glycolysis, lactate secretion, metabolite utilization, and proliferation over the same induction period. Gene expression analysis indicated that high cell seeding density correlated with up-regulation of several genes including cell adhesion molecules of the notch family (NOTCH1 and NOTCH4) and cadherin family (CDH5) related to vascular development.ConclusionsThese results confirm that a distinct metabolic phenotype correlates with cell differentiation of VPCs.
Highlights
Vascular progenitor cells (VPCs) derived from embryonic stem cells (ESCs) are a valuable source for cell- and tissue-based therapeutic strategies
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
The VEGF treatment levels led to variable results, the greatest number of FLK1+ VPCs was consistently and statistically significant in cultures seeded at the highest seeding density (Fig. 2a-b) while cells initially seeded at 1,000 cells/cm2 generated significantly fewer FLK1+ cells
Summary
Vascular progenitor cells (VPCs) derived from embryonic stem cells (ESCs) are a valuable source for cell- and tissue-based therapeutic strategies. Despite our growing understanding of the critical biochemical factors in development, the precise timing and quantitative levels of EC induction/activation for directing vascular fate from ESCs in vitro have remained confounding. This is complicated by the inherent variability in kinetic and autocrine signaling from ESC line-to-ESC line [13]. Increasing evidence supports a role for modified cellular metabolism in the regulation of stem cell self-renewal, specification, and plasticity in cancer and development [19,20,21] Despite this growing understanding of cellular metabolism as a regulator of cell function, the role of cell seeding density in metabolic alterations supporting vascular fate is not defined
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