(117b) Affect of Hypoxia On Cardiomyocyte Differentiation and Its Extracellular Environment

Human embryonic stem cells (hESCs) are pluripotent, having the ability to evolve into various cell lineages. This capability makes hESCs attractive for therapeutic and regenerative medicine applications. The typical differentiation method for hES cells involves the generation of 3-D cell aggregates called embryoid bodies (EBs). These aggregates are allowed to remain in suspension over a period of time wherein the comprised cells undergo spontaneous differentiation.

The stem cell microenvironment influences various signaling cascades that ultimately influence differentiation patterns. Simultaneously hypoxia influences extracellular matrix (ECM) production which invariably affects stem cell fate. Hypoxia has been shown to upregulate cardiac markers in mouse embryonic stem cell derived EBs ultimately leading to an increased fraction of cardiomyocyte formation. Under hypoxic conditions, hypoxia inducible factor 1- alpha is activated leading to activation of vascular endothelial growth factor (VEGF) and erythropoietin.

In this study, we investigate the effects of low oxygen exposure on human embryonic stem cell derived EBs. PCR data for hypoxia inducible factor 1- alpha, extracellular matrix, mesoderm and ectoderm markers are examined to determine the effects of hypoxia on the extracellular matrix proteins and differentiation within EBs. The analyses of gene expression profiles provide evidence that EBs cultured in hypoxic conditions undergo a decrease in collagen expression while fibronectin expression is enhanced. We investigate temporal as well as long term exposure of hypoxia (5% O2) on differentiation and demonstrate that ECM proteins, in particular laminin, collagens, and fibronectin, are affected by hypoxia. We find that prolonged exposure to hypoxia leads to an upregulation of VEGF, an important factor in angiogenesis, but has a negative effect on platelet/endothelial cell adhesion molecule-1 (PECAM-1) expression. Understanding the role of hypoxia on ECM components may allow the synthesis of new synthetic matrices that can lead to increased efficiency in directing hESC differentiation.