(122c) Hypoxia Induced Cardiogenesis May Be Regulated by the Extracellular Matrix

Hypoxia, a characteristic of the in vivo embryonic microenvironment, can influence extracellular matrix (ECM) production which may invariably affect stem cell fate. Hypoxia has been shown to upregulate cardiac markers in murine embryoid bodies (EBs) ultimately leading to an increased fraction of cardiomyocytes. Hypoxia also regulates hypoxia inducible factor-1alpha leading to the activation of vascular endothelial growth factor (VEGF) and erythropoietin.

In this report, we have investigated the effects of low oxygen exposure on embryonic stem cell (ESC) derived EBs as a function of time. PCR data for HIF-1alpha, ECM proteins, mesoderm and ectoderm markers are examined to determine the effects of hypoxia on the ECM and differentiation within EBs. The analyses of gene expression profiles provide evidence that EBs cultured under hypoxic conditions undergo a decrease in collagen expression while fibronectin expression is enhanced. The effect of temporal and long term hypoxic exposure on differentiation is also examined. Prolonged exposure to hypoxia leads to an increase in VEGF but has a negative effect on PECAM-1. Our findings suggest that hypoxia enhances mesodermal and ECM gene expression, specifically, laminin, fibronectin, collagen I and collagen IV gene expression. In addition, EBs cultured under hypoxic conditions result in a 12.4% higher yield of beating EBs. These occurrences increase by 3.6-fold when EBs are initially exposed to hypoxic conditions and later switched normoxic conditions. This finding suggests that a threshold may exist by which hypoxic exposure is no longer effective in inducing cardiac differentiation.

Using flow activated cell sorting to isolate hESC derived cardiomyocytes, we found that culturing EBs under hypoxic conditions resulted in higher cardiomyocyte yields in comparison to normoxic culture. Cardiomyocyte yield was even higher in EBs that experienced hypoxia during the initial 9 days of culture followed by normoxia the remaining 6 ? 11 days of culture suggesting that hypoxia influences cardiac differentiation during early stages of differentiation.

Our studies show that hypoxia influences both hESC ECM and cardiomyocyte differentiation. Understanding the role of hypoxia on the ECM may allow the synthesis of new synthetic matrices that can enhance the efficiency of hESC differentiation.