Enhanced Differentiation to Endodermal Derivatives with Modulated Oxygen Level

Technology      

This invention presents a novel method for differentiating human embryonic stem (hES) cells into β-cells for the treatment of type I diabetes.  The inventors established that modulation of the O2 level to which cells are exposed has a pronounced effect on the outcome of directed differentiation protocols; hence, culture O2 concentration is important for almost every aspect of PSC differentiation. Accurate control of pO2 at hypoxic levels dramatically changes timing and magnitude of differentiation markers and enhances differentiation to mesodermal and endodermal but not ectodermal lineages.  Moreover, low O2 for extended periods after differentiation is complete drastically reduces the number of residual PSC within the differentiated cell population and thus reduces their tumorigenic potential. The method presented in this invention allows accurate control of cellular O2 exposure, making it possible to achieve substantial increases in insulin-producing β-cell yields.  O2 control, alone or combined with other methods, can be applied to future cell therapy protocols to generate and increase the safety of differentiated cells.  

Problem Addressed

Pluripotent stem cells (PCS) hold promise for cell replacement therapy and for studying embryonic development.  However, efficient differentiation of PSC to desired cell types remains a challenge, and potential tumor formation by residual PSC in differentiated populations is problematic.  Prior PSC research has mainly focused on growth factors, signaling molecules, or extracellular matrix (ECM) components to promote targeted differentiation of embryonic stem (ES) cells, and the partial pressure of oxygen (pO2) experienced by ES cells has been largely overlooked.  Most PSC research is performed in high, non-physiological O2, but during embryonic development, cells are exposed to much lower pO2 values.  Effects of low pO2, though poorly understood, are likely to be very important in determining the direction of differentiation pathways.  

Advantages

• Allows accurate control of cellular O2 exposure
• Substantially increases the generation of insulin-producing β-cell cells in sufficient purity and quantity for use in humans  

Publications

"The Effects of Oxygen on Mouse Embryonic Stem Cell Growth, Phenotype Retention, and Cellular Energetics." Biotechnology and Bioengineering 101, no. 2 (2008): 241–254.