| Human embryonic stem cells (hESCs) represent an important source of cells to define the earliest stages of human development while providing a potential source of cells for novel therapies. However, better characterization of hESC differentiation, as well as development of methodologies to aid in their culture and expansion will be vital to realize future clinical applications.; hESCs are known to differentiate into mature cells of defined lineages through the formation of embryoid bodies (EBs). Here we characterize the ability of EBs to support the differentiation of hESCs toward cells of the blood and endothelial lineages. EB-mediated differentiation is an attractive culture method for studying differentiation as EBs are amenable to suspension culture. However, EBs maintained in static suspension cultures are typically non-homogeneous, leading to inefficient cellular development. Here, we demonstrate the use of stirred cultures for producing EBs with a more homogeneous morphology and size, resulting in a 4-fold increase in cell expansion. Importantly, EBs cultivated in these spun conditions retained comparable abilities to produce hematopoietic progenitor cells as those grown in static culture, allowing for the use of a controlled reactor system to better define culture parameters.; One such parameter is oxygen tension, which is likely an important determinant of hematopoietic and endothelial cell differentiation. Genes implicated in hemato-endothelial differentiation are known to respond to oxygen deprivation via stimulation of hypoxia inducible factor proteins (HIFs). Here, we demonstrate the use of a bioreactor to monitor and control the dissolved oxygen concentration in hESC-derived EB cultures (hEBs). Furthermore, using a plate based culture method, multiple oxygen concentration were investigated, as well as time points and duration of hypoxic exposure that would allow for stabilization of HIF-1alpha protein and transcription of target genes. We assessed the effects of these variables on cell metabolism, cell expansion and differentiation.; Together, the results contained in this dissertation define the ability of hEBs to support the differentiation toward cells of the hematopoietic and endothelial lineages. They demonstrate a decoupling of hEB cultivation method and hEB-derived cells' ability to form hematopoietic progenitors. These findings promoted the use of a controlled reactor system to examine the effect of culture variables on hEB differentiation in a monitored, controlled and scalable environment. This work is an important step to better define the conditions necessary to efficiently produce clinically relevant numbers of hESC-derived cells. |
