Human embryonic stem cells (hESCs) represent an important resource for novel cell-based regenerative medical therapies. hESCs are known to differentiate into mature cells of defined lineages through the formation of embryoid bodies (EBs) which are amenable to suspension culture for several weeks. However, EBs derived from hESCs in standard static cultures are typically non-homogeneous, leading to inefficient cellular development. Here, we systematically compare the formation, growth, and differentiation capabilities of hESC-derived EBs in stirred and static suspension cultures. A 15-fold expansion in total number of EB-derived cells cultured for 21 days in a stirred flask was observed, compared to a fourfold expansion in static (non-stirred) cultures. Additionally, stirred vessel mediated cultures have a more homogeneous EB morphology and size. Importantly, the EBs cultivated in spinner flasks retained comparable ability to produce hematopoietic progenitor cells as those grown in static culture. These results demonstrate the decoupling between EB cultivation method and EB-derived cells' ability to form hematopoietic progenitors, and will allow for improved production of scalable quantities of hematopoietic cells or other differentiated cell lineages from hESCs in a controlled environment.

Oxygen tension can provide an important determinant for differentiation and development of many cells and tissues. Genetic regulation of hemato-endothelial commitment is known to respond to oxygen deprivation via stimulation of hypoxia inducible factors (HIFs). Here, we use a closed bioreactor system to monitor and control the dissolved oxygen during differentiation of human embryonic stem cells (hESCs) via formation of embryoid bodies (hEBs). Exposing hESC-derived EBs to ambient oxygen at or below 5% results in stabilization of HIF-1alpha and increased transcription of hypoxic responsive genes. Interestingly, we find that rather than HIF-1alpha expression being stable over prolonged (7-16 days) culture in hypoxic conditions, HIF-1alpha expression peaks after approximately 48 hours of hypoxic exposure, and then declines to near undetectable levels, despite constant hypoxic exposure. This transient stabilization of HIF-1alpha during hESC-derived EB culture is demonstrated for four distinct stages of differentiation. Furthermore, we demonstrate hEB cell expansion is slowed by hypoxic exposure, with increased apoptosis. However, hEB cell proliferation returns to normal rates upon return to normoxic conditions. Therefore, although hypoxia effectively stimulates hypoxic responsive genes, this single variable was not sufficient to improve development of hemato-endothelial cells from hESCs.

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Blood cell products, such as red blood cells and platelets suitable for transfusion, are attractive as a first generation human embryonic stem cell (hESC) derived therapy. Development of hESC-derived transfusion therapies will require a better understanding of how to control the differentiation of human ES cells into functional blood cells in sufficient quantities for clinical use. HESCs are known to produce mature hematopoietic cells during differentiation as embryoid bodies (EBs). Previously we have demonstrated development of both hematopoietic progenitor cells and more differentiated cell types of myeloid, lymphoid and erythroid lineages from hESC. A four-fold increase in total cell number was achieved when ESC-derived EBs were differentiated in stirred vessels compared to conventional static cultures. Spinner cultures generated EBs more uniform in size and density. Static- and spinner cultivated EBs produced equivalent percentages of hematopoietic progenitors when assayed by surface antigen expression (CD34+, CD31+ and CD45+) and colony forming potential. Hence, overall a greater yield of hematopoietic cells was generated in spinner cultures. Here we incorporate pH and oxygen control into the stirred vessel system in order to closely regulate environmental conditions at levels conducive to hematopoietic differentiation. Hematopoietic potential is compared under hypoxic and normoxic conditions. Hypoxic conditions were confirmed in the EB tissue mass by 2-nitroimidazole (hypoxyprobe) staining. We observed that the cellular response to hypoxia, monitored by the presence of HIF 1α protein, is transient. Peak levels of HIF 1α were detected within 48 hours of low oxygen culture, falling to baseline levels within 7 days. Under more severe conditions the kinetics of the hypoxic response were accelerated, HIF 1α expression peaking and subsiding earlier in cultures held at 1% dissolved oxygen compared to 5% dissolved oxygen. We show that by manipulating dissolved oxygen concentration we are able to influence the progress of differentiation. This can at least partly be attributed to the upregulation of hypoxia inducible genes, including VEGF-A and EPO, under low oxygen conditions. Expression levels of VEGF-A are dependent on dissolved oxygen concentration, being most highly expressed under 1% dissolved oxygen conditions. The transitory nature of the cellular hypoxic response suggests that short exposure to low oxygen conditions may be sufficient to gain the full beneficial impact of hypoxic signalling on hematopoietic cell generation without decreases in cell proliferation and increase in cell death associated with extended oxygen deprivation. We propose that control of culture parameters such as dissolved oxygen in conjunction with cytokines can specify the cellular microenvironment within EB to yield robust levels of hematopoietic progenitors. This work demonstrates proof-of-principle for hematopoietic cell production from human embryonic stem cells in a scaleable bioreactor system.