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• W2579223816 abstract "Abstract 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." @default.
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• W2579223816 date "2006-11-16" @default.
• W2579223816 modified "2023-09-29" @default.
• W2579223816 title "Scaleable Production of Hematopoietic Cells Suitable for Transfusion from Human Embryonic Stem Cells in a Stirred Bioreactor System." @default.
• W2579223816 doi "https://doi.org/10.1182/blood.v108.11.728.728" @default.
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