Scalable bioprocess for controlled differentiation of embryonic stem cells to hematopoietic progenitors

Therapeutic application of pluripotent embryonic stem (ES) cells will require advances in cell culture technology that improve our ability to generate target cells. Control of the cell culture environment is of critical importance, as cell fate decisions can be influenced by cell extrinsic factors like cell-cell interactions, soluble cytokines, and physicochemical parameters. However current ES cell differentiation culture systems that make use of static tissue culture plates are limited in terms of measurement and control of the culture environment, and scalability of cell production. In this thesis project, a novel method to differentiate ES cells in scalable, controlled stirred suspension culture was developed. ES cells were differentiated as three dimensional tissue structures termed embryoid bodies (EBs). Successful EB formation was found to depend on the aggregation of ES cells. However cell aggregation, beyond that required for EB formation, was found to impair cell yield. E-cadherin, a cell-cell adhesion molecule, was important in this process. To control cell aggregation, ES cells were encapsulated within agarose microcapsules. ES cells within the same capsule were permitted to aggregate and induce EB formation, but the surrounding agarose matrix prevented developing EBs from contacting and agglomerating with one another. This approach permitted efficient EB formation, growth, and differentiation in stirred culture.;The ability to measure and control the culture environment in stirred suspension bioreactors makes them a valuable tool for investigating exogenous factors and optimizing conditions for target cell generation. Physicochemical factors like oxygen tension and pH are of particular interest because they can influence cell proliferation and differentiation in a cost effective way. Using the novel stirred suspension culture system, the role of oxygen tension in hematopoietic cell generation was investigated. Hematopoeitic progenitor generation was optimal at 4% oxygen tension. The mechanism of hypoxia-enhanced hematopoietic progenitor generation was investigated. By comparing the function and expression of VEGF and its receptors VEGFR1 and VEGFR2 under normoxic and hypoxic culture, activation of VEGFR2 by VEGF was found to have both enhancing and inhibiting effects, depending on the stage of development. VEGFR1, secreted in a soluble form, was found to mediate VEGFR2 signaling by competitively binding VEGF.