Embryonic stem cells (ESCs) are known for their unique property to be maintained almost
indefinitely in an undifferentiated, proliferating state with the potential to give rise to all types of cells.
Current established protocols for the culture and differentiation of ESCs are cumbersome and inefficient
involving three stages: a) maintenance or expansion of undifferentiated ESCs, b) spontaneous
differentiation through formation of embryoid bodies (EBs), and c) dissociation of the EBs and replating
leading to the terminal differentiation to the desired lineages. One of the major challenges in the use of
ESCs for cell therapy is controlling their differentiation pathways. Optimal culture conditions and
requirement as well as precise differentiation mechanisms and cellular interactions within EBs are still
not well characterised resulting in sub-optimal control of homogenous differentiation especially due to
the formation of all three germ layers. Attempts on developing an efficient culture protocol have been
widely reported in order to overcome the limitations. Recent research approaches have shown that
treatment with conditioned medium derived from HepG2, a human hepatocarcinoma cell line enhances
the formation of multipotent mesodermal progenitors from ESCs. This promotes a greater control of ESC
differentiation in a lineage-specific fashion possibly resulting in efficient haematopoietic differentiation.
In this study, we have developed an integrated, single step bioprocess for ESCs hematopoietic
differentiation that: a) uses HepG2-conditioned medium (HepG2-CM), that stimulates mesoderm
formation, b) facilitates three dimensional (3D) culture through encapsulation of undifferentiated ESCs
in hydrogels, c) bypasses EB formation, and d) involves culture in a rotating wall vessel bioreactor that
does not require passaging of the cells and is scalable and automatable. In conclusion, this thesis reports
the development of a novel culture system for the efficient single-step haematopoietic differentiation of
ESC resulting in a reproducible, scalable, high-intensity culture system of mESCs for ex-vivo blood
manufacture.