Sharma, VS; (2016) Size controlled retinal differentiation of human induced pluripotent stem cells in shaking microwells. Doctoral thesis , UCL (University College London).

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Abstract

Human induced pluripotent stem cells (hiPSC) have the potential to provide patient and disease specific cells for research and act as therapeutic agents in unlimited supply. To translate lab-scale research toward clinical applications we need to reduce variability from complex differentiation protocols which often include xenogeneic components. A move toward more defined culture systems will improve predictability and process control as well as reduce risks of exposure to animal pathogens. Cell therapy development, also requires flexibility in scalability as cell numbers for therapies vary greatly between disease indications. Improved control over the microenvironment at the lab scale would offer more defined parameters amenable to scale up with better flexibility, reduced waste and more precision. This thesis describes the use of forced aggregation to improve the initiation of differentiation combined with culture in pre-validated and scalable, 24 microwell plates on shaking platforms, for stem cell differentiation toward retinal lineages. Using an established hiPSC line (MSU001) we show using forced aggregation to form embryoid bodies (EBs) promotes efficient initiation of differentiation. We next determined 2 EB sizes (5K and 10K cells/EB) which improved initiation of retinal differentiation compared to scraped EBs as demonstrated by >3fold increase in expression of early eye field transcription factor Rax at day 3 of culture. The 5K and 10K EBs also facilitated the adaption of an adherent protocol for retinal differentiation to an orbital shaken suspension culture system. Size controlled EBs also enabled the selection of a permissive shaking speed (120rpm) suitable for orbital shaken culture for initiating retinal differentiation with 5K and 10K EBs. Furthermore orbital shaken culture enabled elimination of the undefined xenogeneic ingredients of Matrigel, from the culture system. This thesis demonstrates that combining size control for hiPSC derived EBs and orbital shaken culture is a feasible method for the initiation of retinal differentiation. By facilitating removal of xenogeneic materials from the culture system; the combination of orbital shaken culture with size controlled EBs may be of value to other complex stem cell differentiation systems to improve the initiation of differentiation whilst providing the potential for scalability.

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