Embryonic stem cell self-renewal and differentiation in a scalable bioprocess
Kehoe, Daniel Eugene
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The goal of this research was to produce a scalable bioprocess for the proliferation and differentiation of embryonic stem cell. Experiments on mouse and human embryonic stem cells grown on traditional culture surfaces, in spinner flasks as undifferentiated, and in spinner flasks as differentiated cells were conducted. Several tools were utilized to describe and characterize the cells throughout this process, including bright field microscopy, cell viability assays, PCR, qPCR, flow cytometry, immunostaining, western blot, and confocal microscopy. The results presented here show that mouse Embryonic Stem Cells (mESCs) can be expanded as undifferentiated stem cells in suspension bioreactors with serum and also in defined serum-free media. These cells express pluripotent genes, as well as protein, and have the ability to differentiate to all three germ layers. Neuronal differentiations of mESCs were established where the cells were differentiated as floating bodies, then outgrew the clump, and were seeded on glass. It was observed that there was more neuronal differentiation with retinoic acid (RA) treatments, and the mechanism by which RA stimulates the cells to differentiate was investigated in depth, leading to the conclusion that the retinoic acid receptor(RAR) and the Fibroblast Growth Factor Receptor 1 (FGFR1) work together in the stimulation of neuronal differentiation. The expansion of human Embryonic Stem Cells (hESCs) in spinner flasks presented the most promising results after cells were treated with a rho-associated kinase inhibitor, dissociated with a milder enzymatic cell detachment media, and then incubated with more kinase inhibitor as well as a gelatinous mixture of proteins. These bioreactors were capable of reaching a 6-fold increase in cell number over seven days, and the aggregates expressed several pluripotent stem cell proteins. The differentiation of hESCs in suspension bioreactors to neurospheres was also investigated. Over the two week expansion and differentiation period, a 9-fold increase in cell number was observed and the expression of several neuronal genes and proteins was observed, indicating that hESCs can be differentiated toward neuroepithelial and radial glial cells. These results are important toward supporting the production of large numbers of neuronal cells as well as other types of diffrentiated cells for the purposes of tissue engineering, cell therapies, and drug testing applications.