Mondragon-Teran, P.; (2011) Impact of oxygen tension control on neuronal differentiation of Embryonic Stem Cells (ESC). Doctoral thesis , UCL (University College London).

Abstract

Embryonic Stem Cells (ESC) have the potential to differentiate into any adult cell type even after extended in-vitro culture. These cells have the scope to be a source of specialized cells for pharmaceutical screening and potential cell based transplantation therapies. The successful commercialization of ESCderived products will be highly dependent on the development of cost-effective production bioprocesses. Currently, the most inefficient phase of these bioprocesses is cell differentiation, resulting in low numbers and purity of the target phenotype. Research described in this thesis investigated whether physiological oxygen tensions would influence the yield and purity of neuronal cells during mouse ESC (mESC) differentiation. A chemically defined media, monolayer protocol for the neuronal differentiation of mouse ESC in conjunction with a hypoxia chamber to control the oxygen tension on the growth surface was used. In the first part of the study, it was found that 2% O2 enhanced the yield of cells expressing neuron specific markers (βIII-tubulin and MAP2) as compared with traditional culture conditions (20% O2). Further experiments demonstrated that higher neuronal production was achieved when mES cells were differentiated in the range 4 – 10% O2; an increase in neural rosette diameter was detected within the same oxygen conditions. Embryo development is carried out under hypoxic conditions before week 11 (in humans) of gestation and before 9 days of gestation in the case of mouse embryo. After these indicated stages, blood starts to flow from mother to embryo and oxygen conditions rises from hypoxic to different physiological oxygen values (generally rises from 0% to 2%, 5% or 8% depending of the organ or tissue analyzed). Step changes in oxygen tension during mESC neuronal in vitro differentiation were performed in order to mimic the described in vivo conditions. These results highlight that mimicking in-vivo oxygen tensions during early embryo development can be used to achieve significant enhancements in the yield of neuronal cells from ESC differentiation protocols. Automated mES neuronal differentiation under static oxygen conditions was implemented in order to avoid transient changes from in vitro controlled physiological values to laboratory environmental 20% O2 conditions while manual processing. This lead to an increase of neuronal maturation as observed in the higher MAP2 (mature neuronal marker) expression. Interestingly cell metabolism was also influenced by static oxygen conditions. Results described in this thesis establish the importance of controlling oxygen tension either in a manual or automated way during the in vitro mESC neuronal differentiation. The strategies proposed in this thesis will allow developing higher efficiency and higher purity of neurons from a stem cell bioprocessing perspective.

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