Stem cells hold great therapeutic potential in regenerative medicine due to their self-renewal capacity and their ability to differentiate into a wide range of cell lineages. This thesis is focused on the characterisation of both a pluripotent and a more specialised multipotent stem cell source. These studies involve the optimisation of essential parameters for cell expansion and differentiation. Initially, human mesenchymal stem cells (hMSCs) were used as a model system for cell expansion studies. This was followed by more advanced studies using mouse embryonic stem cells (mESC). The results obtained in this research revealed that hMSCs inoculated at low densities resulted in larger fold expansion than those inoculated at high densities. However, at low (<100 cells/cm^2) and high (10000 cells/cm^2) inoculation densities, the expression of STRO-1, a surface marker present on hMSC was lost very rapidly. The optimal inoculation density was found to be 5000 cells/cm^2. Prolonged in vitro culture of hMSCs caused a reduction in the cell proliferative potential and STRO-1 antigen expression. Therefore to preserve the ‘stemness' properties of hMSCs, expansion needs to be limited to low passage levels. The optimal fetal bovine serum concentration for hMSCs expansion was found to be at 15%. The differentiation of mESC was studied using the embryoid body (EB) system. The results obtained indicated that the size of the EB and activin A, a ligand that activates the nodal signaling pathway have profound effects on mESC lineage commitment and thus on bioprocessing. Quantitative reverse transcription-polymerase chain reaction analysis revealed that activin A enhanced and accelerated endoderm formation within small EBs, and delayed the loss of pluripotency of undifferentiated cells within large EBs. In addition, activin A has an inhibitory effect on neuroectodermal differentiation. Small EBs treated with activin A displayed a minimal neuroectodermal gene expression whereas large treated EBs displayed a higher level of expression. Finally, various in vitro culture systems for mESC differentiation were examined. These include static, rotary suspension and combined culture systems. This research provides an insight into some of the parameters affecting the growth kinetics and developmental characteristics of hMSCs and mESCs. The findings reported may help facilitate the development of successful stem cell bioprocess design essential for the scaling and manufacture of safe, efficacious and cost effective human therapies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:505529 |
| Date | January 2009 |
| Creators | Chu, Pui Kei Carol |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/17422/ |
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