Artificial embryonic stem cell niches were made from murine embryonic stem cells (ESCs) and SAOS-2 osteoblast-like cells (a human osteosarcoma cell line) by constructing aggregates with well-defined architectures with dielectrophoresis between the castellations of interdigitated oppositely castellated electrodes. This combination of the cells was chosen to mimic the bone marrow endosteal niche that harbours haematopoietic stem cells in a quiescent stage, with the aim of transforming the embryonic stem cells into hematopoietic precursor cells. Within aggregates made with dielectrophoresis cells are in very close contact, allowing strong cell-cell interactions to occur. Puramatrix gel was used to immobilize the cells; a concentration of 25% Puramatrix was found to be optimal. Aggregates consisting of only ESCs formed embryoid bodies upon aggregation with dielectrophoresis within 24 hours. The size of the electrodes determines the size of embryoid bodies. Embryonic bodies formed at electrodes with a characteristic size larger than 100 μm tended to split; electrodes smaller than 75 μm gave embryonic bodies which tended to merge. 75 to 100 μm was optimal. When aggregates were made containing both SAOS-2 and ESCs, the reorganization of the two cell types after their aggregation was found to be controlled by the different adhesive-cohesive properties of the two cell types and their initial position. Optimum cell-cell interaction was obtained in an aggregate with a layered architecture with the osteoblasts initially in bottom position, and the ESCs in top position. The study of differentiation in ESCs was made by conducting experiments with Bry ESCs, which mark the onset of differentiation along mesenchymal lineage with the production of GFP. The results indicated that aggregation with dielectrophoresis causes the ESCs to take the first steps towards differentiation along the mesenchymal lineage, and that the differentiation is stronger in aggregates formed at electrodes of 75 μm than at electrodes of 100 and 50 μm. Co-culture with SAOS-2 cells did not lead to differentiation along the mesenchymal lineage. Lastly it was shown that optical tweezers could be combined with dielectrophoresis to move individual cells between niches.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:575248 |
| Date | January 2011 |
| Creators | Agarwal, Sneha |
| Contributors | Markx, Gerard H.; Paterson, Lynn |
| Publisher | Heriot-Watt University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10399/2495 |
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