To some extent formation of embryoid bodies (EBs) from embryonic stem cells (ES) mimics the events during embryonal development and can be a useful model for studying lineage commitment. However, due to morphological and structural features, the effects of direct supplementation of a molecule of interest (MOl) on EBs in cell culture to study differentiation are often limited to the superficial layers. The use of biodegradable microparticles (MPs) to deliver the MOl directly within EBs might be a promising approach for controlling ES differentiation. This study aimed to develop a 3-D model combining cells and MPs loaded with osteo-inductive molecules including simvastatin and BMP2 for ES cell-derived osteogenesis within an EB model. The steps towards achieving this aim involved; i) establishing a reliable approach for producing human and / or mouse ES cell derived EBs; ii) evaluating the osteo-inductive potential for simvastatin iii) establishing a 3-D construct of ES cell derived EBs containing simvastatin loaded MPs followed by evaluation for osteogenesis through studying different markers during formation of /osteoblast- like cells/ bone-like tissue. It was found that mouse and human ES cells could readily form EBs in a controllable manner. Simvastatin pro and! or active drug were found to induce osteogenesis of ES cells in a 2-D environment over a concentration range of 0.1 and 1 ~M. A controlled simvastin release model from MPs loaded with either the prodrug or active drug was fabricated using an emulsion method. The MPs with average diameter of 12-13 urn were designed to be distributed within EBs and provide release of the MOl over a period of three weeks. A reliable method for creating 3-D constructs of ES cell derived EBs containing MPs was tested under static or with centrifugation as well as mass suspension with avidin coated- lyophilised MPs. In this study, improved incorporation of MPs within hES cells derived EBs has been presented by coating MPs with hES cell lysate before EB formation by ~ •.......................-- centrifugation method. EBs containing MPs loaded with osteo-inductive molecules (simvastatin prodrug, active drug or BMP-2) provided a robust and reproducible 3D model for osteogenesis and the lineage commitment of HUES- 7 cells into bone cells. Results were confirmed by positive expression of major osteogenic markers at the gene level (Runx-2, Osterix and osteocalcin) and protein level (Runx-2 and osteocalcin). Extracellular matrix (ECM) formation was confirmed by Picro-sirius red staining for collagen formation and matrix mineralisation by alizarin red staining. In conclusion, the model presented in this study mimics, to some extent, the spatiotemporal presentation of locally released growth factors of early development and can be beneficial for tissue engineering purposes through achieving better presentation of the molecule of interest within the multi-cellular EB model.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:582000 |
| Date | January 2012 |
| Creators | Qutachi, Omar |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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