The injury of articular cartilage represents a great challenge in orthopaedics especially in young patients. To deal with this problem, a number of strategies have been initiated including cell-based tissue engineering for cartilage repair. Human embryonic stem cells (hESCs), which are pluripotent and can differentiate into all cell lineages including chondrocytes, are one potential cell source for such treatments. As several key elements are involved in differentiation based on normal physiology of chondrocyte development, I have investigated the effect of several factors, including low oxygen tension, multilayer-three dimensional culture and the alternative use of bone morphogenic proteins (BMPs), in the differentiation of hESCs toward chondrocytes. An established 14-day differentiation protocol for chondrogenesis of hESCs was set up to generate chondrocytes. This study first tested preconditioning of hESCs in pluripotent culture in 3 sub-ambient O2 levels, followed by directed differentiation towards chondrocytes in parallel in ambient oxygen (20%) and in low oxygen (3%). The results showed that hESCs after preconditioning had a small but significantly favourable response to directed differentiation under lowered oxygen (3%) with up-regulation of COL2A1 and down-regulation of COL1A1. However, clear increases in hypoxia-inducible factor (HIF) proteins were not detected in these low oxygen culture conditions. In addition this study also investigated the effect of chondrogenic differentiation with dimethyloxalylglycine (DMOG), which mimics low oxygen effects by stabilizing HIF proteins. The outcome gave some increases of SOX5, SOX9, ACAN and COL2A1 with decrease of COL1A1. The second set of experiments in multilayer and three dimensional differentiation were able to show chondrogenic tissue formation, but this was accompanied by a lack of up-regulation in chondrogenic-associated markers. The third set of experiments investigated the effect of BMP2, in the differentiation towards chondrocytes, and showed significant increases of COL2A1 and ACAN expression. In vivo implantation in nude rats of the chondroprogenitors derived with BMP2 resulted in satisfactory articular defect repair after 6 weeks of implantation. These results showed that in vivo implantation provided the signals required to complete the full chondrogenic differentiation of the implanted cells. Together my works suggested that although some strategies tested did not achieve a major advantage in differentiation in vitro, the evaluation of these key elements of niche factors improved our understanding of chondrogenesis of hESCs toward chondrocytes. The results from this investigation showed that the chondroprogenitors generated from hES cells did not respond in vitro to low oxygen or to 3D culture to become fully differentiated, but still provided the potential to form cartilage tissue in vivo.
|Date of Award||31 Dec 2015|
- The University of Manchester
|Supervisor||Susan Kimber (Supervisor) & Tim Hardingham (Supervisor)|
- Chondroprogenitor in vivo implantation
- Multi and three dimensional cell cultures
- Human Embryonic Stem Cells
- Low oxygen tension