Strategies for improving the chondrogenic differentiation of human induced pluripotent stem cells for cartilage therapy

Abstract

Articular cartilage is a smooth connective tissue covering the diarthrodial joints. It lacks blood vessels, lymphatics, and nerves, and contains a specialised extracellular matrix (ECM) populated by chondrocytes. Severely damaged cartilage has a poor capability for self-healing due to its avascular structure. Therefore, the replacement of cartilage may be needed. Recently, the use of human induced pluripotent stem cell (hiPSC)-based therapies has gained much attention for cartilage repair as this increases cell populations with high chondrogenic capability. This therapy could provide hope for healing large lesions of the cartilage. In this study, we generated chondroprogenitors from hiPSCs via mesodermal specification and these cells exhibited chondrogenic phenotype. They also had the potential to synthesize cartilaginous extracellular matrix during chondrogenesis in vitro when treated with TGF-β3+BMP4 or TGF-β3+BMP4+BMP7.
Establishing adequate cell culture conditions is essential for developing effective cellular therapies and regulating cell fate in vitro to adapt to the microenvironment of the targeted tissues. Several factors have been shown to influence cell characteristics, such as donor age, cell density, stimulation, and 3D scaffolds. Controlling oxygen tension in vitro is a crucial factor that can impact mesenchymal stem cell (MSC) behaviour and cellular metabolism of chondrocytes. Here, I investigated the influence of hypoxia on the hiPSC-chondroprogenitors (CPs) and adult MSCs in both monolayer and 3D pellet cultures. The results showed that hypoxia impacted cell proliferation, and cartilage matrix formation during chondrogenesis.
We also investigated the effects of cell cycle synchronisation on the chondrogenic potential of hiPSC-CPs and downstream differentiation. Nocodazole is a microtubule polymerisation inhibitor which arrests cells at the G2/M phase. Nocodazole-treated hiPSCs showed greater cell proliferation and chondrogenic differentiation. Moreover, we
synchronised hiPSC-CPs using serum starvation and generated 3D pellets, which increased gene expression and cartilage matrix formation. The results suggested that cell cycle synchronisation could ameliorate the capacity of hiPSCs and chondrogenic cells.
Whilst several biomaterials have been employed for cartilage repair, results are far from achieving native cartilage functions and structure. Buoyancy-driven gradients have been proposed as a promising technique for biomaterial fabrication and tissue engineering providing versatility and accessibility to produce articular tissue constructs. Using this technology, we aimed to create different zones of articular cartilage using hiPSC-CPs coxed with growth factors and supported by buoyancy force-driven gradient polymers. The results were promising with MSCs where the material and growth factor combinations pointed to zone-specific outcomes. hiPSC-CPs were less responsive to the growth factors/biomaterial combination warranting further investigation in the future.
In conclusion, the thesis presented hypothesis-driven data aimed at addressing critical aspects of iPSC chondrogenic differentiation. The findings highlighted the need to pursue prudently the fundamental biology of chondrogenic differentiation and the importance of adopting new technologies to fabricate robustly engineered tissue. Future
studies can build on these findings by optimising the conditions used and scaling up their application.

Date of Award	1 Oct 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Wael Kafienah (Supervisor) & Abdelkader Essafi (Supervisor)