Enhancing Chondrogenesis of Human Pluripotent Stem Cells

Abstract

Osteoarthritis is the most common degenerative condition of the joint, leading to degradation of articular cartilage (AC), chronic pain and immobility. Lack of appropriate therapies that provide tissue restoration and the limited lifespan of joint-replacement implants indicate the need for AC regeneration strategies. Our group established protocols for differentiating human embryonic stem cells (hESCs) into AC progenitors. However, differentiation protocols exhibit run-to-run inconsistencies due to the continued reliance upon growth factors, which is a limiting factor for the use of hESC-based therapies in the clinic. Recently, the use of a single small molecule activator of retinoic acid receptors (RARs) signalling has been shown to be sufficient to guide mesodermal specification and early chondrogenesis of hPSCs. This project aimed to utilise RAR activation, cell sorting approaches and 3D culture systems during hPSC chondrogenic differentiation to enhance the generation of AC chondroprogenitors. An enhanced differentiation protocol, RAPID-E, was established by supplementing cells with TTNPB, a small molecule RAR agonist and administrating BMP2 at specific times. Gene expression analyses indicated a significant upregulation of pro-chondrogenic markers and a downregulation of hypertrophic genes were achieved when compared to the previous protocol. Transcriptomic analyses by RNA-seq indicated that activation of RAR signalling significantly upregulated genes related to limb and embryonic skeletal development in the early stages of the protocol and upregulated genes related to AC development in later stages. Considering cartilage in monolayer dedifferentiate, 3D culture with a scaffold-free method based on pellets in suspension and a cell-laden hydrogel system were tested. The 3D systems resulted in a further upregulation of chondrogenic-related genes, and pellets displayed strong staining for AC-related matrix proteins such as Lubricin, Aggrecan, and Collagen 2. However, although upregulation of hypertrophic gene COL10A1 was not significant, cells in 3D stained positively for it, indicating cells may be maturing towards a hypertrophic population. The occurrence of hypertrophic markers was seen to be modulated by the culture medium, the culture system and in the cell density encapsulated into the gels, indicating the possible generation of a heterogeneous population of off-target cells at the end of the 2D differentiation. Enriching the population for chondrogenic progenitors by sorting the cells for markers reported in the literature could have avoided the off-target differentiation of the cells in 3D. Surface markers were investigated by flow cytometry with promising results for the application of sorting strategies in the future. When fully characterised, this protocol could lay the foundations for cell therapy strategies for osteoarthritis and improve the understanding of AC development in humans.

Date of Award	11 Jul 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Leela Biant (Supervisor), Susan Kimber (Supervisor) & Marco Domingos (Supervisor)