Evaluation of chondrogenic differentiation efficacy in human stem cell populations

Abstract

Cartilage is avascular, aneural and displays a limited capacity to regenerate, supporting the need for a robust alternative cell source for regeneration. Loss of chondrocytes, together with a reduction in the specialised extracellular matrix is often the outcome of articular cartilage injury. Untreated cartilage damage can result in osteoarthritis (OA), the most common chronic pathology of joints. OA is typically seen as an age-related disease and given the current ageing demographic, a significant socio-economic burden. To address cartilage reparation, mesenchymal stromal/stem cells (MSCs) have received increasing attention as a stem cell source for articular cartilage regeneration. Nevertheless, issues including availability, intrinsic potency, cellular ageing and disease remain, limiting the application of aged MSCs for therapy. Human-induced pluripotent stem cell-derived MSCs (hiPSCs-iMSCs) offer an attractive stem cell source displaying a “young” phenotype for an enhanced chondrogenic differentiation efficacy for articular cartilage regeneration. This thesis investigates the process of chondrogenesis in aged MSC populations derived from human bone marrow (hBM-MSCs), human synovium (hSy-MSCs) and in MSC populations derived from hiPSCs (hiPSCs-iMSCs). Experimental studies conducted in this thesis demonstrated the chondrogenic differentiation potential of aged hBM-MSCs and hSy-MSCs populations. Nevertheless, significant variability in chondrogenic phenotype was noted between hBM-MSCs and hSy-MSCs populations, as well as between different donors for the same cell source. Accumulating evidence supports the influence of age and pathological conditions on the phenotypic and functional properties of aged MSCs. Cellular rejuvenation achieved using pluripotency induction offers a promising tool to erase age-related signatures and the acquisition of an unlimited hiPSCs-iMSCs source for articular cartilage regeneration. In this thesis, studies were undertaken to establish a robust hiPSCs population for subsequent in vitro mesengenic induction and chondrogenic differentiation. Upon mesengenic induction, hiPSCs-iMSCs showed a significant loss of pluripotency/epithelial cell markers, an acquisition of MSCs markers and the ability to differentiate along the chondrogenic lineage. Distinct differences in hiPSCs-iMSCs and aged hBM-MSCs derived chondrogenesis were characterised through differential chondrogenic marker gene expression determined using qRT- PCR. Enhanced and rapidly increased II upregulation of the cartilage maturation marker COL10A1 were noted in hBM-MSCs. In contrast, higher levels of the articular cartilage marker gene ACAN were prominent in hiPSCsiMSCs, indicating superior proteoglycan production. MicroRNAs are a class of small single-stranded non-coding RNA which: i) regulate gene expression post-transcriptionally, ii) are known to be dysregulated in disease states, iii) are important in the proliferation and differentiation of stem cells and, iv) have been shown to play an important role in chondrogenesis. This thesis demonstrates, for the first time, the differential expression of miRNA regulators of chondrogenesis between hiPSCs-iMSCs and aged hBM-MSCs populations. Significant upregulation of chondrogenic miRNAs 140, 145, 23a, 26a, 27a, 101 and 148b was detected in hiPSCs-iMSCs derived chondrogenesis. In contrast, no significant changes in miRNAs expression were detected in hBM-MSCs derived chondrogenic populations, apart from miRNA 27a, which was significantly downregulated. Furthermore, significant downregulation of the negative chondrogenic miRNAs 146a/146b expression was observed. miRNA 146a was significantly downregulated in hiPSCs-iMSCs only, whereas miRNA 146b expression was significantly downregulated in hiPSCs-iMSCs and hBMMSCs. Interestingly, a significant reduction over time of miRNA 146b was detectable in hiPSCsiMSCs. In summary, the current studies show i) chondrogenesis was found to be distinct in different human MSC populations and between patient-samples of aged MSC populations, ii) superior chondrogenesis was suggested in hiPSCs-iMSCs, evidenced by higher levels of the articular cartilage marker gene ACAN, and demonstration of the positive regulation of select miRNA regulators of chondrogenesis. An understanding of chondrogenesis and the functional relevance of the examined miRNAs is critical to facilitate and develop optimal differentiation protocols for a pure and functional hiPSCs derived articular cartilage generation. Identification of optimised MSC cell protocols, together with microRNAs pivotal during chondrogenic differentiation, offer approaches for the potential use of microRNA modulation in combination with skeletal stem cells for cartilage regeneration. Elucidation of such approaches will ultimately, help treat cartilage injury and cartilage pathologies such as OA.

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Identifiers

ORCID for Richard Oreffo: orcid.org/0000-0001-5995-6726

ORCID for Rahul Tare: orcid.org/0000-0001-8274-8837

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