Therapeutic Potential of Fetal Cartilage-derived Progenitor Cells for Skeletal Muscle Regeneration

Abstract

Human mesenchymal stem cells (MSCs) have gained a lot of attention in regenerative medicine as a promising cell source that can cause tissue regeneration. The therapeutic efficacy of MSCs mostly comprises differentiation into cells of specific lineages, induction of cell proliferation, angiogenic actions, and immunological modulation, and it is known that they can promote tissue regeneration. However, due to limited therapeutic efficacy, difficulty with laboratory manipulation, and safety concerns, MSCs have limits in clinical usage. We recently demonstrated that human fetal cartilage- derived progenitor cells (hFCPCs) have features similar to MSCs but superior proliferation, differentiation, and tissue regeneration capacity. The hFCPCs produced more mature cartilage and synthesized more extracellular matrix proteins during cartilage differentiation than BMSCs, causing cartilage tissue regeneration. It also developed into corneal cells, resulting in the formation of an artificial cornea, and has shown a healing impact on rabbit cornea damage. Furthermore, hFCPCs produced and secreted more growth factors such as transforming growth factor-β3 (TGF-β3), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor-binding protein (IGFBP), as well as extracellular matrix (ECM) components such as collagen, aggrecan, and fibronectin than hBMSCs in a recent study. This showed that hFCPCs exhibited superior paracrine effects in inducing tissue regeneration and were consequently identified as a more promising cell source than hBMSCs in terms of regenerative medicine. Another therapeutic mechanism of stem cells that contributes to direct tissue regeneration through differentiation is paracrine effects, and manipulating cells to modify and exploit paracrine effects could be a very important method in terms of regenerative medicine. Therefore, the purpose of this study was to confirm and utilize the therapeutic efficacy of hFCPCs in skeletal muscle regeneration through their excellent differentiation capacity and paracrine effects. In chapter I, we optimized the conditions for hFCPC myogenic differentiation and assessed the in vitro and in vivo therapeutic efficacy of myogenic differentiated hFCPCs on muscle regeneration. By optimizing myogenic differentiation under three differentiation circumstances, the differentiation capacity of hFCPCs was compared to that of hBMSCs. As a result, when exposed to muscle differentiation settings comprising basic fibroblast growth factors (bFGF) and dexamethasone, hFCPCs produced mature myotubes and expressed more muscle differentiation markers than hBMSCs. Additionally, in co-culture with atrophy-induced myoblasts, hFCPCs induced more myoblast recovery than hBMSCs. The hFCPCs expressed more HGF, FGF2, VEGF, and IGFBP2, which are implicated in muscle regeneration, than hBMSCs in genetic and protein expression analyses. Myogenic differentiated hFCPCs were found to engraft more than hBMSCs in transplantation experiments on skeletal muscle injury models, induce the activation of satellite cells, and induce skeletal muscle regeneration. As a result, we suggest that hFCPCs can be utilized as a promising cell source for skeletal muscle regeneration based on their excellent muscle differentiation capacity. In chapter II, exosomes secreted from myogenic differentiated hFCPCs were used to utilize the myogenic differentiation ability and muscle regeneration effects of hFCPCs. Exosomes influence cell biology by transferring genetic elements, such as micro-RNA (miRNA) to target cells. We first compared the properties of exosomes secreted from hFCPCs (F-Exo) and hBMSCs (B-Exo) during myogenic differentiation. F-Exo caused higher myogenic differentiation in myoblasts than B-Exo in an experiment in which myoblasts were treated with two types of exosomes. Myoblasts treated with F-Exo displayed increased expression of MyoD, MyoG, and MyHC genes and proteins, as well as superior myotube characteristics. Furthermore, in sarcopenic rats, F-Exo generated higher increases in muscle fiber cross-sectional area and muscle mass than B-Exo. We discovered that F-Exo contained more miR-145-5p than B-Exo when we examined the genetic material in exosomes. As a result, it was determined that F-Exo delivers abundant miR-145-5p to myoblasts and stimulates the Wnt signaling pathway, which is involved in muscle regeneration. The results of this study established a new strategy for muscle regeneration by developing exosomes enriched in miR-145-5p that mimic the excellent myogenic differentiation capacity of hFCPCs. Keywords: Human fetal cartilage-derived progenitor cells, Myogenic differentiation, Skeletal muscle regeneration, Paracrine effects, Exosomes, micro-RNA