Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Dr. Victor Han

Abstract

Human placenta is a readily available source for isolation of adult mesenchymal stem cell (MSC), for potential use in regenerative therapies. MSC fate is influenced by the microenvironment in vivo, and insulin-like growth factors (IGFs) are critical components of the stem cell niche, as they regulate proliferation and differentiation into different lineages including bone, fat, and skeletal muscle. Insulin-like growth factor binding protein-6 (IGFBP-6), relative to other IGFBPs, has high affinity for IGF2 and is believed to be the main modulator of IGF-2 function. However, the role of IGFBP-6 in muscle development has not been clearly defined. In this study, we investigated the role of IGFBP-6 in different stages of muscle commitment and differentiation using human mesenchymal stem cells derived from the placenta (PMSCs). Our central hypothesis is that IGFBP-6 regulates the maintenance of multipotency in PMSCs and also promotes PMSC differentiation into muscle via intracellular and extracellular interactions in both IGF-dependent and independent mechanisms. We used immunoblotting, immunocytochemistry, ELISA, and ALDH-activity to evaluate IGFBP-6 effects on PMSC muscle differentiation. We showed that PMSCs are capable of differentiating into muscle cells when exposed to muscle-specific differentiation medium characterized by the decrease of pluripotency-associated markers (OCT4 and SOX2) and the gain of expression of muscle markers Pax3/7, MyoD, Myogenin, and Myosin heavy chain in a time-dependent manner and eventually forming multi-nucleated fibers. Extracellular supplementation with IGFBP-6 during culture increased muscle differentiation markers levels in early stages. The opposite effects were observed when IGFBP-6 was silenced and was rescued by increasing IGFBP-6. We also showed that IGFBP-6 had impact on muscle differentiation in both IGF-dependent and -independent mechanisms. IGF-1 and IGF-2 had different effects on muscle differentiation with IGF-1 promoting multipotency and early commitment to muscle, whereas IGF-2 promoting muscle differentiation. Muscle differentiation required activation of both AKT and MAPK pathways. Interestingly, we demonstrated that IGFBP-6 could compensate for IGF-2 loss and help enhance the muscle differentiation process by triggering predominantly the MAPK pathway independent of activating either IGF-1R or insulin receptor (IR). These findings indicate complex interactions between IGFBP-6 and IGFs in PMSC differentiation into skeletal muscle. The most prominent effects were observed early in the differentiation process, before muscle lineage commitment. This knowledge on how myogenesis can be manipulated using IGFs and IGFBP-6 will aid in the development of improved muscle regeneration therapies using stem cells from human placenta.


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