The role of TWIST-1 in the regulation of mesenchymal stem cell growth, fracture repair and bone loss.

Abstract

Mesenchymal stem cells (MSC) have the capacity to differentiate into osteoblasts, chondrocytes and adipocytes in vivo. It is well established that the basic Helix-Loop-Helix transcription factor TWIST-1 plays an important regulatory role within the context of bone remodelling and formation in both developmental and adult settings by regulating the proliferation, differentiation and commitment of MSC. Recent studies have demonstrated that elevated levels of TWIST-1 in MSC results in the maintenance of an immature population of cells with enhanced proliferative and adipogenic potential while simultaneously reducing the cells capacity to undergo osteogenesis and chondrogenesis. The aim of this thesis was to define the role of TWIST-1 in MSC commitment and differentiation within the context of bone fracture healing and bone loss during osteoporosis. Microarray results highlighted differential expression of Wnt pathway genes WNT2 and WNT2B in MSC with enforced TWIST-1 expression. Purified MSC isolated from bone marrow aspirates obtained from normal human donors were used to generate over-expressing WNT2 and WNT2B MSC lines using established retroviral based systems. Genetically modified MSC were used to examine the effect of WNT2 and WNT2B on osteogenesis, chondrogenesis, adipogenesis and proliferation of MSC in vitro. Results showed regulation of WNT2 and WNT2B by TWIST-1 through direct interactions between TWIST-1 protein and the proximal promoter regions of WNT2 and WNT2B. Furthermore, functional analysis demonstrated enhanced adipogenesis in MSC with enforced expression of WNT2B. To assess the role of TWIST-1 deficiency in fracture healing and the disease state of osteoporosis in vivo, a heterozygous TWIST-1⁺′⁻ mutant mouse model was used. The role of TWIST-1 deficiency in fracture healing was assessed by induction of a femoral fracture followed by analysis at one, two four and eight weeks post fracture to examine the different stages of fracture healing. Analysis showed enhanced osteoblastic differentiation and accelerated fracture repair in heterozygous TWIST-1⁺′⁻ mutant mice when compared to relevant wild type littermate control mice. The role of TWIST-1 deficiency in osteoporosis was examined by induction of osteoporosis by ovariectomy. Results from this study showed heterozygous TWIST-1⁺′⁻ mice maintained bone volume following ovariectomy induced osteoporosis.