Cartilage is an essential skeletal connective tissue in vertebrates. It comprises extracellular matrix components, especially for collagens and proteoglycans. Once the cartilage is damaged, it has limited self-repair capacity. Thus, by understanding the dynamic cellular process of chondrogenesis and chondrocyte differentiation would be necessary in developing therapeutic approaches for cartilage repair. Currently, there is a great interest in the development of cell therapy to repair damaged tissues. In particularly, human mesenchymal stem cells (hMSCs) are attractive candidates for the treatment of skeletal system disorders because they can be greatly expanded ex vivo and they readily differentiate into chondrocytes upon stimulation. Studies have demonstrated low environmental oxygen tension could affect the chondrogenic differentiation of hMSCs. The three basic helix-loop-helix (bHLH) motif-containing hypoxia inducible factor α (HIF-α) subunits (i.e. HIF-1α, HIF-2α and HIF-3α) are the major oxygen-sensitive transcription factors regulating physiological responses under hypoxia. Of significance, HIF-1α has been reported to induce a hyaline chondrocyte-like phenotype in human articular chondrocytes. The aim of this study was to investigate the roles of all three human HIF-α paralogues in chondrogenesis, particularly for the transcriptional regulation of chondrocyte-specific genes, including type II collagen (COL2A1) and aggrecan (AGC1). The effect of all three human HIF-α paralogues on the chondrogenic differentiation of hMSCs could then be investigated. Self-inactivating lentivirus vector (SIN-LV) shuttle plasmids coding for murine SOX9, wild-type and oxygen-insensitive versions of human HIF-1α and HIF-2α or wild-type HIF-3α were generated. These plasmids were used in luciferase-based promoter assays and to generate SIN-LV particles for overexpression studies. Our data revealed that SOX9, a key transactivator of chondrogenesis, strongly activates the transcription of COL2A1 and AGC1. Ectopic expression of HIF-2α could also induce the transcription of COL2A1 and AGC1. Strikingly, a cooperative transcriptional up-regulation of COL2A1 and AGC1 via the overexpression of HIF-1α and SOX9 was observed. Furthermore, HIF-3α was shown to inhibit the SOX9–dependent transcriptional up-regulation of COL2A1 and AGC1. Here, the multipotency of hMSCs cultured under hypoxia (1% O2 tension) was also illustrated. A pilot study for overexpressing exogenous gene in chondrogenic stimulated hMSC pellets via SIN-LV particles is described. Eventually, a rationale is provided for manipulating HIF-α expression in chondrogenic stimulated hMSC pellet via SIN-LVs encoding HIF-α subunits to study the contribution of HIF-α paralogues on promoting the chondrogenic differentiation of hMSCs. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Sox9, (SRY-type HMG box), has been shown to play a critical role throughout chondrogenesis. Haploinsufficiency of Sox9 in humans leads to a skeletal malformation syndrome known as campomelic dysplasia. To understand the regulation of Sox9 during chondrogenesis, the developing mouse limb was used to identify and characterize regulatory regions within the Sox9 promoter. Luciferase-based reporter assays in mouse revealed a proximal promoter spanning – 2 kb from the transcriptional start site, while mobility shift assays demonstrated that a CCAAT motif is involved in the transactivation of Sox9. Moreover, luciferase-based reporter assays revealed a proximal promoter spanning – 4 kb in Fugu rubripes, and potential regulatory regions spanning the remainder of the promoter. Comparison of mammalian Sox9 upstream intergenic sequences to that of Fugu has identified 5 conserved regions that are contained within 18 kb of upstream Fugu sequence. Analysis of the transcriptional activity of these sequences has led to the identification of regulatory elements within the Sox9 promoter. Several studies also provide evidence of a role for wingless (WNT) and bone morphogenetic (BMP) signaling molecules in the regulation of chondrogenesis. TCF/LEF-LacZ reporter mice show activated canonical WNT signaling distributed throughout the embryonic age (E) 9.5 forelimb. At later stages, LacZ expression becomes confined to distal regions of the limb bud. Previous studies have demonstrated that canonical WNTs inhibit chondrogenesis. Our studies demonstrate that treatment of cultures derived from E11.5 proximal limb buds with the canonical WNT, WNT3a, inhibits chondrogenesis. However, treatment of cultures derived from E9.5 and distal E11.5 limb buds with WNT3a stimulates chondrogenesis. Quantitative PCR (qPCR) also demonstrates that WNT3a modulates a number of genes expressed throughout chondrogenesis. To gain insights into BMP function in the early limb, we have characterized BMP action in sub-populations of cells from the E10.5 limb. Surprisingly, BMPs were found to inhibit cartilage formation in immature cells, while promoting cartilage formation in more mature cells. Transcriptional profiling coupled with qPCR and time course analyses revealed that the extent of induction of Gatas by BMPs was associated with its stimulatory versus inhibitory activity. Further, SOX9 activity was inhibited following over-expression of Gatas.

Sox9, (SRY-type HMG box), has been shown to play a critical role throughout chondrogenesis. Haploinsufficiency of Sox9 in humans leads to a skeletal malformation syndrome known as campomelic dysplasia. To understand the regulation of Sox9 during chondrogenesis, the developing mouse limb was used to identify and characterize regulatory regions within the Sox9 promoter. Luciferase-based reporter assays in mouse revealed a proximal promoter spanning – 2 kb from the transcriptional start site, while mobility shift assays demonstrated that a CCAAT motif is involved in the transactivation of Sox9. Moreover, luciferase-based reporter assays revealed a proximal promoter spanning – 4 kb in Fugu rubripes, and potential regulatory regions spanning the remainder of the promoter. Comparison of mammalian Sox9 upstream intergenic sequences to that of Fugu has identified 5 conserved regions that are contained within 18 kb of upstream Fugu sequence. Analysis of the transcriptional activity of these sequences has led to the identification of regulatory elements within the Sox9 promoter. Several studies also provide evidence of a role for wingless (WNT) and bone morphogenetic (BMP) signaling molecules in the regulation of chondrogenesis. TCF/LEF-LacZ reporter mice show activated canonical WNT signaling distributed throughout the embryonic age (E) 9.5 forelimb. At later stages, LacZ expression becomes confined to distal regions of the limb bud. Previous studies have demonstrated that canonical WNTs inhibit chondrogenesis. Our studies demonstrate that treatment of cultures derived from E11.5 proximal limb buds with the canonical WNT, WNT3a, inhibits chondrogenesis. However, treatment of cultures derived from E9.5 and distal E11.5 limb buds with WNT3a stimulates chondrogenesis. Quantitative PCR (qPCR) also demonstrates that WNT3a modulates a number of genes expressed throughout chondrogenesis. To gain insights into BMP function in the early limb, we have characterized BMP action in sub-populations of cells from the E10.5 limb. Surprisingly, BMPs were found to inhibit cartilage formation in immature cells, while promoting cartilage formation in more mature cells. Transcriptional profiling coupled with qPCR and time course analyses revealed that the extent of induction of Gatas by BMPs was associated with its stimulatory versus inhibitory activity. Further, SOX9 activity was inhibited following over-expression of Gatas.

Sox9, (SRY-type HMG box), has been shown to play a critical role throughout chondrogenesis. Haploinsufficiency of Sox9 in humans leads to a skeletal malformation syndrome known as campomelic dysplasia. To understand the regulation of Sox9 during chondrogenesis, the developing mouse limb was used to identify and characterize regulatory regions within the Sox9 promoter. Luciferase-based reporter assays in mouse revealed a proximal promoter spanning – 2 kb from the transcriptional start site, while mobility shift assays demonstrated that a CCAAT motif is involved in the transactivation of Sox9. Moreover, luciferase-based reporter assays revealed a proximal promoter spanning – 4 kb in Fugu rubripes, and potential regulatory regions spanning the remainder of the promoter. Comparison of mammalian Sox9 upstream intergenic sequences to that of Fugu has identified 5 conserved regions that are contained within 18 kb of upstream Fugu sequence. Analysis of the transcriptional activity of these sequences has led to the identification of regulatory elements within the Sox9 promoter. Several studies also provide evidence of a role for wingless (WNT) and bone morphogenetic (BMP) signaling molecules in the regulation of chondrogenesis. TCF/LEF-LacZ reporter mice show activated canonical WNT signaling distributed throughout the embryonic age (E) 9.5 forelimb. At later stages, LacZ expression becomes confined to distal regions of the limb bud. Previous studies have demonstrated that canonical WNTs inhibit chondrogenesis. Our studies demonstrate that treatment of cultures derived from E11.5 proximal limb buds with the canonical WNT, WNT3a, inhibits chondrogenesis. However, treatment of cultures derived from E9.5 and distal E11.5 limb buds with WNT3a stimulates chondrogenesis. Quantitative PCR (qPCR) also demonstrates that WNT3a modulates a number of genes expressed throughout chondrogenesis. To gain insights into BMP function in the early limb, we have characterized BMP action in sub-populations of cells from the E10.5 limb. Surprisingly, BMPs were found to inhibit cartilage formation in immature cells, while promoting cartilage formation in more mature cells. Transcriptional profiling coupled with qPCR and time course analyses revealed that the extent of induction of Gatas by BMPs was associated with its stimulatory versus inhibitory activity. Further, SOX9 activity was inhibited following over-expression of Gatas. / Medicine, Faculty of / Graduate

published_or_final_version / Biochemistry / Master / Master of Philosophy

Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisor: Mary C. Farach-Carson, Dept. of Biological Sciences. Includes bibliographical references.

published_or_final_version / Biochemistry / Master / Master of Philosophy

The purpose of this study was to develop a fibrin gel system capable of serving as a three dimensional scaffold for the chondrogenesis of rabbit bone marrow mesenchymal stem cells (BM-MSCs) and to examine the effect of two fibrinolytic inhibitors, aprotinin and aminohexanoic acid, on this system. Rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand white rabbits. After chondrogenic potential of BM-MSCs was verified by pellet culture, 2 x 106 cells were pelleted and suspended in fibrinogen (80mg/ml) and then mixed with equal parts of thrombin (5 IU/ml). The specimen were then divided into four groups: aprotinin control (with aprotinin); aprotinin + transforming growth factor (TGF-beta) (with aprotinin and TGF-beta 1); amino control (with aminohexanoic acid); and amino+TGF-beta (with aminohexanoic acid and TGF- beta1). Each of these groups was further divided into three groups depending on the concentration of the inhibitor. Both of the aprotinin groups received 0.0875, 0.175, or 0.35 TIU/ml of aprotinin and both of the aminohexanoic acid groups were supplemented with 2, 4, or 8 mg/ml of aminohexanoic acid. The gels were harvested and analyzed at 7, 14, and 21 days. All of the aprotinin+TGF-beta groups exhibited a significantly higher aggrecan gene expression than control groups whereas only the amino+TGF-â group treated with 8mg/ml was significantly higher than those of the control groups. In addition, the 0.0875 and 0.175 TIU/ml aprotinin+TGF-beta groups exhibited significantly higher levels of expression than the 2 and 4 mg/ml amino+TGF-beta groups. There were no significant differences among the different concentrations of aprotinin or aminohexanoic acid with or without the treatment of TGF-beta. Similar trends were also seen when the glycosaminoglycan (GAG) content was measured and analyzed. These findings suggest that fibrin gels are a suitable environment for the chondrogenesis of BM-MSCs and that aprotinin in combination with TGF-beta1 is the optimal condition for stimulating BM-MSCs to differentiate into chondrocytes.

published_or_final_version / Paediatrics / Master / Master of Philosophy

published_or_final_version / abstract / Mechanical Engineering / Master / Master of Philosophy