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Cartilage is present throughout the human body and works as supporting connective tissue. It assists bones in supporting and connecting the body but differs in structural aspects as bones are more organized at the micro-level than cartilage. Bones can be regenerated if broken but cartilage has less tendency to regenerate after trauma or disease such as osteoarthritis which supports the concept of tissue engineering using stem cells to produce artificial cartilage implants to resurface damaged joints. Mesenchymal stem cells (MSCs) and human-induced pluripotent stem cells (hiPSCs) were used in the current study to address this challenge. The chondrogenic capacity of hiPSC cell lines C19 and OC3 in response to members of the transforming growth factor beta superfamily to derive zonal chondrocytes was investigated. The pellet culture system and 3D scaffold based tissue engineering were used to investigate the impact of transforming growth factor beta in combination with bone morphogenic proteins (BMPs) including BMP2, BMP4, and BMP7 on the constructs phenotype. Chondrogenic markers including type II, type I, and type X collagens, lubricin, aggrecan and Sox9 have been used to evaluate the quality of chondrogenic outcome by quantifying gene expression and histological analysis. The results demonstrated the derivation of chondrocytes of different phenotypes. These phenotypes were mapped to zonal chondrocytes (superficial, deep and hypertrophic). The results demonstrated the possibility of obtaining superficial and hypertrophic chondrocytes but not deep zone chondrocytes. Identical experiments were conducted for comparison using bone marrow mesenchymal stem cells (MSCs). Chondrocytes derived from MSCs treated with various combinations of the transforming growth factor beta superfamily members revealed limited mapping to a particular zonal phenotype. An attempt to ameliorate the phenotypes was sought using 3D bioprinting with limited success. Finally, a cellular reprogramming method was utilised to generate chondrocytes with the aim of controlling their fate and phonotype. Mesenchymal stem cells (MSCs) were treated with different cocktails of small molecules guided by a bioinformatics-based method for cell conversion (Mogrify). Several cocktails targeting the predicted transcription factors were used to drive the conversion process. One cocktail appeared to have limited capacity to upregulate some chondrogenic markers in 2D and 3D cultures. In conclusion, the issues of cartilage related struggles are spreading worldwide and needs to get a permanent solution that can be helpful for every patient. For this purpose the study was conducted to get a better understanding of in vitro chondrogenesis. The study concluded the generation of hypertrophic and superficial chondrocytes through pellet culture system by using BMPs. The aid of 3D bioprinting was also limited because of the limitation in zonal phenotypes. The cocktails of predicted transcriptional factors helped in inducing chondrogenesis. These are not the ideal results but can help the future researchers to develop better strategies and applicable results.

The main aim of this project was to determine the neurotrophic actions of glial cell line-derived neurotrophic factor (GDNF) and neurturin, two novel members of the transforming growth factor-beta superfamily of proteins, on neurons from the peripheral nervous system and to identify their receptors. It is found that GDNF promotes the survival of multiple populations of chicken sensory and autonomic neurons in culture throughout development. Whereas sympathetic, parasympathetic and propioceptive neurons become less responsive to GDNF with age, enteroceptive and sensory cutaneous neurons become more responsive to GDNF. GDNF mRNA is expressed in the tissues innervated by these neurons, and developmental changes in its expression in several tissues mirror the changing responses of the innervating neurons to GDNF. These results have changed the previous notion that GDNF is a highly specific neurotrophic factor for motoneurons and dopaminergic neurons. It is shown that neurturin, which is structurally related to GDNF, also promotes the in vitro survival of embryonic chicken sensory and autonomic neurons. Thus, GDNF and neurturin compose a novel subfamily of homologous neurotrophic factors with a similar pattern of activity. The cloning of chicken GDNF receptor-α (GDNFR-α) and a novel receptor termed neurturin receptor-α (NTNR-α) is reported. GDNFR-α and NTNR-α are homologous receptors linked to the membrane via a glycosyl- phosphatidylinositol linkage. It is shown that ectopic co-expression in neurons of GDNFR-α with RET (rearranged during transfection), a transmembrane receptor tyrosine kinase, confers a survival response to GDNF, but not neurturin, and that co-expression of NTNR-α with RET confers a survival response to neurturin, but not GDNF. GDNFR-α and NTNR-α mRNAs are widely expressed in the nervous system, including GDNF and neurturin responsive neurons, and in non-neuronal tissues. These findings indicate that GDNF and neurturin promote neuronal survival by signalling via similar multicomponent receptors that consist of a common transducing receptor tyrosine kinase and a member of a newly emerging family of glycosyl-phosphatidylinositol-linked receptors that confer ligand- specificity.

Bone Morphogenetic Proteins (BMPs) belong to the Transforming Growth Factor Beta superfamily of growth factors. While BMP signaling has been shown to induce skeletogenic differentiation of mesenchymal stem cells and be involved in the formation of ectopic bone and ossification, BMPs also are involved in the differentiation of many other tissues including neurogenic tissues. Prior studies from our laboratory showed that BMP-2 induction of chondrogenic differentiation of the C3H10T1/2 murine mesenchymal stem cell line was associated with increased oxidative metabolism. This study was performed to test the hypothesis that BMP-2 promotes increased oxidative metabolism during the differentiation of other types of cells. Using the neurogenic cell line, SH-SY5Y, this study examined whether cellular differentiation induced by BMP-2 also was associated with increased oxidative phosphorylation in non-mesenchymal stem cells. SH-SY5Y cells were grown in growth medium (DMEM/F12 with 10% FBS, 1% Penicillin/Streptomycin, L-glutamine, and non-essential amino acids). Cells were plated appropriately at differing seeding densities (Day 0), treated one time with ± 200ng/mL BMP-2 two days after plating and analyzed on Day 4. Oxidative metabolism was measured using a Seahorse XF Analyzer that measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). BMP-2 induction of neural cell differentiation was assayed by quantifying dendrite communication between neurons and decreased proliferative capacity as assessed by overall culture DNA contents. Mitochondrial density after BMP-2 treatment was examined using vital mitochondrial labeling. Groups treated with BMP-2 contained significantly less DNA content than control groups (p=0.006). BMP-2 treated cells had on average more dendritic interactions with one or two and more processes than control groups (p=0.396; p=0.872), while there were a larger percentage of cells not treated with BMP-2 that had zero dendritic interactions (p=0.470). All parameters of oxidative metabolism were increased in cells treated with BMP-2. More specifically, basal respiration and ATP production were significantly increased in BMP-2 treated cells (p=0.031; p=0.010). SH-SY5Y cells were significantly affected by BMP-2 treatment in both DNA content and oxidative phosphorylation. The diminished DNA content with BMP-2 treatment is consistent with the known decrease in cellular proliferation that is associated with neural cell differentiation. While there were small increases in dendritic interactions these were not significant and are inclusive for demonstrating BMP-2’s effect on neural differentiation. Upregulation of oxidative phosphorylation indicated after BMP-2 treatment was validated by the substantial increases in metabolic parameters associated with BMP-2 treatment. Through the utilization of the neural cell line, SH-SY5Y, this research suggests a more generalized functionality of BMP-2 in upregulation of oxidative metabolism as well as differentiation non-specific to bone and chondrocyte cell lineage.

Normally growth suppressive in epithelial cells, the transforming growth factor beta superfamily has been shown to be disrupted in many types of cancers. Bone Morphogenetic Protein (BMP), a member of the transforming growth factor beta superfamily has been found to be intact and growth suppressive in a variety of cancers, but it has been found to be targeted for mutation in the colon cancer predisposition syndrome Juvenile Polyposis, and little is known about its role in colon cancer. In this dissertation, we attempt to understand what role the BMP signaling pathway plays in colon cancer. We aimed to do this by: a) determining if the BMP pathway is intact in microsatellite stable and unstable colon cancer cell lines and tissues, b) determining if the Ras pathway affects BMP signaling, and c) examining SMAD4-independent means of BMP signaling. We found that while the BMP pathway was intact in many of the colon cancer cell lines examined, the RAS pathway interfered with transmission of SMAD signaling to the nucleus. BMP2 was shown to increase the stability of p21, and the RAS/ERK pathway acts to dull the growth suppressiveness and decrease the stability of p21. Additionally, we found that in the absence of SMAD4, which is targeted for mutation in Juvenile Polyposis and some types of colon cancers, that BMP treatment becomes growth proliferative over time by decreasing cellular levels of PTEN through the RAS/ERK pathway. Our results suggest that while the BMP pathway is often intact in colon cancer cell lines, its effects are modulated by other signaling pathways

Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-beta superfamily that when exogenously administrated exerts a potent trophic action on dopaminergic (DA) cells. Although we know a lot about its signalling mechanisms and pharmacological effects, physiological actions of GDNF on the adult brain remain unclear. Here, we have used morphological and molecular techniques, and an experimental model of Parkinson's disease in rats, to investigate whether GDNF constitutively expressed in the adult mesostriatal system plays a neuroprotective role on midbrain DA cells. We found that although all midbrain DA cells express both receptor components of GDNF (GFRalpha1 and Ret), those in the ventral tegmental area (VTA) and rostromedial substantia nigra (SNrm) also contain GDNF but not GDNFmRNA. The levels of GDNFmRNA are significantly higher in the ventral striatum (vSt), the target region of VTA and SNrm cells, than in the dorsal striatum (dSt), the target region of DA cells in the caudoventral substantia nigra (SNcv). After fluoro-gold injection in striatum, VTA and SNrm DA cells show triple labelling for tyrosine hydroxylase, GDNF and fluoro-gold, and after colchicine injection in the lateral ventricle, they become GDNF-immunonegative, suggesting that GDNF in DA somata comes from their striatal target. As DA cells in VTA and SNrm are more resistant than those in SNcv to intracerebroventricular injection of 6-OHDA, as occurs in Parkinson's disease, we can suggest that the fact that they project to vSt, where GDNF expression is significantly higher than in the dSt, is a neuroprotective factor involved in the differential vulnerability of midbrain DA neurons.

Testosterone is known to act differentially on skeletal muscle from different regions. Two genes likely to mediate the testosterone effect are insulin-like growth factor-I (IGF-I), an important growth regulator acting in an autocrine and paracrine way, and androgen receptor (AR), as receptor density could account for differential muscle growth. Another muscle-specific gene that may play a role in differential muscle growth is myostatin, a member of the transforming growth factor-beta superfamily, shown to be a negative regulator of skeletal muscle mass. The objective of this study was to quantify and compare the expression of these three genes in two different skeletal muscles in sheep. East Friesian x Dorset sired ram lambs born from Dorset ewes were used in a 2 x 4 factorial experiment. Eighteen sets of twins were assigned to four age groups corresponding to 77, 105, 133 and 161 days of age and one individual from each set was castrated at birth. Total RNA was extracted from samples of semitendinosus (ST) and splenius (SP) muscles collected at the time of slaughter. Insulin-like growth factor I mRNA was measured using competitive reverse-transcription-polymerase chain reaction (RT-PCR). Androgen receptor and MSTN mRNA were measured by ribonuclease protection assay (RPA) with standard curves. Splenius muscle weight was greater than semitendinosus muscle weight in rams compared with wethers at 105, 133 and 161 days (p = 0.05, p = 0.04 and p = 0.02). The difference in IGF-I mRNA levels between the two muscles was higher in rams than in wethers at 133 and 161 days (p < 0.05) and the difference in AR mRNA levels was higher in rams than in wethers at 105, 133 and 161 days (p < 0.05), with higher expression in the SP. No difference was found in the MSTN mRNA level between the two muscles in rams and wethers at any age. These results show that locally produced IGF-I and the regulation of AR expression are important for sexually dimorphic muscle growth patterns.

Myostatin is a new member of the transforming growth factor-beta superfamily of secreted growth and differentiation factors. Myostatin is almost exclusively expressed in skeletal muscle and act as a negative regulator of skeletal muscle growth. Because anti-myostatin antibodies will be useful in investigating the mechanism of action of myostatin, an experiment was designed to produce polyclonal anti-chicken-myostatin antibodies. A PCR amplified prepro-myostatin composed of the prodomain plus mature myostatin and a C- terminal fragment containing only the mature myostatin were cloned into an expression vector, and these proteins were expressed in E. coli. The recombinant proteins were fractionated by SDS-PAGE, then the myostatin bands were cut out and electro-eluted to obtain purified myostatins. Rabbits were immunized with the purified myostatins to produce polyclonal anti-myostatin antibodies. IgGs were separated from the rabbit sera using Protein A affinity chromatography. Antibody binding characteristics were then examined using Western blot analysis of various chicken tissues. Both polyclonal antibodies demonstrated a strong affinity to both form of recombinant myostatin in Western blot analysis. The anti-mature myostatin antibody showed binding affinity to proteins at 50, 30 and 20 kD in skeletal muscle and liver. No specific binding in skeletal muscle was demonstrated by the anti-mature myostatin antibody, suggesting that the above proteins are non-myostatin proteins that have affinity to anti-mature myostatin antibody. In contrast, the anti-prepro-myostatin antibody showed affinity to a 37 kD band only in skeletal muscle in addition to the 50, 30 and 20 kD bands. Since the molecular weight of the myostatin prodomain is known to be close to 37 kD, it is postulated that the 37 kD protein specifically recognized by the antiprepro- myostatin in skeletal muscles is likely to be a myostatin prodomain with further validation of its binding specificity, the anti-prepro-myostatin antibody generated in this study will potentially be useful in investigating the mechanism of action of myostatin.