Introduction: Intervertebral disc (IVD) degeneration is suggested to begin from the nucleus pulposus (NP). Evidence from various studies highlights mesenchymal stem cells (MSC), in most cases using bone marrow derived MSC, as a potential stem cell source for NP regeneration. However MSC can be isolated from many sources with various characteristics. There are indications that fetal or close to fetal tissue sources contain MSC with relatively undifferentiated phenotype with respect to MSC from adult sources. Moreover, umbilical cord (C)-MSC may have better chondrogenic differentiation potential than bone marrow (B)-MSC. We hypothesize CMSC are different from BMSC, and more efficient than BMSC in stimulating NP regeneration. Methods: MSC were isolated from human bone marrow and umbilical cord with corresponding ethical approval. BMSC and CMSC were characterized for cell surface marker expression profile and differentiation potential.. RT-PCR of interest genes in NP cells isolated from scoliosis and degenerate discs was performed to search for NP degeneration indicators. Conditioned media (CM) was collected from confluent MSC monolayer, and used for stimulation of four batches of degenerated NP cells isolated from human degenerative intervertebral discs. Cell proliferation and cytotoxicity were assessed by MTT assay. Proteoglycan content were measured by DMMB assay. Gene expression of a series of degeneration related molecules including ACAN, SOX9, CDH2, CD55, KRT19, KRT18, FBLN1 and MGP, and fibrosis related molecules, including MMP12, HSP47, COL1A1, COL3A1 and FN1, of NP cells in MSC-CM were determined by real- time RT-PCR. All results were normalized to the control cells in basal medium. The expression of discogenic, chondrogenic and osteogenic markers on BMSC and CMSC were compared by RT-PCR. Results and Conclusion: CMSC were similar to BMSC and fulfilled the minimum criteria of MSC, however the expression of CD146, CD106 and Stro-1 was different, and BMSC had a spontaneous osteogenesis tendency while CMSC expressed chondrogenic marker even without TGF-beta stimulation. BMSC demonstrated a paracrine effect on modulating human degenerated NP cells towards a non-degenerative phenotype in stimulating cell proliferation, slightly enhancing proteoglycan production, upregulating KRT19 while downregulating MMP12. Compared with BMSC, a higher paracrine effect of CMSC was disclosed in modulating the phenotype of NP cells in all aspects tested, and an intrinsic higher expression on CMSC of ‘potential NP markers’, including KRT19, KRT18 and CD55, but lower expression of osteogenic markers, including RUNX2 and ALPL, was revealed, which indicate a higher potential of CMSC for future clinical application to treat IVD degeneration diseases. KRT19 and MMP12 were also confirmed to be the highest differentially expressed candidate genes between cultured scoliosis and degenerated human NP cells, indicating a high indicator potential of NP degeneration. Furthermore, a subpopulation was detected in the degenerated NP cells that possessed macrophage-like phenotype and activities, which may play a role in the pathogenesis of IVD degeneration. In conclusion, studies in this thesis highlighted CMSC as a superior source than BMSC for IVD repair. Further investigations into the active agents in the conditioned media and the signalling pathway may help to elucidate the mechanism of the effect. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Skeletal system plays a crucial role in our life. Skeletal diseases and disorders unlike cancer, are not fatal, but affect the quality of our life. Cell-based therapeutic strategies to generate targeted desired cell types for repair or replacement of damaged skeletal tissues are ideal regenerative medicines. Because of the heterogeneous cell types generated from embryonic and mesenchymal stem cells, the ability of progenitor population to differentiate into a target cell type appear to be a better alternative for tissue regeneration. Osteo-chondroprogenitors uniquely co-expressing Sox9 and Runx2 with dual differentiation potential to become chondrocytes and osteoblasts is a progenitor cell which is suitable for cell based therapy of bone disease. Therefore, developing effective strategies to generate sufficient quantities of osteo-chondroprogenitors are essential. Toward this, we took advantage of two lineage conversion approaches. The first strategy was to interrogate the ability of osteoblasts to be reprogrammed into induced pluripotent stem (iPS) cells and another one was to use defined transcription factors to induce chondrocyte lineage from skin fibroblasts. The selection of osteoblasts is based on the fact that it is originally derived from osteo-chondroprogenitor lineage and the stochastic events of iPS induction might revert osteoblasts first to their progenitor state before becoming pluripotent. The second approach is based on a previous report using three transcription factors (Sox9, Klf4 and c-Myc) to reprogramme skin fibroblasts into chondrocyte lineage. Our aim is to examine whether osteo-chondroprogenitors would be formed during the two reprogramming processes using Sox9-EGFP knock-in mice as a reporter. We reasoned that osteoblasts can be reprogrammed into iPS cells by four Yamanaka’s factors with pluripotency as shown by their ability to form teratomas and contribute to chimeric embryos. However base on the limitation of selector marker of osteo-chondroprogenitor we still cannot capture this progenitor during iPS reprogramming. And because of the pluripotency potential, pluripotent reprogramming approach also brings high risk of teratoma formation. Therefore our second objective was performed to examine whether osteo-chondroprogenitors would be formed during lineage reprogramming. Transient appearance of Sox9-EGFP/Runx2+ve cells was observed in the intermediate stage of over 14 days of chondrocyte lineage induction from skin fibroblasts by Sox9, klf4 and c-Myc. Cells expressing Sox9-EGFP/Runx2+ve showed typical molecular markers of osteo-chondroprogenitors. In vitro and in vivo differentiation assays demonstrated that Sox9-EGFP/Runx2+ve cells can differentiate predominantly into osteoblasts and chondrocytes. Taken together our data indicate that cells with osteo-chondrogenic potential could be generated by defined transcription factors-mediated reprogramming processes. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

When human mesenchymal stem cells (hMSCs) are cultured inside a 3D collagen meshwork, they become a potential tissue engineering bone graft alternative. However, the in vitro osteogenesis rate of hMSCs is slow, leading to a low mineral deposition. To enhance the osteogenic differentiation of hMSCs, low intensity pulsed ultrasound (LIPUS) was employed as an external stumulus. The present study demonstrated the feasibility of employing daily LIPUS exposure for enhancing osteogenesis in vitro. Exposure of seven consecutive days LIPUS, each of 30 minutes duration, did not affect the cell viability, and the organization of hMSCs within the collagen meshwork was not disturbed. The calcium deposition within the collagen meshwork was enhanced after seven days of exposure. The osteoinductivity was also upregulated at the early period of culture. In order to optimizing the enhancement effects of LIPUS, various ultrasound parameters, including intensity, exposure duration and exposure repetition were investigated. Results showed the LIPUS enhancement effects are dose dependent, LIPUS exposure should be longer than 10 minutes/day in order to elicit a significant effect. Calcium deposition was higher when LIPUS exposure was done twice per day instead of one. Although individual variation exists, optimal LIPUS intensity range was between 60-120 mW/cm2 ISATA (Spatial Average Temporal Average Intensity). The interaction mechanism between LIPUS and cells was also investigated. Microbubbles were added to the culture during LIPUS exposure to find out whether cavitation is involved in the interaction. Flow sensor primary cilium was also studied in order to verify that ultrasound is transduced through fluid flow. Results showed cavitation may not be a contributing factor to osteogenesis, and primary may be involved in the transduction of LIPUS stimulation. This study demonstrated that osteogenesis of hMSCs encapsulated in collagen constructs could be enhanced by LIPUS. The LIPUS parameters were also optimized. The LIPUS interaction pathways were also being better understood. This thesis study will be a paradigm for cellular mechanotransduction studies and put an important step forward for therapeutic ultrasound. / published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by persistent airway obstruction that is only partially reversible. It is the fourth leading cause of death and is predicted to be the third by 2030. The progression of the disease involves chronic inflammation, oxidative stress, excess protease activity, increased lung cell apoptosis and accelerated lung aging, but the exact pathogenesis is still unclear. The major cause of COPD is cigarette smoking(CS). Although COPD is associated with increasing social and economical burden, there have been few advances in pharmacological therapy of COPD. Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells which can be isolated from a broad range of sources including bone marrow (BM) and adipose tissue. Administration of BM-derivedMSCs (BM-MSC) or adipose tissue-derived MSCs was reported to attenuate CS-induced emphysema in murine models. Induced pluripotent stem cell-derived MSC (IPSC-MSC) are MSCs differentiated from induced pluripotent stem cells(IPSCs), which are pluripotent cells generated by somatic cell reprogramming in vitro. IPSC-MSCs have several advantages over BM-MSC, including more abundant sources and high capacity of doubling without loss of differentiation potency. A general exploration and comparison on the effects of human IPSC-MSC and BM-MSC treatments were carried out in a 56-day CS-exposed rat model. Compared to BM-MSC, IPSC-MSC showed a higher capacity to reside in lung tissue. The two treatments shared similar efficacy to attenuate CS-induced lung cell apoptosis, to restore CS-induced reduction of lungIL-10and to alleviate CS-induced elevation of systemic TGF-β1. In addition, IPSC-MSC was found to cause reduction in CS-induced elevation of systemic oxidative stress and reversal of CS-induced reduction of lung adiponectin. Furthermore, in order to understand the possible paracrine mechanism involved, human airway epithelial cells were treated with IPSC-MSC or BM-MSC-conditioned medium in a cell culture system in the presence of cigarette smoke medium (CSM). Potentiation rather than attenuation of CSM-induced release of pro-inflammatory cytokine IL-8, MCP-1 and IL-6 was observed with IPSC-MSC or BM-MSC conditioned medium. It is currently unknown whether cultured IPSC-MSCs or BM-MSCs will release pro-inflammatory mediators into the conditioned medium or not. In order to study CS-induced oxidative stress and inflammation in a short time frame, anacute (5-day) CS-exposed rat model was established in juvenile and adult groups. An age-dependent alteration of CS-induced oxidative and inflammatory responses was demonstrated in this model. In summary, our in vivo rat model provides a platform for elucidating the effects of stem cell treatment in CS-induced oxidative stress and inflammation, leading to lung damage. Our findings suggest that treatment of IPSC-MSC or BM-MSC might be able to slow down CS-induced disease progression, possibly through anti-oxidant, anti-inflammatory and anti-apoptotic properties. However, caution should be taken as our in vitro data revealed that conditioned medium from MSCs may provoke pro-inflammatory responses. Further studies on the regulation of the activity of MSCs in vivo will be needed before developing IPSC-MSC into cell therapies for COPD to halt the progression over time. / published_or_final_version / Medicine / Master / Master of Philosophy

Degenerative disc disease (DDD) is a medical condition whereby the intervertebral discs (IVD) of the human spine degenerates and may cause pain which significantly affects the quality of one掇 life. Its prevalence has sparked off much research in deciphering its causes and developing new treatments. Recently, attempts to treat this degenerative problem have turned to seeking answers from regenerative medicine. One approach is to deliver mesenchymal stem cells (MSCs) with or without carriers to the nucleus pulposus (NP) in degenerative disc to restore both its structural and functional properties. However, the optimal conditions and signals for inducing MSCs differentiation toward NP-like phenotype have not been identified. This work aimed to develop injectable microspheres with matrix microenvironment mimicking that of native NP tissue for MSCs delivery. Firstly, it was aimed to establish a collagen microencapsulation based 3D culture system for maintenance of the phenotype of nucleus pulposus cells (NPCs) and remodeling of the collagen matrix to one that was similar to the native NP. Secondly, it was aimed to optimize a decellularization protocol for complete removal of the encapsulated NPCs with minimal loss of remodeled extracellular matrix. Thirdly, it was aimed to investigate whether this acellular matrix produced by NPCs was inductive for MSCs discogenic differentiation. Finally, it was aimed to evaluate the efficacy of the MSC-seeded acellular matrix microspheres in a pilot rabbit disc degeneration model. It demonstrated that NPCs maintained their phenotype, survived within the collagen microspheres and produced NP-like ECM such as glycosaminoglycan (GAG) and collagen type II. GAG production of NPCs was found to positively correlate with the dosage of TGF-? within a short period, initial collagen concentration and cell seeding density. An optimized decellularization protocol with 50mM SB-10, 0.6mM SB-16 and 0.14% Triton X-200 was established to completely remove the encapsulated NPCs with partial retention of the GAG-rich matrix. The decellularized microspheres were able to be repopulated with human MSCs (hMSCs) or rabbit MSCs (rMSCs). Within the NPC-derived acellular matrix, the repopulated hMSCs were able to partially exhibit NPC phenotype with upregulated expression of a few NPC markers and NP-like ECM according to histological, biochemical, immunohistological and real-time PCR results. In the pilot in vivo evaluation study, preliminary results showed that intra-discal injection of MSCs reseeded acellular NPC-matrix microspheres maintained a better water content than the control MSC-microspheres without the NPC-derived acellular matrix. This work reconstituted in vitro a NP-like 3D matrix and provided preliminary evidence on discogenic differentiation of MSCs in such a matrix environment. This work laid down the foundation to future development of stem cell-based therapies for DDD. Further studies should focus on deciphering the soluble and insoluble composition of such a NP-like matrix environment and understanding the molecular mechanism of the cell-matrix interactions involved. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Heart failure (HF) following myocardial infarction (MI) is the leading cause of mortality and morbidity worldwide. Existing medical and interventional therapies can only reduce the cardiomyocytes (CMs) lost during MI. They are unable to replenish the permanent loss of CMs and this contributes to progressive pathological left ventricular (LV) remodeling and HF. Cell-based therapies using adult stem cells or embryonic stem cells (ESCs) and their cardiac derivatives have frequently been explored as a potential therapeutic approach to restore cardiac function in HF. The objectives of this thesis are to evaluate the efficacy and safety of different approaches of stem cell based therapy to improve cardiac function using small and large animal MI models. In Chapter 3, we studied the functional consequences of direct intramyocardial transplantation of ESCs and ESC-derived cardiomyocytes (ESC-CMs) in a murine model of acute MI. LV ejection fraction (LVEF) and maximal positive or negative pressure derivative (dP/dt) improved 4 weeks after transplantation of either ESCs or ESC-CMs. Nevertheless there was a higher incidence of inducible ventricular tachyarrhythmia (VT) and higher mortality in animals transplanted with ESC-CMs than those with ESCs. At a single cell level, ESC-CMs exhibited immature electrophysiological properties such as depolarized resting membrane potential (RMP), longer action potential duration (APD) and automaticity. In Chapter 4, we tested the hypothesis that genetic modification of these immature electrophysiological properties of ESC-CMs by overexpression of Kir2.1 gene encoding the ion channels for IK1, may alleviate the pro-arrhythmic risk. In this study, Kir2.1 channels expression could be controlled with the administration of doxycycline (DOX). The DOX-treated ESC-CMs were more mature with hyperpolarized RMP and shorter APD than their counterparts without DOX treatment. A similar improvement in LV systolic function was observed 4 weeks after both DOX treated and untreated ESCCMs transplantation, although those animals transplanted with DOX-treated ESC-CMs had a significantly lower incidence of spontaneous and inducible VT. Histological analysis in both studies suggested that the major mechanisms of improvement in cardiac function were related to angiogenesis and low apoptosis rate of native cardiomyocytes mediated via paracrine effects. Importantly, very limited retention of ESC-CMs was observed 4 weeks after transplantation. Cell-based patches that use different bioengineering techniques have been proposed to improve cell retention and survival following transplantation. In Chapter 5, the efficacy of a passive epicardial patch was tested in a chronic large animal MI model with HF created with catheter-based coronary embolization. The implantation of an epicardical patch over the infarcted LV region was performed 8 weeks after MI in pigs with impaired LVEF. At week 20, pigs implanted with epicardical patches had significantly thicker LV wall thickness at the infarction sites, smaller LV dilation and better LV systolic function compared with control animals. The expression of MMP-9 was significant lower in the epicardical patch group at the peri-infarct zones. These findings suggested that a passive epicardial patch can improve LV function in HF and provides important proof-of-principle data to support its use as a platform for delivery of cell-based therapies after MI. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The growth plate is a cartilaginous structure located at the proximal and distal ends of immature long bones, which contributes to longitudinal growth through the process of endochondral ossification. Cartilage has a limited ability to regenerate and in children, injury to the the growth plate can result in limb length discrepancies and angular deformity, due to formation of a bone bridge at the damaged site which disturbs structure and function of the growth plate. Current treatments of the abnormalities arising from growth plate arrest involve surgical correction once the deformities have manifested. To date, there is no biological based therapy for the repair of injured/damaged growth plate cartilage. Mesenchymal stem cells (MSC) are self renewable mulitpotential progenitor cells with the capacity to differentiate toward the chondrogenic lineage. Since their discovery, significant interest has been generated in the potential application of these cells for cartilage regeneration. In this study, the ability of autologous bone marrow mesenchymal stem cells to regenerate growth plate cartilage in a sheep model was examined. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1330837 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The growth plate is a cartilaginous structure located at the proximal and distal ends of immature long bones, which contributes to longitudinal growth through the process of endochondral ossification. Cartilage has a limited ability to regenerate and in children, injury to the the growth plate can result in limb length discrepancies and angular deformity, due to formation of a bone bridge at the damaged site which disturbs structure and function of the growth plate. Current treatments of the abnormalities arising from growth plate arrest involve surgical correction once the deformities have manifested. To date, there is no biological based therapy for the repair of injured/damaged growth plate cartilage. Mesenchymal stem cells (MSC) are self renewable mulitpotential progenitor cells with the capacity to differentiate toward the chondrogenic lineage. Since their discovery, significant interest has been generated in the potential application of these cells for cartilage regeneration. In this study, the ability of autologous bone marrow mesenchymal stem cells to regenerate growth plate cartilage in a sheep model was examined. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1330837 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Thesis (MPhil)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The progress that has been made over the last decade in the field of medical technology, has made it possible to treat medical conditions today, that was considered incurable before. In the medical field there are three milestones in particular which has led to important new discoveries. These are the charting of the human genome, the development of cloning techniques, and the discovery that stem cells could be used in the treatment of a great number of illnesses, as well as the regeneration of sick or damaged tissue. The development of these therapies are, however, morally controversial. The main reason for this is the fact that in most cases, these techniques involve research on, or the use of, embryonic cells. The reason why many people believe that it is morally problematic to use embryo tissue for research and/or therapeutic purposes, is because embryonic cells have the potential to develop into fully independent human persons. It is, however, also this characteristic of these cells which makes them so suitable for use in stem cell therapies: Because certain types of stem cells, especially stem cells that are found in embryos in an early stage of development, have not yet differentiated into specific cell types, they can be used to repair any cell type in a person with a compatible tissue type. The reason for this is that undifferentiated stem cells have the potential to change into any cell type found in the human body. The question that arises when a decision about the moral acceptability of these therapies has to be made is whether one can say that an existing person who happens to be ill, has a higher moral standing than an embryo. The only way in which the use of embryo tissue could be morally justified, would thus be if it could be proved that the moral standing of an embryo is not equal to the moral standing of a person. The other important consideration that has to be taken into account when the moral acceptability of these therapies has to be taken into account is the fact that it is possible to harvest stem cells from a number of sources. Some of these sources of stem cells are less controversial than others. The discussion of the moral problems arising from the use of embryo tissue for therapeutic purposes, would thus, in this thesis, focus to a large extent on determining what the moral status of the embryo might be. The different positions with respect to the moral standing of the embryo will be discussed in the light of arguments for, as well as against the use of embryo tissue for therapeutic purposes. An explanation will also be given of therapies for which the use of embryo tissue might be needed, at present as well as in the future. The potential gains for people suffering from certain conditions, could possibly serve as a justification for destroying embryos for therapeutic uses. The main purpose of this thesis is to be able to give morally justifiable reasons for the therapeutic use of embryo tissue. The specific conditions that would have to be met to make these therapies morally justifiable will also be explained. / AFRIKAANSE OPSOMMING: Die vordering wat die afgelope dekade gemaak is ten opsigte van mediese tegnologie, het tot gevolg gehad dat dit vandag moontlik is om siektetoestande te behandel wat voorheen as ongeneeslik beskou is. Daar is veral drie belangrike mylpale wat in die mediese veld tot belangrike nuwe ontdekkings gelei het, naamlik die kartering van die menslike genoom, die ontwikkeling van kloningstegnieke, en die ontdekking dat stamselle gebruik kan word vir die behandeling van 'n groot aantal siektetoestande, asook die regenereering van siek of beskadigde weefsel. Die ontwikkeling van hierdie terapieë is egter moreel kontroversieel. Die rede hiervoor is dat hierdie tegnieke in die meeste gevalle navorsing op, of die gebruik van embrionale selle behels. Die rede waarom baie mense van mening is dat dit moreel problematies is om embrionale weefsel vir navorsing enlofterapeutiese doeleindes te gebruik, is omdat embrionale selle die potensiaal het om te ontwikkel tot volwaardige persone. Dit is egter ook hierdie eienskap van hierdie selle wat hulle so geskik maak vir terapeutiese doeleindes: Omdat sekere tipes stamselle, veral stamselle wat verkry word van embrio's wat in 'n vroeë stadium van ontwikkeling verkeer, nog nie gedifferensieer is wat seltipe betrefnie, kan hulle gebruik word om enige seltipe in die liggaam van 'n persoon met 'n verenigbare weefseltipe te herstel. Die rede hiervoor is dat ongedifferensieerde stamselle die potensiaal het om in enige seltipe wat in die menslike liggaam voorkom, te verander. Die vraag wat ontstaan wanneer daar besluit moet word oor die morele aanvaarbaarheid van hierdie terapieë, is of daar gesê kan word dat 'n reeds bestaande persoon wat siek is, 'n hoër morele status sou hê as 'n embrio. Die enigste manier waarop die gebruik van embrionale selle moreel regverdigbaar sou wees, sou dus wees indien daar bewys kan word dat die morele status van 'n embrio nie gelykstaande is aan die morele status van 'n persoon nie. Die ander belangrike oorweging wat in ag geneem moet word wanneer die morele aanvaarbaarheid van hierdie terapieë beoordeel moet word, is dat dit moontlik is om stamselle te verkry uit 'n verskeidenheid bronne. Sommige van hierdie bronne van stamselle is moreel minder kontroversieel as ander. Die bespreking van die morele problematiek rondom die gebruik van embrionale weefsel VIr terapeutiese doeleindes in hierdie tesis, sal dus tot 'n groot mate fokus op die bepaling van die morele status van die embrio. Die verskillende standpunte oor die morele status van die embrio sal bespreek word in die lig van argumente vir, sowel as teen die gebruik van embrionale weefsel vir terapeutiese doeleindes. Daar salook 'n verduideliking gegee word van watter tipe terapieë waarvoor die gebruik van embrionale weefsel nodig sou wees, tans en in die toekoms moontlik sou wees. Die potensiële baat wat siek persone uit hierdie terapieë sou kon vind, sou moontlik ook as 'n regverdiging vir die vernietiging van embrio's vir terapeutiese doeleindes kon dien. Die uiteindelike doel van hierdie tesis is om moreel regverdigbare redes te kan gee vir die terapeutiese gebruik van embrionale weefsel. Die spesifieke voorwaardes wat nagekom sou moes word om hierdie terapieë moreel regverdigbaar te maak, salook verduidelik word.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy