D.Tech. (Biomedical Technology) / Stem cells are defined as undifferentiated cells that can proliferate and have the capacity of both self-renewal and differentiation to one or more types of specialised cells (Bishop et al., 2002). The two types of stem cells are embryonic and adult stem cells. Adult stem cells have been isolated from adipose tissue in abundance and with ease (Mvula et al., 2010) and these cells have been differentiated into smooth muscle cells (SMCs) with the enhancement of low intensity laser irradiation and the growth factors (de Villiers et al., 2011). Smooth muscles play an important role in diseases like cancer, hypertension, asthma and others (Rodriguez et al., 2006). Studies have shown that low intensity laser irradiation (LILI) can increase proliferation of cells, cellular attachment, differentiation and production of transforming growth factor-beta 1 (TGF-β1) in cells indicating that in vitro LILI can modulate the activity of cells and tissues (Khadra et al., 2005). Further studies have also discovered that LILI enhances wound healing (Fiszerman and Markmann, 2000). LILI has been successfully used for pain attenuation and to induce wound healing in non-healing defects (Hawkins and Abrahamse, 2005). LILI has been shown to increase viability and proliferation of adipose derived stem cells (ADSCs) (Mvula et al., 2008 and Mvula et al., 2010). Growth factors such as retinoic acids (RA) have been shown to have major influences on cells. They are involved specifically in apoptosis, cell proliferation, differentiation and maturation (Duong and Rochette, 2011; Gudas and Wagner, 2011). Co-culturing is used to achieve several cellular processes including proliferation, differentiation and migration (Kim et al., 2012). When two types of cells are cultured together, they are exposed to a number of complex environmental factors such as cytokines, extracellular matrix components, cell interactions, mechanical stimuli, signalling transcriptional pathways and transcriptional factors such as growth factors. v These factors are able to affect migration, proliferation and differentiation of one cell type into another (Zhang et al., 2012). The aim of this study was to investigate the differentiation potential of ADSCs when co-cultured with (SMCs) and to determine the role of LILI on the co-cultured cells. Short and long term biological effects were monitored on these cells following exposure to LILI and addition of growth factors. The study used commercial and isolated human ADSCs and SMCs (SKUT-1) cells. After growing cells to semiconfluency for ADSCs and confluency for SMCs, they were co-cultured in a ratio of 1:1 using the established methods supplemented with and without growth factors (TGF-β1and RA) and then exposed to LILI. The cellular morphology, viability and proliferation activities of the irradiated cells were then assessed using direct inverted and differential interference contrast microscopy (DIC), trypan blue test, adenosine triphosphate luminescence, optical density analysis, and carboxyfluorescein diacetate succinimdyl ester (CFSE) methods. In particular the expression of the specific markers of both ADSCs, β1 Integrin (CD29) and Thy-1 (CD90) and SMCs, Myosin Heavy Chain (MHC) were investigated through immunoflourescent microscopy and flow cytometric analysis. Up and down regulation of genes involved in the human mesenchymal stem cell array were analysed through Reverse Transcriptase Polymerase Chain Reaction (RTPCR)...

Cardiovascular disease is a leading cause of death worldwide and becomes increasingly prevalent in the elderly population. Independent of etiopathogenesis, heart failure (HF) is the final common stage of numerous heart diseases. Cardiac stem cell (CSC) therapy has emerged as a promising cell-based strategy for treatment of HF. However, cell replacement is not able to fully restore a structurally damaged myocardium in advanced and end-stage HF. The objective of this project was to test the following hypotheses: that a bioengineered heart extracellular matrix (ECM) with preserved intact geometric structure could be generated using decellularization by coronary perfusion; and that autologous CSCs, to repopulate this ECM, could be isolated and expanded from the adult heart, with the caveat that autologous CSCs are depleted and impaired by both aging and chronic dilated cardiomyopathy. This will help to develop a possible therapeutic approach for advanced HF, using a combination of CSCs and engineering technique. Resident CSCs were isolated from explant-derived cells (EDCs) and expanded into cardiosphere-derived cells (CDCs) via cardiosphere formation. The CDCs expressed CSC markers (c-kit and Sca-1), pluripotent markers (Oct3/4 and Sox2), and the cardiac lineage-committed marker (Nkx2.5), and showed clonal expansion, self-renewal, and cardiomyogenic potential in vitro. In tissue engineering experiments, CDCs survived and proliferated within biomaterial alginate scaffolds for up to 7 weeks. An engineered bioartificial ECM scaffold was successfully produced from a whole rat heart using retrograde coronary perfusion and possessed an intact 3D architecture with functionally perfusable vascular network. Compared with ventricles, cultures derived from atria produced significantly higher number of c-kit+ and Sca-1+ CSCs (c-kit: 13% vs. 3.4%; Sca-1: 82% vs. 53%, respectively) and exhibited greater clonogenic and proliferative capacity. CDCs could be grown from young and aged mice, but the yield of CSCs significantly declined with age, as did cell migration and differentiation potential. In comparison to wild-type mice, atrial-CDCs from dystrophic mice showed no significant differences in CSC subpopulations and characteristics, despite confirmation of cardiac dysfunction using MRI. In conclusion, CDCs could be considered to be a viable cell candidate for cardiac therapy and may be used to treat HF at various stages, in combination with myocardial tissue engineering.

Breast cancer remains the leading cause of cancer-related death in women worldwide. Furthermore, it has been demonstrated that the treatment-resistant ER-PR-HER2/neu- sub-type is more common among women of African descent, necessitating the search for novel chemotherapies for this form of the disease. The secondary metabolites produced by marine algae represent a rich source of structurally unique compounds with chemotherapeutic potential, particularly in South Africa, whose oceans allegedly host 15 % of the total number of species in the world. Indeed, a recent study reported the isolation of a range of novel compounds from South African red and brown algae of the Plocamium, Portiera and Sargassum genera which displayed cytotoxicity against oesophageal cancer cells in vitro. The molecular mechanisms mediating this toxicity were unknown, as was the effect of these and similar compounds on metastatic ER-PR-HER2/neu- breast cancer cell lines or breast cancer stem cells. The current study aimed to address these questions by screening a library of twenty-two novel marine algal compounds for the ability to inhibit MDA-MB-231 and Hs578T breast cancer cells, while having no adverse effects on non-cancerous MCF12A breast epithelial cells. While twelve of these were toxic in the micromolar range against breast cancer cells, only the polyhalogenated monoterpenes RU004 and RU007, and the tetraprenylated quinone sargaquinoic acid (SQA) were identified as hit compounds based on the criteria that their cytotoxicity was specific to breast cancer and not healthy breast cells in vitro. On the other hand, the halogenated monoterpene RU015 was found to be highly toxic to both breast cancer and non-cancerous breast cell lines, while the halogenated monoterpene stereoisomers RU017 and RU018 were non-toxic to either of these cell lines. The mode of action of RU004, RU007, RU015 and SQA, together with the previously characterized carotenoid fucoxanthin (FXN), was assessed in terms of the type of cell death induced and the effect on cell cycle distribution of these compounds. Flow cytometric analysis of the extent of Hoescht 33342 and propidium iodide staining along with PARP cleavage studies suggested that SQA induced apoptosis in MDA-MB-231 cells. On the other hand, the highly toxic compound RU015 appeared to induce necrosis as evidenced by 50 kDa PARP cleavage product in MDA-MB-231 cells. The flow cytometry profiles of MDA-MB-231 and Hst578T cells treated with the hit compounds RU004 and RU007 were suggestive of the induction of apoptosis by these compounds. Cell cycle analysis by flow cytometry with propidium iodide staining revealed that both SQA and FXN induced G0-G1 arrest together with an increase in the apoptotic sub-G0 population, which agreed with previous reports in the literature. The molecular mechanism of action of SQA and FXN were further investigated by the identification of specific signal transducer molecules involved in mediating their anti-cancer activities. SQA was found to require the activity of numerous caspases, including caspase-3, -6, -8, -9, -10 and -13, for its cytotoxicity and was demonstrated to decrease the level of the antiapoptotic protein Bcl-2. On the other hand, FXN was shown to require caspase-1, -2, -3, -9 and - 10 for its toxicity. This, together with the ability to decrease the levels of Bcl-2, pointed to the involvement of the intrinsic pathway in particular in mediating the activity of FXN. The screening of algal compounds against non-cancerous breast epithelial cells carried out in this study, together with the investigation into their mechanisms of action, represent one of the few reports in which characterization of algal metabolites goes beyond the initial cytotoxicity assays. Finally, in order to assess the potential anti-cancer stem cell activity of the marine algal compounds, a subset of these was screened using a mammosphere assay technique developed in this study. The cancer stem cell (CSC) theory proposes that cancers arise from and are maintained by a specific subpopulation of cells able to undergo asymmetric cell division and termed CSCs. These CSCs are capable of anchorage-independent growth in serum-free culture conditions, such as those in the mammosphere assay. Using this assay, the novel halogenated monoterpene stereoisomers RU017 and RU018 were demonstrated to possess putative anti- CSC activity as evidenced by their ability to completely eliminate mammosphere formation in vitro. Furthermore, since RU017 and RU018 were non-toxic to both breast cancer and healthy breast cells, it appeared that the activity of the compounds was potentially specific to the CSCs. The results require further validation, but represent the first report of selective anti-CSC activity.

Since Yamanaka and Takahashi first described the isolation of induced pluripotent stem cells (iPSCs) in 2006, researchers have invested a vast amount of time and resources into trying to understand the process of reprogramming. However, the exact mechanisms underlying the induction of somatic cells to pluripotency is still incompletely understood. With this in mind, a screening approach was undertaken to identify shRNA that enhance the reprogramming process. A retrovirus based system was used to knock down candidate genes during reprogramming of mouse embryonic fibroblasts (MEF) containing doxycycline-inducible reprogramming factors and a Nanog-GFP reporter, which is activated when cells become iPSCs. The initial round of screening with over 150 shRNA vectors successfully identified several shRNAs that enhance reprogramming. One of these shRNA vectors exhibited both faster reprogramming kinetics as determined by activation of the Nanog-GFP reporter 2 to 3 days earlier and increased reprogramming efficiency giving rise to >5 fold more GFP+ colonies when compared with a control. Cell surface marker analysis with flow cytometry demonstrated that changes in CD44 and ICAM1 expression, which occur preceding Nanog-GFP expression, were also accelerated. Validation of this shRNA determined that the enhanced reprogramming phenotype is the result of an unknown off-target effect. Microarray and RNA-sequencing analysis was carried out to identify the off target gene with a view to investigate the functional importance of this knock down and its role in establishing the pluripotency transcriptional network during reprogramming.

Abstract Bone marrow mesenchymal stromal cells (MSCs) and mononuclear cells (BM-MNCs) have shown great therapeutic potential in various clinical settings. Although intravascular transplantation of the cells constitutes the optimal delivery route, massive pulmonary entrapment, with the threat of embolization, remains a major obstacle for using this type of therapy. Because pulmonary entrapment is at least partially mediated by adhesion molecules, cell surface modification could enhance pulmonary passage. We used a porcine model of allogeneic MSC and autologous BM-MNC transplantation and radionuclide labelling to track the cells. The role of the transplantation route on lung entrapment, biodistribution, safety and BM-MNC targeting to the injured brain was studied. Effects of pronase detachment on the lung passage of MSCs were studied in porcine and murine models; a rat model of acute limb injury was used to further evaluate tissue targeting. Treatment with pronase to detach cell surface molecules and the effect on stem cell potential was assessed in vitro. Intra-arterial administration of MSCs diminishes their lung deposition; intravascular transplantation did not cause pulmonary embolisms. Intra-arterially transplanted BM-MNCs did not reach the brain in significant numbers. Transient proteolytic modification of MSCs with pronase decreased lung accumulation and tissue targeting without affecting their therapeutic characteristics. Intra-arterial transplantation increases lung passage of MSCs. Although thromboembolic events were not observed, further studies are warranted to ensure the safety of this route of MSC delivery. Pronase detachment is a promising method to enhance the potential of systemic MSC therapies. / Tiivistelmä Luutytimen mesenkymaaliset kantasolut (MSC) ja mononukleaariset solut (BM-MNC) ovat osoittautuneet tehokkaiksi useissa kliinisissä käyttöaiheissa. Solujen systeeminen annostelu verenkiertoon olisi käytännön kannalta paras soluterapian toteutukseen, mutta solujen merkittävä taipumus jäädä keuhkoihin loukkuun ja veritulppariski muodostavat haasteen. Keuhkohakeutumisen tiedetään ainakin osin johtuvan solujen pintamolekyyleistä ja näiden muokkaaminen voisi parantaa solujen keuhkoläpäisevyyttä. Tutkimuksessa käytettiin koe-eläimenä sikaa, jolle istutettiin systeemisesti allogeenisia mesenkymaalisia kantasoluja tai autologisia luuytimen mononukleaarisia soluja; solujen kudoshakeutumisen seuranta toteutettiin isotooppileimauksella- ja kuvannuksella. Tutkimuksessa arvioitiin annostelureitin vaikutusta keuhkoläpäisevyyteen, solujen kudojakautumista, toimenpiteen turvallisuutta sekä mononukleaarisolujen hakeutumista vaurioituneeseen aivokudokseen. Pronaasikäsittelyn vaikutusta mesenkymaalisten kantasolujen keuhkoläpäisevyyteen arvioitiin sika- ja hiirimallissa; rotan raajavauriomallia käytettiin lisäksi pronaasin kudoshakeutumisvaikutusten arvioimiseen. Pronaasikäsittelyn vaikutuksia solujen pintarakenteisiin ja toiminnallisuuteen arvioitiin in vitro- kokeissa. Mesenkyymalisten kantasolujen annostelu valtimonsisäisesti paransi solujen keuhkoläpäisevyyttä; tutkimuksissa käytetyt solut eivät aiheuttaneet keuhkoveritulppia. Valtimonsisäisesti annostellut mononukleaarisolut eivät hakeutuneet vaurioituneeseen aivokudokseen sikamallissa. Pronaasikäsittely muovasi solujen pintaproteiineja palautuvasti ja tämä lisäsi huomattavasti mesenkymaalisten kantasolujen keuhkoläpäisevyyttä ja kudoshakeutumista vaikuttamatta solujen toiminnallisuuteen. Mesenkymaalisten kantasolujen annostelu valtimonsisäisesti voi parantaa solujen keuhkoläpäisevyyttä. Tutkimuksessa ei todettu keuhkoveritulppaa tai muita tromboembolisia tapahtumia, mutta lisätutkimuksia tarvitaan MSC- terapian turvallisuuden takaamiseksi. Pronaasikäsittelyn tulokset mesenkymaalisten kantasolujen systeemisen annostelun parantamisessa olivat lupaavia.

The cornea is the transparent, avascular and highly innervated outer anterior layer of the eye. The cornea is a very delicate structure and any traumatic insult may lead to damage and limbal stem cell deficiency (LSCD), leading to chronic discomfort, visual impairment and ultimately blindness. The resultant issues can have a significant effect on patients and reduce their quality of life. Whilst conservative and therapeutic management of these problems play a part in the treatment of corneal injuries often surgery is indicated. However, surgical repair of damaged corneas may be limited by the availability of suitable donor tissue and donor site morbidity. Corneal grafts or penetrating keratoplasty (PK) or donor limbal grafts may lead to surgical complications such as corneal scarring, infection and graft rejection. First described in 1908 by A. Maximow, stem cells offer the opportunity to produce functional cell specific tissues from undifferentiated “primordial” cells. By using stem cells from human adult or deciduous tooth pulp, repair and regeneration of the cornea may be possible. Furthermore, it may lead to development of new and innovative treatments of other corneal disorders and injuries. The aim of the investigations detailed in this thesis was to characterize dental pulp stem cells (DPSC), help establish their use in regenerative medicine and help enhance the repair and regeneration of damaged corneal epithelium. Using various laboratory techniques including PCR, western blot and immunostaining it was determined that DPSC possess adequate potency and plasticity to be differentiated into a number of cell-lines. Co-culture of DPSC with human cornea demonstrated that stem cells were attracted to the tissue and migrate towards it and attach to the surface of the limbal explant. Additionally, using soft contact lenses it has been shown that DPSC can be successfully transferred from culture to human cornea in vitro. Expression of terminally differentiated corneal epithelium markers such as cytokeratin 3 & 12 further supports the concept that DPSC were transdifferentiated into epithelial progenitor cells. Once transferred onto the corneal surface, DPSC supported corneal epithelium regeneration, allowed corneal epithelial like cells to grow and avert conjunctivalisation and thus maintained cornea transparency. Further studies are needed to provide a better understanding of the DPSC’s role in corneal regeneration, but it is clear that DPSC are promising candidates for this novel and non-invasive method of corneal epithelium regeneration.

Doctor of Philosophy / Department of Anatomy and Physiology / Mark L. Weiss / Umbilical cord matrix or Wharton’s Jelly is a mucous connective tissue ensheathing the cord blood vessels and contains mesenchymal-like stem cells. Previously, we have shown that pig umbilical cord matrix stem (pUCMS) cells transplanted into normal rat brain were recovered up to 6 weeks post-transplantation, where a sub-population of pUCMS cells exhibited neuronal morphology and expressed a variety of neuronal markers. Here, approximately 150 pUCMS cells were transplanted into non-immunesuppressed rats that previously received a brain lesion by neurotoxin, 6-hydroxydopamine (6-OHDA), which specifically affects midbrain dopaminergic neurons, leading to pathologic findings similar to that of Parkinson’s disease (PD). The pUCMS cells proliferated up to 8 weeks post-transplantation and there was a significant increase in the percentage and number of pUCMS cells expressing tyrosine hydroxylase (TH), which is a marker for dopaminergic cells. We conclude that 1. Xenotransplants of pig UCMS cells are not rejected by rats at least up to 8 weeks after transplantation and 2. The pig UCMS cells proliferate and differentiate after transplantation into PD model rats. The surface antigen and gene expression profile of human umbilical cord matrix stem (hUCMS) cells resemble that of mesenchymal stem cells. Apomorphine-induced rotatory behavior was used to analyze the motor deficits of the PD model rats. In different experiments 1000, 2500 and 25000 hUCMS cells were transplanted into the brain of non-immunesuppressed PD model rats. There was a dose-dependent decrease in apomorphine-induced rotations; the maximum benefit was found in the rats that received 1000 hUCMS cells. The graft cells were recovered at 2 days and 1 week, but not at 6, 10 or 12 weeks post-transplantation. Quantitative assessment of host TH-positive midbrain dopaminergic neurons revealed a positive correlation between the behavioral improvement and TH-positive cell number in the low-density (1000 cells) transplant group, showing that the hUCMS cells may play a role in rescuing damaged host dopaminergic neurons and promote improvement of motor deficits in PD-model rats. In summary, hUCMS cells appear to be mesenchymal stem cells that can be harvested in great numbers from a non-controversial, inexhaustible source. Human UCMS cells show therapeutic benefit in PD model rats, but the mechanism by which they promote improvement is presently unknown.

Background: Stem cells are a promising therapy for regeneration following myocardial infarction (MI). Another therapy currently under investigation for MI is glucagon-like peptide-1 (GLP-1), a natural incretin hormone that has cardio-protective properties, although a short half-life in vivo. GLP-1 CellBeads are a novel therapy, combining stem cells and GLP-1. Human mesenchymal stem cells (MSCs) were immortalised, engineered to secrete a fusion protein of GLP-1 and encapsulated in alginate. We have previously demonstrated that GLP-1 CellBeads significantly reduce infarct size and improve ejection fraction post-MI, but the underlying mechanisms are unclear. The therapy was assessed in an in vivo pig MI model and an in vitro cardiomyocyte ischaemia model. Methods: GLP-1 CellBeads were delivered to coronary artery branches in pigs, creating micro-infarcts, as determined by echocardiography. Cell-free beads (Beads) and CellBeads containing hMSCs without GLP-1 (Beads-MSC) were delivered as controls (n=3-5/group). Pigs were sacrificed one and four weeks post-MI. Tissue was analysed for: apoptosis, collagen, cardiomyocyte cross sectional area and myofibroblasts. The localised response around the beads was also measured using immunohistochemistry. Atomic force microscopy (AFM) was used to examine the ultra-structure of the collagen scar. The expression profiles of genes involved in collagen remodelling were measured using qRT-PCR. Viability of MSCs was measured using GFP-tagging and confirmed using qRT-PCR. To examine effects on apoptosis in vitro, human adult cardiomyocytes underwent ischaemia for 1 hour before incubation with: media conditioned with MSCs or MSC+GLP-1, GLP-1, Exendin-4 or media. Apoptosis and viability were measured at 24 and 48 hours respectively. Results: In the in vivo pig model, significant increases in apoptosis were observed in the infarct of all groups one week post-MI, with no differences between treatments. Despite decreased numbers of myofibroblasts, significantly more collagen was observed in MSC treated groups, with increased collagen fibril periodicity and a more organised collagen scar. The altered scar structure was reflected in differences in gene expression between groups, with an accelerated healing response in the MSC groups. However, significantly fewer myofibroblasts were observed in the MSC treated groups. Viability of MSCs was confirmed up to four weeks post-infusion, with GLP-1 secretion confirmed up to one week. In the in vitro ischaemia model, MSC+GLP-1 conditioned media significantly reduced cardiomyocyte apoptosis 24 hours post-ischaemia, compared to media alone. All agonists (GLP-1, MSC media and MSC+GLP-1 media) significantly improved viability compared to media alone 48 hours post-ischaemia. Conclusions GLP-1 CellBeads have a beneficial effect on healing following MI by significantly decreasing infarct size and improving ejection fraction post-MI. these benefits are associated with decreased cardiomyocyte apoptosis and altered collagen scar formation. The CellBeads act as local hubs for regeneration and are viable up to one month post-infusion. The effects observed are due to a combination of the GLP-1 and paracrine factors released from the hMSCs.

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