Rozprawy doktorskie na temat „Stem cells”

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology and Laboratory Medicine." Discipline: Pathology and Laboratory Medicine; Department/School: Medicine.

Thesis (Ph.D.)--University of Florida, 2005.
Typescript. Title from title page of source document. Document formatted into pages; contains 97 pages. Includes Vita. Includes bibliographical references.

Thesis (M.S.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed August 11, 2009). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 128-134).

Human embryonic stem cells (hESCs) are able to proliferate indefinitely without losing their ability to differentiate into multiple cell types of all three germ layers. Due to these fascinating properties, hESCs have promise as a robust cell source for regenerative medicine and as an in vitro model for the study of human development. In my PhD study, I have investigated the neural differentiation process of hESCs using our established protocol, identified characteristics associated with each stage of the differentiation and explored possible signalling pathways underlying these dynamic changes. It was found that neural differentiation of hESCs could be divided into 5 stages according to their morphology, marker expression and differentiation potencies: hESCs, neural initiation, neural epithelium/rosette, neuronal progenitor cells and neural progenitor/stem cells (NPSCs) and 4 of these stages have been studied in more detail. At the neural initiation, hESCs firstly lose TRA-1-81 expression but retain SSEA4 expression. This transient cell population shows several similar properties to the primitive ectoderm. After neural-tube like structure/neural rosette formation, neural progenitor cells appear as typical bipolar structures and exhibit several properties of radial glial cells, including gene expression and pro-neuronal differentiation. The neural progenitor cells are able to grow in culture for a long time in the presence of growth factors bFGF and EGF. However, they gradually lose their bipolar morphology to triangular cell type and become pro-glial upon further differentiation. In addition, the state of neural progenitor and stem cells can be distinguished by their differential response to canonical Notch effector, C protein-binding factor 1. It was also found that delta like1 homolog (DLK-1) is temporally upregulated upon initial neural differentiation, but becomes undetectable after the neural progenitor stage. Overexpression of DLK-1 in NPSCs enhances neuronal differentiation in the presence of serum by blocking BMP and Notch pathways. These results show that neural differentiation of hESCs is a dynamic process in which cells go through sequential changes, and the events are reminiscent of the in vivo neurodevelopment process. Moreover, I have characterized stably transfected nestin-GFP reporter hESC lines and found that the cell lines maintained the features of hESCs and the expression of GFP is restricted to the neural lineage after differentiation. Therefore, these reporter lines will be useful for the study of factors that regulate neural differentiation and for the enrichment of neural progenitors from other lineages. Taken together, this study has demonstrated that hESCs are a good in vitro model to study the mechanisms and pathways that are involved in neural differentiation. The availability of hESCs allows us to explore previously inaccessible processes that occur during human embryogenesis, such as gastrulation and neurogenesis.

Primary keratinocytes form 3 types of colony with different morphologies termed holoclones, meroclones and paraclones, thought to be derived from stem, early and late stage precursor cells respectively (Barrandon and Green, 1987b, Rochat et al., 1994). Cancer cell lines produce colonies with morphologies analogous to those of holoclones, meroclones and paraclones, and consequently holoclone morphology is used as a surrogate marker for stem cell colonies. The aim of this study was to elucidate the relationship between clonogenicity, colony morphology and stem cells. Colonies formed by primary prostate epithelial cells and prostate cancer cell lines (DU145, PC3, LNCaP) were characterised. The proportions of colonies were not altered significantly by modification of culture conditions. In contrast to cancer cells, primary prostate epithelial cells form only two types of colony, termed types 1 and 2, which are analogous to holoclones and paraclones. Only type 1 colonies were highly proliferative, able to self-renew and express putative stem cell markers. Paradoxically, cells from DU145 meroclones formed holoclones and had self-renewal capacity (by serial cloning and xenografting). It is concluded that the major difference between holoclone and meroclone colonies from the cancer cell line DU145 is the proportion of stem cells within each colony, not the presence or absence of stem cells. Phage display was used to look for targets on the surface of cells in Type 1 colonies. Various experimental protocols were tested, but no targets were identified.

Science and technology have utterly transformed human life in the past few generations. But big changes arrive faster and faster these days. Now we’re right on the verge of some extraordinary stuff. New techniques will circumvent ethical concerns, and maybe lead us to the Medical Holy Grail: the ability to grow new tissue and organs from the patient’s own cells, virtually eliminating the possibility of rejection. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/31100

Thesis (MTech (Nursing))--Cape Peninsula University of Technology, 2017.
Stem cell transplantation has become one of the standard methods of treatment for patients with malignant and benign blood disorders. The multidisciplinary team interacting with these patients and their families, must be knowledgeable concerning the appropriate quality health care. The objectives of the study were to explore the knowledge of the members of the health care team in terms of the processes that need to be adhered to with stem cells transplantation, as well as exploring the perceptions amongst the health care team members and their reactions towards patients undergoing stem cell transplantation. An exploratory research design with a qualitative approach was employed. Data collection took place at two stem cell transplant units in the Western Cape, using non-probability purposive sampling technique. The health care team members included a medical doctor, dietician, physiotherapist, social worker, radiographer and nursing staff. Data was collected by face-to-face personal interviews which were transcribed and analysed by using coding and thematic analysis. The majority of the professional participants could identify the processes for stem cell transplantation, which affirmed their knowledge. The non-professional health care team member, could also identify the types of methods and processes of stem cell transplantation. Participants stated that the health care team members had passion for this treatment option. Some participants felt it to be emotionally challenging to work in the environment, especially with paediatric patients and the dying. However, some health care team members could detach themselves emotionally from the patients. The team stated that the stem cell transplanted patients need special care to overcome all challenges experienced, but were positive about treatment. It is evident that management of stem cell transplanted patients is complicated and the health care team members must have knowledge, skills and the appropriate attitude to practice in these units. This study emphasised how vital it is that stem cell transplantation be included in the training programs of the multidisciplinary team. Health care practitioners in the field must stay abreast with stem cell research in order to effectively conduct health promotions for patients and staff. In addition, hematology and transplant awareness campaigns should also be conducted in order to educate society and suggest referrals if necessary.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering." Discipline: Biomedical Engineering; Department/School: Medicine.

The relationship between cellular proliferation and differentiation is a major topic in cell biology. What we know comes from models of somatic cell differentiation, where it is widely viewed that cycling and differentiation are coupled, antagonistic phenomena linked at the G1 phase. The extension of this view to stem cells, however, is unclear. One potential possibility is that stem cells also tightly link their G1 phase with their differentiation, indicating a similarity between the differentiation of stem cells and the differentiation of more mature somatic cells. On the other hand, stem cells may utilize different mechanisms or adaptations that confer on them some aspect of uniqueness or "stemness." In this case, stem cells will not exhibit the same coupling with the cell cycle as in many somatic cell models. In this thesis, we examined mouse embryonic stem cells (mESCs), a stem cell that is pluripotent and rapidly cycling with a highly condensed G1 phase. Direct extension of the somatic view posits that elongation of their G1 phase to somatic lengths by cyclin-dependent kinase (CDK) activity inhibition should induce or increase differentiation of these stem cells. Evidence supporting this claim has been contradictory. We show that elongation of the cell cycle and elongation of G1 to somatic lengths is fully compatible with the pluripotent state of mESCs. Multiple methods that lengthen the cell cycle and that target CDK activity or that trigger putative downstream mechanisms (i.e. Rb and E2F activity) all fail to induce differentiation on their own or even to facilitate differentiation. These results indicates that the model of linkage between the G1 phase and differentiation in mESCs is incorrect and leads us to propose that "stemness" may have a physiological basis in the decoupling of cell cycling and differentiation. In summary, we provide evidence that there is a resistance of mESCs to differentiation induced by lengthening G1 and/or the cell cycle. This could allow for separate control of these events and provide new opportunities for investigation and application.

Graft-versus-host disease (GvHD) remains the main complication associated with haematopoietic stem cell transplantation (HSCT). GvHD is caused by allo-reactive donor T cells mounting an attack against specific target tissues. CD4+CD25HiFoxp3+ regulatory T cells have been shown to modulate GvHD in vitro and also in vivo animal models. More recently early stage clinical trials have described the successful use of Treg to reduce the incidence of GvHD following HSCT. The aim of this study was to investigate further the suppressive mechanisms by which Treg are able to modulate GvHD and assess the influence of Treg on the beneficial graft-versus-leukaemia (GvL) effect therefore providing further insight into the use of Treg in the therapeutic management of GVHD. Data presented in this thesis demonstrates the successful isolation and expansion of a highly pure Treg population which maintained suppressive capacity throughout culture. We also confirmed that Treg retain suppressive capacity following cryopreservation resulting in reduced workload and increased consistency when used for in vitro functional studies. We also provide the first human in vitro evidence that Treg are able to prevent cutaneous GvH reaction by blocking the migration of effector T cells into the target tissues. The presence of Treg during allo-stimulation caused reduced effector cell activation, proliferation, IFNγ secretion and decreased skin homing receptor expression. Further investigation into the Treg modulation of dendritic cells demonstrated, for the first time in experimental in vitro human GvHD, that this was due to ineffective effector T cell priming in the presence of Treg caused by impairment of dendritic cell functions. Comprehensive phenotypic and functional analysis of Treg treated moDC showed their decreased antigen processing ability and allostimulatory capacity, resulting in a less severe GvH reaction in the skin explant model. Furthermore, this work has revealed that despite Treg impairing in vitro GvL mechanisms at a cellular level there was no association observed between increased Treg levels and the incidence of relapse in a small clinical cohort of HSCT patients. In conclusion this study has provided further insight into the mechanisms by which Treg are able to modulate GvHD. This would inform future clinical trials using Treg as a therapeutic alternative to current GvHD treatment and prophylaxis.

Liver development involves the differentiation and interaction of both endoderm and mesoderm cell types. The role of the liver in drug metabolism makes it an important area of medical research. Mimicking embryonic liver development in vitro using human ESCs is a strategy used to differentiate liver cell types. These can then be used as a model playing a role in the development of drugs and the study of their hepatotoxicity and would also have potential for use in cell therapy and regenerative medicine. Differentiated hepatocyte-like cells were found to have more in common with liver cells than those of other organs, including the secretion of albumin and activity of proteins important in drug metabolism, CYP3A and CYP2D6. However the hepatocyte-like cells were found to more closely resemble fetal rather than adult hepatocytesOrganoid differentiation resulted in cells types which in vivo are both endoderm and mesoderm derived cells of the liver. Culture in this 3D system allowed the spontaneous acquisition of polarity by these cells and their formation into structures reminiscent of liver architecture. After treatment with the toxin 4,4′-diaminodiphenylmethane a cell type and structure specific dose response was observed which matches that described in vivo.

Haemopoietic recovery after high dose chemotherapy (HOC) in the treatment of haematological disease may be slow and/or incomplete. This is generally attributed to progressive haemopoietic stem cell failure, however we hypothesize that HOC induced defective haemopoiesis may be in part due to poor stromal function. Although chemotherapy is known to damage mature bone marrow stromal cells in-vitro, the extent to which marrow mesenchymal stem cells (MSC) are damaged by HOC in-vivo and in-vitro is unknown. To firstly address this question the physical characteristics and functional properties of marrow MSC derived from patients who have received chemotherapeutic treatment for various haematological diseases were investigated. Subsequently a suitable in-vitro treatment culture model was developed and the effects of chemotherapy exposure in-vitro using cell culture and proteomic techniques were shown. Results of this study demonstrate proliferative and phenotypic changes to patient MSC caused by HOC regimens. In contrast, the differentiation capacity, and ability to form functional marrow stroma after exposure to HOC in-vivo was equal to that of patient MSC studied prior to HOC. Chemotherapeutic exposure to MSC cultures in-vitro have confirmed changes seen in-vivo, with abnormalities evident in MSC proliferation, differentiation and also in their ability to support haemopoietic stem cell migration and repopulation after treatment. A reduced C044 expression on MSC, after exposure to cyclophosphamide, was also observed and the importance of this molecule shown through con-focal microscopy demonstrating its interaction with C034+ haemopoietic stem cells. Using proteomic techniques differences in protein expression by MSC, relating to changes in their function after chemotherapy exposure, were also observed after treatment with cyclophosphamide or melphalan. Finally the role of keratinocyte growth factor as a potential cyto-protective agent to MSC against damage caused by chemotherapy exposure was tested. Results indicated there was significant evidence to indicate that KGF was able to preserve reductions in C044 expression levels after MSC exposure. It was concluded that marrow MSC sustain prolonged injury due to recurrent courses of HOC in-vivo and in-vitro. However, the clinical importance of the chemotherapy induced defects we have observed must be determined through the initiation of prospective randomized trials of the effects of MSC co-transplantation on haemopoietic recovery in the setting of HOC with and without haemopoietic stem cell rescue.

The immunosuppressive property of mesenchymal stem cells (MSC) has been utilised to ameliorate autoimmune reactions such as graft-versus-host disease. However, variation exists in primary MSC isolated due to differences in donor age and tissue of origin. Alternatively, human embryonic stem cells (hESC) can be differentiated to homogeneous populations of MSC (hESCMSC), thus providing an unlimited source of MSC for cell therapy. In this study, the immunomodulatory properties of two hESC-MSC lines, hESC-MSC1 and hESC-MSC2, were compared with adult bone marrow-derived MSC (BM-MSC) and neonatal foreskin fibroblast (Fb). hESC-MSC were able to suppress the proliferation of anti-CD3/CD28-stimulated CD4+ T cells in contact and transwell systems. The immunosuppression was demonstrated by both the carboxyfluorescein diacetate succinimidyl ester (CFSE) and [3H]- thymidine proliferation assays. However, hESC-MSC were less potent and twice the number of adherent hESC-MSC (as measured by IC50) compared to BM-MSC and Fb were required to suppress T cell proliferation by 50%. Supernatants collected from transwells of MSC or Fb with T cells were shown to suppress T cell proliferation, suggesting that suppressive factors were only produced in the presence of activated T cells. Among several candidates, endothelial monocyte-activating polypeptide-II (EMAP-II) was identified as a potential suppressive factor. T cells also induced indoleamine-2,3- dioxygenase (IDO) expression in MSC and Fb. IDO led to the depletion of tryptophan, an essential amino acid, and/or the production of tryptophan metabolites (kynurenines), thereby inhibiting T cell proliferation. Interestingly, blocking of IDO with 1-methyltrytophan reversed the suppressive effect, implicating IDO as a potential mediator in T cell suppression. Concomitantly, several candidate suppressive factors in the supernatants have also been identified using antibody arrays. However, their functions require validation. In conclusion, hESC-MSC share similar suppressive properties as BM-MSC and represent a potential cell source for clinical purposes.

Despite the remarkable beneficial effects of disease-modifying agents in relapsing-remitting multiple sclerosis (MS) patients, progressive forms of (P)MS still lack effective treatments. This stark contrast is partially dependent on the difficulties researchers have found in tackling the complex pathophysiology of this phase of disease, in which chronic inflammation within the central nervous system (CNS) is coupled by ongoing neurodegeneration and demyelination. Cell transplantation is among the most promising therapeutic approaches in regenerative medicine, combining tissue trophic and immunomodulatory effects of the graft with its intrinsic potential for cellreplacement. These are all attributes that can be harnessed to treated patients with PMS. As such, within this thesis, I have focused my attention on investigating how cellular therapies could be used to (i) prevent neuronal damage, (ii) modulate the chronic activation of the immune system and (iii) replace the damaged myelin in PMS. Olfactory Ensheathing Cells (OECs) are a special population of glial cells known to exert neuroprotective mechanisms and capable of promoting neuroprotection. Using in vitro models of neuron-like cells, I have demonstrated that OECs exert their neuroprotective effect by reducing Cx43-mediated cell-to-cell and cell-toextracellular environment communications. Despite this important finding, the immunomodulatory and remyelinating potential of OECs is still limited. As such, I decided to study a complementary stem cell approach that conjugates these attributes with ease in clinical applicability. Induced Neural Stem Cells (iNSCs) are a source of autologous, stably expandable, tissue specific and easily accessible stem cells, which have the potential to differentiate into the three main neural lineages. Mouse iNSCs were characterized in vitro and in vivo and their immunomodulatory potential was initially studied. This work uncovered a novel mechanism that underpins the potential of iNSCs to interact with the chronic CNS compartmentalised activation of the innate immune system. Specifically, I found that iNSCs are able to sense extracellular metabolites, which accumulate in the chronically inflamed CNS, and to ameliorate neuroinflammation via succinate-SUCNR1-dependend mechanisms. To characterize the potential for tissue replacement and remyelination of such a promising cell line, I have also analysed how iNSCs grafts differentiate in an experimental model of focal demyelination. I found that iNSCs are able to integrate and differentiate into remyelinating oligodendrocytes (OLs) in chronic demyelinated CNS. These data suggest that iNSCs are indeed an effective source of stem cell transplantation, being able to modulate inflammation and to effectively replace lost tissue in mouse models of PMS. Altogether the evidences gathered in this thesis are important new steps in the field of cell transplantation, which will be pivotal in the march forward for future clinical applications in chronic demyelinating CNS disorders.

Despite major advances in pharmacological and surgical treatments of cardiovascular diseases (CVDs), clinical outcomes of patients with severe CVDs remain very poor. Most of medication and interventions currently available are only playing roles of preventing further damage to myocardium, declining the risk of on-going cardiovascular events, lifting the cardiac pumping efficiency and lower early mortality rates, none of these treatments can regenerate or repair damaged cardiac tissue or restore heart function. As a result, several new strategies have been explored to overcome limitations of current therapeutic approaches. One prospective is to replace dead cardiac vascular cells with young and green cells to repair or regenerate damaged heart myocardium. Several types of stem cells, including bone marrow hematopoietic stem cells, mesenchymal stem cells (MSCs), embryonic stem cell (ESCs)and induced pluripotent stem cells (iPSCs),have been tested as the candidates for treatment of CVDs. Among a myriad of types of stem cells, bone marrow derived MSCs(BM-MSCs) has received great attention based on several unique properties such as easy isolation and expansion, stable genetic background and low immunogenicity. However, the therapeutic efficacy of BM-MSCs derived from aging or diseased donors is impaired. The differentiation potential of BM-MSCs is gradually reduced with the increased culture time. Thus, it is urgent to identify some novel alternative sources for MSCs. Moreover, the potential mechanisms of MSCs therapy have not been understood totally. This thesis is designed to investigate the therapeutic efficacy and potential mechanisms of several novel types of MSCs, including hESC-MSCs and hiPSC-MSCs and Rap1-/--BM-MSCson several types of CVDs, including pulmonary arterial hypertension (PAH), dilated cardiomyopathy (DCM)and myocardial infarction (MI). In Chapter 4, it disclosed that hESC-MSCs have a better therapeutic efficacy than BM-MSCs in attenuation of PAH induced by monocrotaline in mice. The greater therapeutic potential of hESC-MSCs on PAH was not only attributed to the higher capacity of differentiation into de-novo vascular cells, but also attributed to higher cell survival rate and greater paracrine effects post-transplantation. In Chapter 5, it demonstrated that compared with BM-MSCs, iPSC-MSCs have a better therapeutic effect on doxorubicin-induced cardiomyopathy. Several potential mechanisms of action were involved in iPSC-MSCs-based therapy for cardiomyopathy. It demonstrated that iPSC-MSCs transplantation not only attenuated the generation of reactive oxygen species(ROS)and the level of inflammation, but also restored depletion of cardiac progenitor cells and promoted endogenous myocardial regeneration against doxorubicin induced cardiomyopathy. Moreover, mitochondrial transfer and paracrine actions of iPSC-MSCs played critical roles in the rescue for doxorubicin-induced cardiomyopathy. In Chapter 6, it uncovered that compared with wild type BM-MSCs,Rap1-/--BM-MSCs transplantation achieved a better benefit to MI induced by ligation of left anterior descending (LAD)coronary artery. Rap1-mediated NF-κB activity plays a key role in regulation MSCscytokine secretion profiles. The absence of Rap1 in MSCs leads to reduced pro-inflammatory cytokines secretion and enhanced MSCs survival capacity, thus yielding a better therapeutic efficacy. In conclusion, findings presented in this thesis provide important new insights regarding different novel types of MSCs, including those derived from ESC and iPSC. They have distinct mechanisms of action from BM-MSCs and provide superior therapeutic efficacy in various form of severe CVDs, including PAH and DCM. The safety and efficacy of these novel types of MSCs for treatment of CVDs deserve further investigations.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy

Functional mesenchymal stem cells (MSCs) derived from human induced pluripotent stem cells (iPSCs) may represent an unlimited cell source with superior therapeutic benefits for tissue regeneration to somatic tissue, such as bone marrow (BM)-derived MSC. In the first part of this project, I investigated whether the differential expression of ion channels in iPSC-MSCs was responsible for their higher proliferation capacity than that of BM-MSCs. The expression of ion channels for K+, Na+, Ca2+ and Cl- currents was assessed by reverse transcription-polymerase chain reaction (RT-PCR). The functional role of these ion channels were then verified by patch clamp experiments to compare the electrophysiological properties of iPSC-MSCs versus BM-MSCs. I detected significant mRNA expression of ion channel genes including KCa1.1, KCa3.1, KCNH1, Kir2.1, SCN9A, CACNA1C and Clcn3 in both human iPSC-MSCs and BM-MSCs; while Kir2.2 and Kir2.3 were only observed in human iPSC-MSCs. Furthermore, I identified five types of currents (BKCa, IKDR, IKir, IKCa and ICl) in iPSC-MSCs, while only four of them (BKCa, IKDR, IKir and IKCa) were observed in BM-MSCs. The rate of cell proliferation was 1.4 fold faster in iPSC-MSCs as compared to BM-MSCs. Interestingly, the proliferation rate of human iPSCMSCs was significantly reduced when inhibiting IKDR with shRNA and hEAG1 channel blockers, 4-AP and astemizole. Though to a lesser extent, the proliferation rate of human BM-MSCs also decreased by IKDR blockage. These results demonstrated that hEAG1 channel plays a crucial role in controlling the proliferation rate of human iPSC-MSCs but to a lesser extent in BM-MSCs. Next, I examined whether forced expression of a transcription factor- myocardin in iPSC-MSC using viral vectors (adenovirus or lentivirus) can further enhance their trans-differentiation to cardiomyocytes and improve their electrophysiological properties for cardiac regeneration. My results on RT-PCR and immunofluorescent staining revealed that myocardin induced the expression of several cardiac and smooth muscle cell markers, including α-MHC, cTnT, GATA4, α-actinin, and cardiac MHC, smooth muscle cell markers MYH11, calponin, and SM α-actin, but not the more mature cardiac markers such as β-MHC and MLC2v in iPSC-MSCs. These findings indicate that forced expression of myocardin in iPSC-MSC resulted in partial trans-differentiation into cardiomyocytes phenotype. Furthermore, I also discovered that myocardin altered the electrophysiological properties of iPSC-MSCs when examined by RT-PCR and patch clamp experiments. Forced expression of myocardin in iPSC-MSC enhanced the expression of Kv4.3, SCN9A and CACNA1C, but reduced that of KCa3.1 and Kir 2.2 in iPSC-MSCs. Moreover, BKCa, IKir, ICl, Ito and INa.TTX were detected in iPSC-MSC with ectopic expression of myocardin; while only BKCa, IKir, ICl, IKDR and IKCa were noted in iPSC-MSC transfected with green florescence protein. Furthermore, as measured by multi-electrode arrays recording plate, the conduction velocity of the neonatal rat ventricular cardiomyocytes cocultured iPSC-MSC monolayer was significantly increased after ectopic expression of myocardin. Taken together, I have demonstrated that hEAG1 channel is important in the regulation of iPSC-MSC proliferation and forced expression of myocardin in iPSC-MSC resulted in their partial transdifferentiation into cardiomyocytes phenotype and improved the electrical conduction during integration with mature cardiomyocytes.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy

Introduction: Stem cell biology has received much interest because of its potential in both therapeutic application and in vitro modeling of diseases. In particular embryonic stem cells have good proliferative and differentiative abilities, but their use is still associated to ethical concerns and problems related to their teratogenic potential. Adult stem cells have also been described to be pluripotent both in vitro and in vivo. However, their use is limited because they are difficult to isolate and expand, particularly in a clinical setting. In this scenario, it would be advantageous to obtain a cell population with high selfrenewal and differentiation capacities, without ethical problems. In 2007 our group described that amniotic fluid stem (AFS) cells could be derived selecting amniocytes using c-Kit antibody. AFS cells have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Aim: This work aiming at characterize the myogenic potential of mouse AFS cells using a mouse model of spinal muscular atrophy and in particular at analyzing their ability to differentiate into satellite cells and colonize the muscle stem cell niche. Materials and Methods: Mouse AFS cells were obtained by amniocentesis and selected for the marker c-Kit with immunomagnetic beads. Freshly isolated AFS cells were analyzed for the expression of different markers (CD90, CD45, CD44, CD34, CD31, Flk1, SCA1, CD105) by flow cytometry and the expression of Oct4, Sox2, c-Myc, Klf4 and Sca-1 by qRT-PCR at different embryonic stages. For the treatment of HSA-Cre, SmnF7/F7 mutant mice, GFP+ AFS cells were injected via the tail vein and animals were sacrificed one and fifteen months after transplantation. Clinical aspects were observed and analyzed after transplantation to evaluate AFS cells’ effects. Several muscles were stained with hematoxylin and eosin, Masson’s trichrome and analyzed by immunofluorescence with anti-GFP and anti-dystrophin antibodies. To demonstrate the ability of AFS cells to replenish the muscle niche, staining for satellite cell markers and secondary transplantation were performed. The myogenic potential of AFS cells was also evaluated with transplantation after in vitro expansion. Results: Mouse AFS cell number changes during the course of gestation. At E12.5 these cells express hematopoietic markers (CD45, CD34, SCA1), mesenchymal markers (CD90, CD105) together with Flk1, CD31 and CD44. Gene expression analysis showed that mouse AFS cells express at low levels Oct4 and Sox2 and at high levels c-Myc and Klf4, whereas they are negative for the expression of myogenic genes. Mild muscular mutant HSA-Cre, SmnF7/F7 mice die at the age of 10 months and show evident clinical complications such as kyphosis and muscle shrinkage. After transplantation with GFP+ AFS or bone marrow (BM) cells mice survival rate increased by 75% and 50% respectively. Animals treated with AFS cells recovered more than 75% of force compared to the untreated. One month after transplantation, muscles obtained from AFS-treated mice displayed 37% of GFP+ fibers, with very low number of regenerating myofibers (<1%) and normal dystrophin expression. Fifteen months after transplantation BM-treated mice displayed a high number of central nucleated fibers and consistent infiltration of interstitial tissue and no GFP+ myofibers, while AFS-treated mice had a normalized phenotype, close to the same age WT mice, and 58% of the myofibers were GFP+. Similar results were obtained with transplantation of mouse AFS cells expanded in culture. Discussion: Mouse AFS cells are a heterogeneous population, and their phenotype changes during the course of gestation. At E12.5 they express mesenchymal, hematopoietic and endothelial markers, but most importantly don not express myogenic factors, indicating that no myogenic progenitor cells are present in this stem cell population. When injected in a muscular mutant mouse model, AFS cells showed a myogenic potential, even after long-term transplantation, suggesting an interesting therapeutic potential. They indeed were able to differentiate into satellite cells localizing in the muscle stem cell niche and expressing Pax7, a7integrin and SM/c-2.6, exclusively markers of satellite cell population. Moreover, AFS cells could contribute to the formation of new myofibers even after in vitro expansion.
Introduzione: Negli ultimi anni lo studio delle cellule staminali ha suscitato molto interesse, sia per il grande potenziale di queste cellule nelle terapie e applicazioni cliniche, sia come modello di studio in vitro per diversi tipi di malattie. In particolare, le cellule staminali embrionali hanno una elevata capacità proliferativa e di differenziazione, ma il loro utilizzo è ancora associato a problematiche etiche. Anche le cellule staminali adulte possiedono grandi potenzialità differenziative sia in vitro che in vivo, tuttavia il loro utilizzo è limitato in quanto difficili da isolare ed espandere, soprattutto in ambito clinico. In questo scenario sarebbe vantaggioso poter ottenere una popolazione di cellule con elevata capacità di proliferazione e differenziazione, senza dover affrontare però problemi di tipo etico. Nel 2007 il nostro gruppo ha isolato una popolazione di cellule staminali dal liquido amniotico (cellule AFS), utilizzando come marcatore il recettore c-Kit. Queste cellule hanno capacità clonogenica e possono essere dirette a differenziare in una vasta gamma di tipi cellulari appartenenti a tutti e tre i foglietti germinativi. Obiettivo: Questo lavoro mira a caratterizzare il potenziale miogenico delle cellule staminali del liquido amniotico di topo utilizzando un modello murino di atrofia spinale muscolare. In particolare è volto ad analizzare la capacità delle cellule AFS di dare origine a cellule staminali muscolari e colonizzare la nicchia staminale del muscolo scheletrico. Materiali e Metodi: Le cellule AFS sono state ottenute mediante amniocentesi e selezionate per la positività al marcatore c-kit con metodo immmunomagnetico. Appena isolate le cellule AFS sono state analizzate per l'espressione di diversi marcatori (CD90, CD45, CD44, CD34, CD31, Flk1, SCA1, CD105) tramite citometria a flusso; inoltre, attraverso qRT-PCR è stata analizzata l'espressione di Oct4, Sox2, c-Myc, Klf4 e Sca-1 delle cellule AFS isolate a diversi stadi embrionali. Per la terapia di topi transgenici HSA-Cre, SmnF7/F7, le cellule AFS GFP+ sono state iniettate per via sistemica attraverso la vena caudale; gli animali sono stati poi sacrificati a uno e a quindici mesi dopo il trapianto. Sono stati osservati e analizzati alcuni parametri clinici per valutare l’effetto del trapianto cellulare. Diversi muscoli sono stati raccolti ed analizzati con ematossilina e eosina, tricromica di Masson e mediante immunofluorescenza con anticorpi anti-GFP e anti-distrofina. Per dimostrare la capacità delle cellule AFS di colonizzare la nicchia staminale del muscolo, sono state eseguite delle immunofluorescenze per i marcatori specifici delle cellule satelliti e sono stati eseguiti dei trapianti secondari. Il potenziale miogenico delle cellule AFS è stato valutato anche con trapianto dopo espansione in vitro. Risultati: Il numero medio di cellule AFS presenti nel liquido amniotico varia nel corso della gestazione murina; all’età di 12.5 giorni queste cellule sono circa l’1% del totale ed esprimono marcatori ematopoietici (CD45, CD34, SCA1), marcatori mesenchimali (CD90, CD105) unitamente a Flk1, CD31 e CD44. L’analisi di espressione genica ha dimostrato che le cellule AFS esprimono a bassi livelli Oct4 e Sox2 e alti livelli di c-Myc e Klf4, mentre, nonostante la composizione mista di questa popolazione, non è stata rilevata espressione di marcatori o fattori di trascrizione tipici dei precursori muscolari. I topi HSA-Cre, SmnF7/F7 mediamente muoiono all'età di 10 mesi e durante il corso della loro vita mostrano evidenti complicazioni cliniche come una pronunciata cifosi e atrofia a livello muscolare. Dopo il trapianto con cellule AFS GFP+ o con cellule del midollo osseo, il tasso di sopravvivenza di questi animali aumenta rispettivamente del 75% e 50%. Gli animali trattati con cellule AFS hanno recuperato più del 75% della forza rispetto agli animali non trattati. Un mese dopo il trapianto, i muscoli di topi trattati con cellule AFS presentano il 37% di fibre GFP+, un numero molto basso di miofibre rigeneranti (< 1%) ed una normale espressione di distrofina. Quindici mesi dopo il trapianto, gli animali trattati con cellule del midollo osseo mostrano un elevato numero di fibre centro nucleate, un’importante infiltrazione di tessuto interstiziale e nessuna miofibra GFP+, mentre i topi trattati con cellule AFS hanno un fenotipo molto simile a quello di topi sani della stessa età, e il 58% delle miofibre è GFP+. Risultati simili sono stati ottenuti trattando lo stesso modello animale con cellule AFS dopo espansione in cultura. Discussione: Le cellule AFS isolate dal liquido amniotico di topo sono una popolazione eterogenea; queste cellule esprimono marcatori mesenchimali, ematopoietici e marcatori endoteliali. Va evidenziato che, nonostante la composizione mista di questa popolazione staminale, non esistono precursori muscolari al suo interno, e quindi qualunque differenziamento in senso muscolare di queste cellule è dovuto ad una differenziazione delle cellule AFS e non ad una maturazione di cellule già pre-commited. Quando vengono iniettate in un modello di atrofia muscolare, le cellule AFS mostrano un grande potenziale miogenico, anche a lungo termine, dimostrandosi una interessante fonte cellulare per scopi terapeutici. Queste cellule infatti sono state in grado di differenziare in cellule satelliti localizzandosi nella nicchia delle cellule staminali muscolari ed esprimendo Pax7, a7integrina e SM/c-2.6, tutti marcatori esclusivi delle cellule satelliti. Inoltre, le cellule AFS possono contribuire alla formazione di nuove miofibre anche dopo espansione in cultura, aumentando così lo spettro di possibili applicazioni terapeutiche.

Haematopoietic stem cells (HSCs) are capable of differentiation into all mature haematopoietic lineages, as well as long-term self-renewal and are consequently able to sustain the adult haematopoietic system throughout life. Currently, in the mouse, HSCs are understood to first appear in the aorta-gonad-mesonephros (AGM) region at embryonic day 11 via a process of maturation from precursors (pre-HSCs). This maturation within the AGM region involves the complex interplay of signalling between cells of the niche and maturing precursor cell populations, but is relatively little understood at a molecular level. Recently our understanding of the AGM region has been refined, identifying the progression from E9.5 to E10.5 and the polarity along the dorso-ventral axis as clear demarcations of the supportive environment for HSC maturation. In this thesis, I investigated the molecular characteristics of these spatio-temporal transitions in the AGM region through the application of RNA-sequencing. This enabled the identification of molecular signatures which may underlie the supportive functionality of the niche. I further compared these expression signatures to the transcriptional profile of an independent cell type, also capable of supporting HSC maturation, the OP9 stromal cell line. By combining this transcriptional information with an ex vivo culture system, I screened a number of molecules for their ability to support HSC maturation from early precursors, leading to the discovery of a novel regulator of HSC maturation: BMPER. Further characterisation of this molecule enabled the identification of its specific cellular source and the proposal that through its action as an inhibitor of BMP signalling it facilitates the maturation of precursors into HSCs. These results lend further detail and support to the role of BMP signalling in the regulation of HSC maturation as well as demonstrating the potential of these transcriptional profiles to yield novel mechanistic insight.

Small cell lung cancer (SCLC) is a highly aggressive malignancy with extreme mortality and morbidity. Although initially chemo- and radio-sensitive, almost inevitable recurrence and resistance occurs. SCLC patients often present with metastases, making surgery not feasible. Current therapies, rationally designed on underlying pathogenesis, produce in vitro results, however, these have failed to translate into satisfactory clinical outcomes. Recently, research into cancer stem cells (CSCs) has gained momentum and form an attractive target for novel therapies. Based on this concept, CSCs are the cause of neoplastic tissue development that are inherently resistant to chemotherapy, explaining why conventional therapies can shrink the tumour but are unable to eliminate the tumour completely, leading to eventual recurrence. Here I demonstrate that SCLC H345 and H69 cell lines contain a subset of cells expressing CD133, a known CSC marker. CD133+ SCLC sub-population maintained their stem cell-like phenotype over a prolonged period of culture, differentiated in appropriate conditions and expressed the embryonic stem cell marker Oct-4 indicating their stem-like phenotype. Additionally, these cells displayed augmented clonogenic efficacy, were chemoresistant and tumorigenic in vivo, distinct from the CD133- cells. Thus, the SCLC CD133 expressing cells fulfil most criteria of CSClike definition. The molecular mechanisms associated with CD133+ SCLC chemoresistance and growth is unknown. Up-regulated Akt activity, a known promoter of resistance with survival advantage, was observed in CD133+ SCLC cells. Likewise, these cells demonstrated elevated expression of Bcl-2, an anti-apoptotic protein compared to their negative counterpart explaining CD133+ cell chemoresistance phenotype. Additionally, CD133+ cells revealed greater expression of neuropeptide receptors, gastrin releasing peptide (GRP) and V1A receptors compared to the CD133- cells. Addition of exogenous GRP and arginine vasopressin (AVP) to CD133+ SCLC cells promoted their clonogenic growth in semi-solid medium, illustrating for the first time neuropeptide dependent growth of these cells. A novel peptide (peptide-1) was designed based on the known structure of the substance P analogues that have shown benefit in animal models and in early clinical trials. This compound inhibited the growth of SCLC cells in in vitro with improved potency and stability compared to previous analogues and reduced tumorigenicity in vivo. Interestingly, peptide-1 was more effective in CD133+ cells due to increased expression of neuropeptide receptors on these cells. In conclusion, my results show that SCLC cells retain a sub-population of cells that demonstrate CSC-like phenotype. Preferential activation of Akt and Bcl-2 survival pathways and enhanced expression of neuropeptide receptors contribute to CD133+ SCLC chemoresistance and growth. Therefore, it can be proposed that CD133+ cells are the possible cause of SCLC development, treatment resistance and disease recurrence. Despite being chemoresistant, CD133+ cells demonstrated sensitivity to peptide-1. The identification of such new analogue that demonstrates efficacy towards resistant CD133+ SCLC cells is a very exciting step forward in the identification of a potential new therapy for resistant disease.

Stem cell therapies and tissue engineering strategies are required for the clinical treatment of respiratory diseases. Previous studies have established protocols for the differentiation of airway epithelium from stem cells but have involved costly and laborious culture methods. The aim of this thesis was to achieve efficient and reproducible maintenance and differentiation of embryonic stem cells to airway epithelium, in 2D and 3D culture, by developing appropriate bioprocessing technology. Firstly, the 2D differentiation process of human and murine ES cells into pulmonary epithelial cells was addressed. The main finding in was that the proportion of type II pneumocytes, the major epithelial component of the gas-exchange area of lung, differentiated with this method was higher than that obtained in previous sudies, 33% of resultant cell expressed the specific marker surfactant protein C (SPC) compared with up to 10%. Secondly, the maintenance and differentiation was carried out in 3D. A protocol was devised that maintained undifferentiated human ES cells in culture for more than 200 days encapsulated in alginate without any feeder layer or growth factors. For ES cell differentiation in 3D, a method was devised to provide a relatively cheap and simple means of culture and use medium conditioned by a human pneumocyte tumour cell line (A549). The differentiation of human and murine ES cells into pulmonary epithelial cells, particularly type II pneumocytes, was found to be upregulated by culture in this conditioned medium, with or without embryoid body formation. The third step was to test whether this differentiation protocol was amenable to scale-up and automation in a bioreactor using cell encapsulation. It was possible to show that encapsulated murine ES cells cultured in static, co-culture or rotating wall bioreactor (HARV) systems, differentiate into endoderm and, predominantly, type I and II pneumocytes. Flow cytometry revealed that the mean yield of differentiated type II pneumocytes was around 50% at day 10 of cultivation. The final stage of the work was to design and produce a perfusion system airlift bioreactor to mimic the pulmonary microenvironment in order to achieve large scale production of biologically functional tissue. The results of these studies thus provide new protocols for the maintenance of ES cells and their differentiation towards pulmonary phenotypes that are relatively simple and cheap and can be applied in bioreactor systems that provide for the kind of scale up of differentiated cell production needed for future clinical applications.

Glioma is a malignant tumor with a high mortality rate within few years of diagnosis. Due to limited effect of currently available cancer therapies, alternative therapies are investigated. Studies have shown that mesenchymal stem cells (MSCs) may have a therapeutic potential in glioma treatment. The objective of this in-vitro study was to examine the effect of three different human dental MSCs (D-MSC) on rat glioma cells. We utilized three experimental set ups in order to study the effect of D-MSC on glioma cell survival: unstimulated conditioned medium, stimulated conditioned medium, and direct co-culture. Unstimulated conditioned medium showed an inhibitory effect of 10-30 % on cell survival. Stimulated conditioned medium showed no statistically significant inhibition of glioma cell survival. Direct co-culture immunofluorescence microscopy revealed propidium iodide absorption, indicating cell death, in glioma. Results indicate that D-MSC has a negative influence on glioma proliferation but secreted/expressed factors mediating this effect are unidentified. This is a screening of D-MSCs potential therapeutic effect in treating glioma.

Although the current system of blood transfusion is relatively safe and established within the UK, periodic shortages of certain blood groups and residual risks of emerging transfusion transmitted infections (TTIs) make an industrial manufacture process for the generation of red blood cells more desirable. The generation of red blood cells from human embryonic stem cells has been completed in vitro but the major challenge lies in making the process highly scalable and economically viable. Initially human embryonic and induced pluripotent stem cells were trialled for use on the project however these were found to be inconsistent, a major issue in cellular therapies. They were replaced with CD34+ cord blood stem cells which are morphologically and physiologically divergent. In order to assess their suitability for a GMP-complaint manufacturing process an ultra-scale down (microfluidic) approach was taken to assess the cells’ reactions to the changeable physical environment associated with scale-up procedures. Cellular responses to hypoxia and shear stress were evaluated at successive time-points in the step-wise haematopoietic differentiation process and recommendations made for optimum scale-up conditions. Conversely further challenges in the manufacture of red blood cells from stem cells were uncovered regarding the differences between stem cell derived red blood cells and their adult equivalents.

If nuclear organisation regulates gene expression, then it may have a role in gene silencing as a cell commits to differentiation. It is therefore important to determine the nuclear organisation of stem cells. Here I ask if the nuclear architecture of human and mouse embryonic stem (ES) cells is different from that of differentiated cells? I have examined the position of chromosomes 18 (gene-poor) and 19 (gene-rich) and show that their radial nuclear organisation, seen in differentiated cells, is already established in human ES cells. However, I show that the position of centromeres is different in ES cells and differentiated cells. I have investigated the location of two gene dense regions, 11p15 and 6p21 (which contains the pluripotent gene OCT4) and the 12p13 region (containing NANOG), in differentiated and human ES cells. I show that although the position of gene dense regions is maintained through differentiation, specific genes involved in pluripotency change position either within the nucleus or relative to their chromosome territories. In parallel I have established data on nuclear reorganisation during differentiation of murine ES cells towards an ectodermal lineage. I show the relocalisation of chromosome territories and genes involved in neuronal development during differentiation, which is accompanied by substantial clustering of centromeric heterochromatin. Using mutant mouse ES cells I show that clustering still occurs in cells the lack DNA methylation, the DNA binding protein MeCP2 and the Suv39h histone methyltransferase. I conclude that although some of the basic principles of nuclear organisation are already established in ES cells, spatial reorganisation of the genome is evident during differentiation.

Extracellular matrix (ECM)-rich basal laminae are an important component of other stem cell niches and thus are likely to form part of the NSC niche. Interactions with the ECM are mediated manly by cell surface heterodimers called integrins. β1 integrins have been implicated in maintaining human epidermal stem cells, and their elevated expression has allowed the enrichment of human prostate epithelial stem cells from transit amplifying populations. My work has focused on the role of the ECM and its receptors in the mammalian CNS stem cell niche. Initial experiments examined the conditions necessary to grow human NSCs under clonal conditions, before using these findings to demonstrate that these cells express higher levels of integrin α6β1 then progenitor or differential cell types. This led to the question of the role of integrins in NSC behaviour, which was investigated in vitro with human and murine tissue using integrin activating/blocking antibodies together with ECM molecules. Laminin-211, an α6β1 ligand, was found to increase NSC survival in an integrin-dependent manner. However, activation of integrin β1 did not recapitulate these results, indicating that integrin β1 is necessary, but not sufficient, for laminin to increase survival. Finally, in utero injections of the integrin activating/block antibodies into the lateral ventricle of embryonic day 15 (E15) embryos were conducted in order to examine the role of integrins in the in vivo stem cell niche. In conclusion, the findings indicate that integrins are highly expressed by neural stem cells and that integrins function in vitro and in vivo to influence stem cell behaviour.

MSCs are the most studied subtype of adult stem cells and have been derived from most postnatal organs and tissues. MSCs are defined as having the capacity to self-renew and to differentiate into both mesodermal and non-mesodermal lineages, and are immunosuppressive. For these properties, MSCs have been considered ideal candidates for regenerative medicine and have been used in several clinical trials. The difficulty, however, to preserve the potency of the cells during culture expansion and to monitor differentiation are obstacles in their use in the clinic and have emphasized the need to investigate molecular pathways underlying stem cell fate-decisions during differentiation in more detail. Hippo pathway was recently identified in Drosophila melanogaster and mammals, and controls organ size by regulating cell proliferation, apoptosis and differentiation. It is composed of a core of serine/threonine kinases and scaffold proteins that when activated, phosphorylate and inhibit yes-associated protein (YAP) transcriptional co-factor. Inactivation of YAP in some stem and progenitor cells by this pathway is required for their differentiation. In contrast, failure to inhibit YAP enhances proliferation and may cause oncogenic transformation. In the present study, the expression of multiple YAP variants was confirmed in human and mouse MSCs. In both human and mouse, YAP was inhibited in response to cell-contact inhibition and remained unchanged during in vitro chondrogenic differentiation. Overexpression of human (hYAP1) variant in C3H/10T1/2 cells did not appear to affect colony formation, cell cycle distribution or cell size, but increased cell proliferation, induced cell transformation and reduced the in vitro differentiation capacity of the cells towards the chondrogenic, adipogenic and osteogenic lineages. The effects of hYAP1 overexpression are hypothesized to be either a result of a nuclear co-factor function or indirectly via protein interactions in the cytoplasmic compartment. Hippo pathway and YAP are possible pharmacological targets for modulation of MSC function.

In adult life mesenchymal stem cells (MSCs) reside primarily in the bone marrow and are defined according to their ability to self-renew and differentiate into tissues of mesodermal origin. Due to their immuno-modulatory properties and ability to form cartilage and bone, MSCs have clinical potential, for the treatment of autoimmune diseases and tissue repair. This project determines the chemokine receptor profile on murine bone marrow MSCs at early and late passage and on human MSCs derived from a range of fetal tissues including fetal blood, bone marrow, amniotic fluid and placenta. The overwhelming result from this analysis is the consistency across species and tissue source with respect to chemokine receptor profiles. In addition it is clear that expression of specific chemokine receptors defines sub-populations of MSCs. Currently, clinical trials using MSCs have relied on continued in vitro culture in order to obtain sufficient numbers for treatment. Here, MSCs have been shown to lose external chemokine receptor expression and associated chemotactic ability, whilst growing in size upon continued culture. All cultured MSCs investigated in this thesis were shown to be a heterogeneous population of stem cells and progenitors that contained ‘true’ MSCs within its number. This thesis investigates a pharmacological approach to mobilise endogenous MSCs from the bone marrow, increasing their numbers in the blood. It has previously been reported that administration of VEGF-A over 4 days followed by a single dose with a CXCR4 antagonist (AMD3100) causes selective mobilisation of MSCs into blood. The VEGF biology of this response has been interrogated. MSCs were shown to express high levels of VEGFR-1 and lower levels of VEGFR-2 on the cell surface but do not express VEGFR-3. By blocking VEGFR-1 with mAbs during VEGF-A165 treatment, a ten-fold increase in MSC mobilisation in response to AMD3100 was recorded, while treating with VEGFR-2 blocking mAbs had no effect. Using VEGF isoforms specific for VEGFR-1 and VEGFR-2 (PlGF and VEGF-E respectively), it was determined that MSC mobilisation was dependant on activation of VEGFR-2 and not VEGFR-1. PαS cells are a subset of MSCs found in the murine bone marrow that are PDGFRα+, Sca-1+, CD45-, Ter119-. Further characterisation of mobilised mMSCs by flow cytometric analysis of PαS cells, now provides a way to investigate the biology of MSCs, both in their steady state in vivo and in models of injury and inflammation. Molecular mechanisms lying downstream of VEGFR-2 have been explored and it has been shown that MMPs play a critical role in mobilisation. The use of drugs to mobilise MSCs into the blood may provide a cost effective, non-invasive treatment to promote tissue repair.

The cornea is the anterior most structure at the front of the eye and its most important function is to transmit light to the retina for a clear vision. The external surface of the cornea is composed of an epithelium which is continuous with the surrounding conjunctiva. The corneal limbus forms the narrow transition zone between the corneal and conjunctival epithelia and is believed to harbour the cornea stem cells. Limbal stem cell (LSC) deficiency (LSCD) is a painful and blinding condition of the eye. The recent technique of ex vivo expansion of healthy autologous limbal epithelia for transplantation is the mainstay of cellular therapy for this condition. Successful outcome of ex vivo expanded LSC transplantation is dependent on the quality of the transplanted tissues. The main aims of this thesis are; i) to characterise human LSC from both primary LEC (LEC) and from a human telomerase-immortalised corneal epithelial cell (HTCEC) line, ii) to enrich for LSC by utilising the side population (SP) discrimination assay. Further, adult stem cells are promising candidates for promoting donor-specific tolerance and adult stem cells have immunosuppressive mechanisms to protect them from immunological reactions which are damaging to the survival of the transplanted tissues. Therefore, another aim of the study was iii) to investigate the immunobiological aspects of LSC including HLA expression and cellular migration which can promote the success of cellular transplantation. LSC markers were characterised in both LEC and HTCEC. Using an optimised protocol, SP were identified in LEC and HTCEC. Isolated limbal mesenchymal stem cells (MSC) fulfil the minimum requirement of a human MSC. These limbal MSC (LMSC) exhibited plasticity, could maintain the expression of limbal markers and demonstrated viable growth on a biological substrate, qualities making them a suitable alternative to cultured limbal explants for clinical transplantation. SP cells in HTCEC and LMSC expressed known common limbal markers, stem cell antigens and the chemokine CXCR4. The presence of CXCR4 and CXCL12-mediated cellular migration were demonstrated in HTCEC. Further, HTCEC constitutively expressed HLA Class I antigens, while HLA Class II expressions were induced by Interferon-γ stimulation. In this study, HTCEC HLA-typing was presented and that HTCEC were in many ways comparable to LEC, therefore suitable as a LSC replacement or as a robust model for further understanding of LSC biology.

Background: Vascular endothelial dysfunction plays a major role in the pathogenesis of atherosclerosis. As such, the study of endothelial cells has sought to identify causal pathways and novel therapeutic approaches to promote vascular repair. Induced pluripotent stem (iPS) cell technology may be a particularly useful tool, and could be used to derive endothelial cells and their progenitors from individuals with endothelial dysfunction to explore these pathways and develop novel strategies for vascular regeneration. Whilst iPS cells are conventionally obtained from the reprogramming of dermal fibroblasts, it was hypothesised that endothelial cells could also be reprogrammed, and that these pluripotent cells would have enhanced capacity for endothelial differentiation and vascular regeneration. Objectives: To generate iPS cells from human fibroblasts and endothelial cells and to assess their potential for endothelial differentiation and vascular regeneration. Methods and Results: A) Reprogramming: Dermal fibroblasts and endothelial outgrowth cells from blood were obtained from healthy donors (n=5) and transfected with episomal vectors containing six reprogramming factors: Sox2, Klf4, Oct3/4, L-Myc, Lin28 and Shp53. Successfully reprogrammed fibroblast-derived iPS (fiPS) and endothelial cell-derived iPS (eiPS) arose as colonies, and were isolated and expanded. Reprogrammed cells expressed pluripotency markers SSEA3, SSEA4, TRA 1 60, Oct3/4 and NANOG, and developed into all three germ layers following embryoid body formation. B) Endothelial differentiation: iPS and ES cell lines were aggregated into embryoid bodies in stem cell growth media containing mesoderminducing cytokines. Embryoid bodies were then disaggregated and cultured in endothelial medium supplemented with VEGF. After seven days, a population of CD31+ cells was isolated and further cultured. Mature endothelial cell antigen expression was confirmed by flow cytometry. CD31+ cells were similar to mature endothelial cells in functional assays of proliferation, migration, nitric oxide production and angiogenesis. C) Comparison of fiPS versus eiPS: eiPS differentiated into endothelial cells with greater efficiency than fiPS (21±3% versus 3±2%, P < 0.05). fiPS-derived endothelial cells and eiPS-derived endothelial cells expressed similar levels of endothelial markers CD146, CD31, VEFGR2 and CD34 compared to control endothelial cells. When grown on Matrigel, they formed tubule-like structures with a similar number of vessel connections. In vivo, endothelial cells derived from fiPS and eiPS increased neovasculogenesis in a nude mouse model: vessel density was increased after implantation of endothelial cells from fiPS and eiPS by 3.50 vessel counts (P≤0.001) and 3.47 vessel counts (P≤0.001) respectively, when compared to controls. By comparison control endothelial cells did not increase vessel density compared to control (P > 0.05). Conclusions: Endothelial cells can be isolated from blood and reprogrammed to form pluripotent stem cells with enhanced capacity to differentiate into endothelial cells than those derived from dermal fibroblasts. Endothelial cells derived from both sources promote angiogenesis in vivo, and have major potential for therapeutic applications in vascular regeneration.

Trabalho apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Licenciado em Análises Clínicas e Saúde Pública
Esta monografia teve como objectivo, verificar o estado da arte no que se refere à utilização de células estaminais no tratamento de lesões e de doenças degenerativas do sistema nervoso central. Foram abordados ao longo deste trabalho de revisão, alguns aspectos fundamentais, descritos em diversos artigos científicos de âmbito internacional que definem o estado actual do desenvolvimento de terapias de recuperação do sistema nervoso central com base nas células estaminais, bem como as várias formas como estas poderão ser aplicadas. A terapia celular com recurso a células estaminais é uma área de investigação com bastante potencial para o desenvolvimento de tratamentos para as lesões e doenças degenerativas do sistema nervoso central. Este tipo de tratamento é de particular importância, sobretudo devido à baixa capacidade de regeneração do sistema nervoso central. Possibilita a recuperação através de vários mecanismos, podendo repor ou melhorar a função do sistema nervoso lesado, tanto através da reposição celular, bem como, através da promoção da protecção neural libertando substâncias terapêuticas, e pode envolver a utilização de células de origem neural ou não neural. Já foi demonstrada a eficácia terapêutica na utilização de várias fontes celulares. Contudo, de acordo com a literatura consultada, nenhum dos resultados mostrou uma importância clínica que justifique a sua aplicação na medicina actual. Por outro lado também apoiam a ideia de que, embora ainda existam muitos obstáculos a ultrapassar, a recuperação do sistema nervoso central lesado é uma hipótese bem mais realista do que anteriormente se poderia pensar. This work had as aim to check “the state of the art” in what concerns the use of stem cells to treat lesions and degenerative diseases of the central nervous system. This review work covers some fundamental aspects, mentioned in several scientific articles of international extent, that define the present stage of a cell therapy treatment as to recover the central nervous system, and the different ways of how they can be applied. Cell therapy using stem cells is an area of medical investigation with a very good potential in the development of therapeutics to treat lesions and degenerative diseases of the central nervous system. This kind of approach is of particular importance, especially due to the low capacity of regeneration of the central nervous system. It allows the central nervous system to recover by means of several mechanisms, being able to restore or to improve the function of the injured central nervous system, both through cell reposition, as well as with the release of therapeutic substances, and it can involve the use of cells of neural or non neural origin. It has already been shown the therapeutic efficiency of stem cells from various sources. Even so, according to the articles consulted, none of the results showed a clinical relevance that justifies the application of stem cells in the current medicine. Nevertheless, the results also support the idea that, even with many hurdles to overcome, the recovery of the injured central nervous system may be more realistic than previously thought.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 3, 2009) Vita. Includes bibliographical references.