No description available.

During differentiation stem cells are exposed to a range of microenvironmental chemical and physical cues. In this study, human multipotent progenitor cells (hMLPCs) were differentiated from umbilical cord into oligodendrocytes and astrocytes. Chemical cues were represented by a novel defined differentiation medium containing the neurotransmitter norepinephrine (NE). In traditional 2 dimensional (2D) conditions, the hMLPCs differentiated into oligodendrocyte precursors, but did not progress further. However, in a constructed 3 dimensional (3D) environment, the hMLPCs differentiated into committed oligodendrocytes that expressed MBP. When co-cultured with rat embryonic hippocampal neurons (EHNs), hMLPCs developed in astrocytes or oligodendrocytes, based on presence of growth factors in the differentiation medium. In co-culture, physical cues provided by axons were essential for complete differentiation of both astrocytes and oligodendrocytes. This study presents a novel method of obtaining glia from human MLPCs that could eliminate many of the difficulties associated with their differentiation from embryonic stem cells. In addition, it reveals the complex interplay between physical cues and biomolecules on stem cell differentiation.

Indiana University-Purdue University at Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction. Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.

No description available.

No description available.

<p>Recent studies have implicated that a small sub population of cells within a tumour, termed cancer stem cells (CSCs), have an enhanced capacity for tumour formation in multiple cancers and may be responsible for recurrence of the disease after treatment. Further work has suggest that CSCs are radioresistant relative to other cell types composing tumours, in several solid cancers. The genetic and phenotypic heterogeneity of malignant CSCs, as well as the difficulty associated with culturing these cells in vitro, limits the capacity to study the response of CSCs to ionizing radiation. Further, the absence of normal known counterparts for many CSCs has made it difficult to compare the radiation responses of CSCs with the normal stem cells required for post radiotherapy tissue regeneration. Here we have shown that transformed human embryonic stem cells (t-hESCs), showing features of neoplastic progression, produce tumours resistant to radiation relative to their normal counterpart. We further show that t-hESCs have a reduced capacity for radiation induced cell death via apoptosis and exhibit altered cell cycle arrest in vitro, relative to hESCs. We found that decreased levels of p53ser15, following DNA double strand break induction, is associated with this radiation resistance.</p> / Master of Science (MSc)

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial Colony Forming Cells (ECFCs) are highly proliferative endothelial progenitor cells with clonal proliferative potential and in vivo vessel forming ability. While endothelial cells have been derived from human induced pluripotent stem cells (hiPS) or human embryonic stem cells (hES), they are not highly proliferative and require ectopic expression of a TGFβ inhibitor to restrict plasticity. Neuropilin-1 (NRP-1) has been reported to identify the emergence of endothelial precursor cells from human and mouse ES cells undergoing endothelial differentiation. However, the protocol used in that study was not well defined, used uncharacterized neuronal induction reagents in the culture medium, and failed to fully characterize the endothelial cells derived. We hypothesize that NRP-1 expression is critical for the emergence of stable endothelial cells with ECFC properties from hES cells. We developed a novel serum and feeder free defined endothelial differentiation protocol to induce stable endothelial cells possessing cells with cord blood ECFC-like properties from hES cells. We have shown that Day 12 hES cell-derived endothelial cells express the endothelial markers CD31+ NRP-1+, exhibit high proliferative potential at a single cell level, and display robust in vivo vessel forming ability similar to that of cord blood-derived ECFCs. The efficient production of the ECFCs from hES cells is 6 logs higher with this protocol than any previously published method. These results demonstrate progress towards differentiating ECFC from hES and may provide patients with stable autologous cells capable of repairing injured, dysfunctional, or senescent vasculature if these findings can be repeated with hiPS.

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

Development of safe and efficient approaches for gene delivery in human embryonic stem cells (hESc) and particularly in human induced pluripotent stem (hiPS) cells, which can be derived in a person-specific manner, is considered to be imperative for harnessing their full potential in both the basic and applied research. The aim of this study was to evaluate the potential of human artificial chromosome (HAC) for gene delivery and expression in hESc and hiPS cells. HAC offers many potential advantages including the provision for carrying large genes with corresponding regulatory elements to obtain long-term regulated gene expression. In addition, they can replicate and segregate independently without integration into the host cell genome. To develop HAC in hiPS cells, the first part of the study was aimed at generating hiPS cells utilising the Herpes Simplex Virus (HSV)-1 amplicon system. With the use of EBNA-1/OriP retention elements incorporated into the HSV-1 amplicon vectors, hiPS cells completely free of vector and transgenes sequences were successfully derived from human embryonic fibroblasts. The hiPS cells exhibited proliferation and differentiation potential similar to that of hESc. In the second part of the study, development of HAC in hESc and hiPS cells was assessed by utilising the HSV-1 amplicon system to deliver the HAC DNA. Analysis of the hESc confirmed the presence of functional HAC which replicated the behaviour of the host chromosomes. Additionally, HAC generation did not lead to impairment in the developmental potential and pluripotency of hESc. The hiPS cells supported HAC at low frequency but DNA also integrated into the host chromosomes. The HAC system, therefore, needs further refinements to improve the frequency of HAC formation and reduce the chromosomal integration of HAC constructs in hiPS cells. Overall, these findings provide a simple and safe way of pluripotency induction and genetic modification of pluripotent stem cells using the HSV-1 amplicon system and represent an important advance towards patient specific gene and cell therapy.

O diabetes mellitus do tipo 1 (T1D) é uma doença autoimune mediada por células T e caracterizada pela destruição seletiva das células ? pancreáticas produtoras de insulina. Células estromais mesenquimais (MSCs) e células tronco hematopoéticas (HSCs) são os principais componentes do nicho hematopoético na medula óssea. Estas células vêm sendo utilizadas nos últimos anos em transplantes autólogos para tratamento do T1D. O objetivo geral do presente trabalho foi avaliar o perfil de expressão gênica global de MSCs e HSCs de pacientes com T1D e compará-lo com células isoladas de indivíduos saudáveis através da técnica de microarray e programas específicos de bioinformática. As MSCs e HSCs foram isoladas da medula óssea de pacientes com T1D antes e após o tratamento com imunossupressão em altas doses seguida pelo transplante autólogo de células tronco hematopoéticas (AHSCT). As MSCs apresentaram valor elevado de expressão absoluta de diversas moléculas potencialmente relacionadas com suas funções de suporte à hematopoese. MSCs de pacientes diabéticos apresentaram perfil de expressão gênica global distinto das isoladas de indivíduos saudáveis, com hiper-regulação da sinalização via proteína G e hiporregulação da atividade transcricional. O receptor ?3 adrenérgico, assim como a sinalização simpática, foram hiper-expressos nas células dos pacientes. Genes que codificam moléculas que suportam a hematopoese e regulados pelo sistema nervoso simpático, VCAM1 e CXCL12, foram hiporregulados em nossa análise. Após o AHSCT, houve atenuação do perfil de expressão diferencial das MSCs dos pacientes, entretanto elas permaneceram com hiperatividade da sinalização via proteína G e déficit da atividade transcricional. As HSCs apresentaram altos níveis de expressão absoluta de diversas integrinas e receptores de citocinas e fatores de crescimento, potencialmente relacionados com funções na hematopoese. HSCs de pacientes com T1D apresentaram perfil de expressão gênica global distinto das de indivíduos saudáveis, com hiper-regulação de genes associados com a atividade transcricional. Os fatores de transcrição TCFL2 e p53, que têm papel fundamental na regulação do ciclo celular das HSCs, foram diferencialmente expressos entre as HSCs de pacientes diabéticos e controles. Assim, nossos resultados de expressão gênica global apontaram alterações intrínsecas nas HSCs e MSCs de pacientes diabéticos que podem estar relacionadas com a falha terapêutica dos transplantes autólogos. A implicação dessas alterações no desenvolvimento e patogênese do T1D permanece desconhecida e a realização de ensaios funcionais poderá esclarecer o significado biológico das mesmas. / Type 1 diabetes mellitus (T1D) is a T cell-mediated autoimmune disease, characterized by selective destruction of insulin-producing pancreatic ? cells. Mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) are the main components of hematopoietic niches. In the last years, these cells are being used in autologous transplantation settings for T1D treatment. The main goal of this study was to evaluate the global gene expression profile of MSCs and HSCs from T1D patients, by using microarrays and bioinformatics specific programs. MSCs and HSCs were isolated from bone marrow of T1D patients before and after treatment with high dose immunossupression followed by hematopoietic stem cell transplantation. MSCs showed high absolute expression values of several molecules potentially related to their function of hematopoiesis support. MSCs from T1D patients exhibited distinct gene expression profile from control MSCs and presented up-regulation of the G protein-coupled receptor signaling pathway and down-regulation of transcriptional activity. The ?3 adrenergic receptor, as well the sympathetic nervous system signaling were up-regulated on patient´s cells. Genes that codify molecules which support hematopoeisis and are regulated by the symphatic nervous system, VCAM1 and CXCL12, were downregulated on our analysis. After AHSCT, there was an attenuation of the differential expression profile of MSCs from T1D patients, however they remained with G proteincoupled receptor signaling pathway hyperactivity and transcriptional activity deficit. HSCs exhibited high absolute expression values of integrins, cytokine receptors and growth factors, molecules potencially related to hematopoietic functions. HSCs from T1D patients showed distinct expression profile from control HSCs and demonstrated up-regulation of genes related to transcriptional activity. The transcription factors TCFL2 and p53, which have important role in regulating HSC cycle, were differentially expressed between HSCs from T1D patients and controls. Thus, our global gene expression analysis has revealed intrinsic alterations on MSCs and HSCs from T1D patients that could be related to the autologous transplant therapeutic failures. The implications of these alterations on the development and pathogenesis of T1D remain unknown and functional assays could unravel their biological meaning.