[Truncated abstract] Hepatic progenitor cells (HPC) are becoming increasingly recognized as facultative stem cells capable of regenerating the liver during chronic liver injury and also as targets of malignant transformation. Similar markers are expressed by hepatocellular carcinoma (HCC) and HPC, and a precursor-product relationship is well established. This thesis focuses on the ways in which the HPC population can be controlled under circumstances of chronic liver injury, and in this manner, reduce the risk of progression to HCC reduced. The major aim of Chapters 3 to 5 was to elucidate the effect of interferon α (IFNα) therapy on HPC. Chronic hepatitis C affects approximately 250 million individuals world wide. Approximately 80% of infections progress to chronicity, which places the individuals at greater risk of developing HCC. The gold standard of treatment of chronic hepatitis C is a combination of pegylated IFNα and ribavirin. ...The results were surprising. While IFNγ exerted a pro-apoptotic and antiproliferative effect on HPC in vitro, administration of IFNγ to CDE-fed mice for 14 days increased fibrosis, enhanced inflammatory infiltration and exacerbated the HPC response, with concurrent hepatocyte cell death. In addition, increased morbidity and mortality were observed in the IFNγ-treated mice compared to control. IFNγ treatment was found to prime the liver for the HPC response by recruiting inflammatory cells and altering the hepatic cytokine profile, both of which may facilitate an increased HPC response. Numbers of activated HSC were also increased in the IFNγ-treated, CDE-fed mice, correlating with the increased fibrosis seen in these animals. This data contradicts the current experimental use of IFNγ for treatment of fibrosis. Based on our results, we suggest that IFNγ promotes HPC proliferation in the CDE model, by encouraging inflammatory infiltration and hepatocyte damage and this initiates pro-fibrotic events. Concurrent proliferation of HPC and activated HSC further supports the view that there is a close relationship between the two cell types, and thus, a link between the HPC response and fibrosis. In conclusion, findings documented in this thesis suggest that administration of IFNα and IFNγ can contribute to shaping the HPC response. IFNα therapy may reduce HCC risk in chronic hepatitis C patients by bringing the HPC population under control. In contrast, IFNγ treatment can exacerbate the HPC response, liver fibrosis and parenchymal damage, illustrating the need to approach this method of fibrosis treatment with caution.

In experimental models of demyelination such as cat optic nerve injected with antibody to galactocerebroside, stepwise and ultimately full repair occurs, starting with recruitment of oligodendrocyte progenitor cells (OP) from surrounding tissue and culminating in remyelination by young competent oligodendrocytes. Endogenous repair of demyelination can also occur in the adult human central nervous system, as evidenced by remyelinated shadow plaques in MS, but ultimately fails in this disease, leading to areas of chronic demyelination where surviving axons are both dysfunctional in terms of conduction and vulnerable to ongoing damage. In order to meaningfully investigate this failure of remyelination in the human situation, an essential prerequisite is to be able to reliably identify the neuroglial cells, and in particular, oligodendrocyte lineage cells, involved in the repair pathway in situ in post mortem tissue. While some marker antigens have been shown to remain demonstrable despite autolytic change and through differing fixation levels, others are far more sensitive and only reliable in freshly obtained tissue with light fixation. For instance, the surface antigens NG2 and PDGFαR, which have been widely used in experimental studies as a marker for OP both in vivo and in vitro, have been shown to be adversely affected by both fixation and autolysis. To this end, the cat optic nerve demyelination model, in which the reparative oligodendrocyte lineage stages have been antigenically defined, was extended to normal optic nerve including lightly fixed tissue. Here, NG2, PDGFαR and the oligodendrocyte lineage transcription factors Olig1 and Olig2 were able to be demonstrated and then correlated with the existing antigenic phenotypes. Subsequently, normal human optic nerve, optimised for both morphological preservation and antigen retention, was used to develop an in vivo staining profile for all neuroglia including OP, that was then applied to conventionally prepared, normal and MS tissue. It was found that, with careful attention to technical parameters such as post mortem interval and details of fixation, OP and other stages of the remyelinating oligodendrocyte lineage could be identified in such material, resulting in meaningful insight into the repair status of the three MS samples studied.

Diss. (sammanfattning) Göteborg : Göteborg University, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves: 147-162). Also available on the Internet.

No description available.

abstract: Traumatic brain injury (TBI) is a significant public health concern in the U.S., where approximately 1.7 million Americans sustain a TBI annually, an estimated 52,000 of which lead to death. Almost half (43%) of all TBI patients report experiencing long-term cognitive and/or motor dysfunction. These long-term deficits are largely due to the expansive biochemical injury that underlies the mechanical injury traditionally associated with TBI. Despite this, there are currently no clinically available therapies that directly address these underlying pathologies. Preclinical studies have looked at stem cell transplantation as a means to mitigate the effects of the biochemical injury with moderate success; however, transplants suffer very low retention and engraftment rates (2-4%). Therefore, transplants need better tools to dynamically respond to the injury microenvironment. One approach to develop new tools for stem cell transplants may be to look towards the endogenous repair response for inspiration. Specifically, activated cell types surrounding the injury secrete the chemokine stromal cell-derived factor-1α (SDF-1α), which has been shown to play a critical role in recruiting endogenous neural progenitor/stem cells (NPSCs) to the site of injury. Therefore, it was hypothesized that improving NPSC response to SDF-1α may be a viable mechanism for improving NPSC transplant retention and migration into the surrounding host tissue. To this end, work presented here has 1. identified critical extracellular signals that mediate the NPSC response to SDF-1α, 2. incorporated these findings into the development of a transplantation platform that increases NPSC responsiveness to SDF-1α and 3. observed increased NPSC responsiveness to local exogenous SDF-1α signaling following transplantation within our novel system. Future work will include studies investigating NSPC response to endogenous, injury-induced SDF-1α and the application of this work to understanding differences between stem cell sources and their implications in cell therapies. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2015

abstract: Several debilitating neurological disorders, such as Alzheimer's disease, stroke, and spinal cord injury, are characterized by the damage or loss of neuronal cell types in the central nervous system (CNS). Human neural progenitor cells (hNPCs) derived from human pluripotent stem cells (hPSCs) can proliferate extensively and differentiate into the various neuronal subtypes and supporting cells that comprise the CNS. As such, hNPCs have tremendous potential for disease modeling, drug screening, and regenerative medicine applications. However, the use hNPCs for the study and treatment of neurological diseases requires the development of defined, robust, and scalable methods for their expansion and neuronal differentiation. To that end a rational design process was used to develop a vitronectin-derived peptide (VDP)-based substrate to support the growth and neuronal differentiation of hNPCs in conventional two-dimensional (2-D) culture and large-scale microcarrier (MC)-based suspension culture. Compared to hNPCs cultured on ECMP-based substrates, hNPCs grown on VDP-coated surfaces displayed similar morphologies, growth rates, and high expression levels of hNPC multipotency markers. Furthermore, VDP surfaces supported the directed differentiation of hNPCs to neurons at similar levels to cells differentiated on ECMP substrates. Here it has been demonstrated that VDP is a robust growth and differentiation matrix, as demonstrated by its ability to support the expansions and neuronal differentiation of hNPCs derived from three hESC (H9, HUES9, and HSF4) and one hiPSC (RiPSC) cell lines. Finally, it has been shown that VDP allows for the expansion or neuronal differentiation of hNPCs to quantities (>1010) necessary for drug screening or regenerative medicine purposes. In the future, the use of VDP as a defined culture substrate will significantly advance the clinical application of hNPCs and their derivatives as it will enable the large-scale expansion and neuronal differentiation of hNPCs in quantities necessary for disease modeling, drug screening, and regenerative medicine applications. / Dissertation/Thesis / Masters Thesis Bioengineering 2016

abstract: Neurodegenerative diseases such as Alzheimer’s Disease, Parkinson’s Disease and Amyotrophic Lateral Sclerosis are marked by the loss of different types of neurons and glial cells in the central nervous system (CNS). Human Pluripotent Stem Cell (hPSC)-derived Neural Progenitor Cells (hNPCs) have the ability to self-renew indefinitely and to differentiate into various cell types of the CNS. HNPCs can be used in cell based therapies and have the potential to reverse or arrest neurodegeneration and to replace lost neurons and glial cells. However, the lack of completely defined, scalable systems to culture these cells, limits their therapeutic and clinical applications. In a previous study, a completely defined, robust, synthetic peptide- a Vitronectin Derived Peptide (VDP) that supports the long term expansion and differentiation of various embryonic and induced pluripotent stem cell (hESC/hIPSC) derived hNPC lines on two dimensional (2D) tissue culture plates was identified. In this study, the culture of hNPCs was scaled up using VDP coated microcarriers (MC). VDP MC were able to support the long term expansion of hESC and hiPSC derived hNPCs over multiple passages and supported higher fold changes in cell densities, compared to VDP coated 2D surfaces. VDP MC also showed the ability to support the neuronal differentiation of hNPCs, and produced mature neurons expressing several neuronal, neurotransmitter and cortical markers. Additionally, alzheimer’s disease (AD) relevant phenotypes were studied in patient hIPSC derived hNPCs cultured on laminin MC to assess if the MC culture system could be used for disease modelling and drug screening. Finally, a microcarrier based bioreactor system was developed for the large scale expansion of hNPCs, exhibiting more than a five-fold change in cell density and supporting more than 100 million hNPCs in culture. Thus, the development of a xeno-free, scalable system allows hNPC culture under standard and reproducible conditions in quantities required for therapeutic and clinical applications. / Dissertation/Thesis / Masters Thesis Bioengineering 2017

Existem evidências na literatura que demonstram a existência de células indiferenciadas na capsula adrenal, e que o ACTH poderia estimular estas células. Porém não se sabe quais os genes e vias que desencadeiam esta resposta. Através de animais Cre-Lox induzível por Tamoxifeno, silenciamos o gene Pomc em camundongos adultos e avaliamos o efeito do ACTH nessas células. Foram utilizadas placas de PCR array para análise de genes relacionados com células progenitoras em amostras obtidas pela técnica de rolamento, e validação por PCRq com amostras microdissecadas da zona capsular/subcapsular da adrenal. Após caracterização dos animais com o gene Pomc silenciado e tratamentos com ACTH observamos o aumento da expressão de genes relacionados com as vias Wnt, Igf1 e Notch. Esses dados corroboram evidencias descritas na literatura que mostram a importância dessas vias no desenvolvimento e manutenção do córtex adrenal, e sugerem o envolvimento do ACTH nesses processos que envolvem as células progenitoras do córtex adrenal. / There is evidence in the literature demonstrating the existence of stem cells in the adrenal capsule, and that ACTH could stimulate these cells. However, it remains unknown which genes and pathways that trigger this response. By using a tamoxifen-inducible Cre-Lox mice strain, we knocked-out Pomc gene in adult mice and evaluated the effect of ACTH in these cells. PCR array technique was used to determine the expression level of key genes related to progenitor cells in samples obtained by the technique of \"rolling bearing\". Also, we validated the data by qPCR using samples from microdissected capsular areas of the adrenal gland. After characterization of animal model, the results show that treatment with ACTH increase the expression of genes related to Wnt, Igf1 and Notch pathways. These data corroborate with the literature, reinforcing the importance of these pathways in the development and maintenance of the adrenal cortex, and also suggesting the involvement of ACTH in these processes involving the progenitor cells of the adrenal cortex.

Em vista das limitações éticas em torno da obtenção de células-tronco de fetos humanos, o cão é uma alternativa para estes estudos. Além disso, a terapia celular proporciona novas expectativas para o tratamento na espécie. Realizamos o estudo comparativo das células isoladas de saco vitelino, fígado e medula óssea de fetos caninos. As células foram analisadas microscopicamente e ultra estruturalmente. O imunofenótipo das células foi avaliado através de marcadores. Caracterizamos a plasticidade, o cariótipo e o potencial teratogênico destas células. Após expansão as células progenitoras formaram colônias com morfologia fibroblastóide. As células progenitoras do saco vitelino e medula óssea são compostas por: células com alta proporção núcleo-citoplasma e células com citoplasma rico em organelas, enquanto que as células progenitoras do fígado eram semelhantes à célula epitelial e células ricas em organelas. As células-progenitoras dos três tecidos fetais foram positivas para os anticorpos nestina e vimentina, mas negativas para CD45 e CD13. Células progenitoras de medula óssea foram positivas para o marcador CD44. Células progenitoras do fígado e medula óssea expressaram a proteína citoqueratina-18, enquanto as do saco vitelino expressaram ve-caderina. Células positivas para Oct3/4 foram detectadas em todas as células progenitoras. As células-progenitoras do saco vitelino e medula óssea diferenciaram-se em tecidos ósseo, cartilaginoso e muscular; já as do fígado para tecido ósseo e muscular. Nenhum tipo celular diferenciou-se em adipócitos. As células progenitoras da medula óssea diferenciaram em células semelhantes a neurônios. Sugere-se a presença de progenitores semelhantes a células mesenquimais e epiteliais. Todas as células mantiveram o cariótipo estável e não formaram tumores. Células progenitoras de medula óssea apresentaram maior capacidade de proliferação e diversidade de diferenciação. Sugere-se que estas células são possíveis candidatas para a terapia celular. / The use the human fetuses for stem cells isolation have ethical limitations. In this context the dog is an excellent candidate to fetal stem cells. Furthermore, these cells can be used in cell therapy of canine diseases We aimed at isolation and comparative characterization of progenitor cells from yolk sac, liver and bone marrow of canine fetuses. Cells were characterized using stem cells antibodies. Differentiation assays as well as karyotype analysis were performed. Teratogenic properties this cells were evaluated. After establishment of primary culture, best proliferation potential was observed in bone marrow progenitor cells. Bone marrow and liver progenitor cells were more efficient in CFU-F assay, then yolk sac progenitor cells. Evidenced by TEM cells with a high nuclear-to-cytoplasmic ratio and cells with cytoplasm rich in organelles. Cells isolated from liver showed epithelial-like morphology and cytoplasm rich in organelles. The yolk sac, liver bone marrow cells reacted positively with nestin and vimentin, being negative to CD45 and CD13 antibodies. Additionally bone marrow progenitor cells were positive to CD44. Bone marrow and liver progenitor cells reacted positively with cytokeratin 18. Yolk sac progenitor cells were positive to ve-cadherin. A few Oct3/4 positive cells were found in yolk sac, liver and bone marrow. Yolk sac and bone marrow progenitor cells showed successful osteogenic, chondrogenic, myogenic differentiation. Differentiation liver progenitor cells were able to bone and muscle cells. The bone marrow progenitor cells were able to produce neuron-like cells. None of progenitor cells showed adipogenic differentiation. The study suggests the presence of mesenchymal-like and epithelial-like progenitor cells. All the karyotype remained and failed to induce the formation of tumors. Stem cells from bone marrow showed high diversity of differentiation than other cell types. It is suggested that these cells are possible candidates for cell therapy.