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3D differentiation enhances the efficiency of differentiation of human induced pluripotent stem cells to insulin producing cellsRotti, Pavana Gururaj 01 December 2014 (has links)
Type 1 Diabetes (T1D) is an autoimmune disorder in which the pancreatic β-cells are destroyed by the body's immune system. The reduced number of β-cells leads to inadequate insulin secretion and high glucose levels in the body. The requirement of insulin injection throughout life and lack of donors for islet transplantations has prompted a search for more accessible and available sources of insulin producing cells that can be transplanted in T1D patients. To that end, the discovery of induced pluripotent stem (iPS) cells has provided a potential source of precursors for cell therapy for T1D. iPS cells are reprogrammed somatic cells which can be transplanted back into the patient from whom the somatic cells were initially derived, thus potentially avoiding immune rejection when transplanted. As a potential therapy for T1D, we aim to derive insulin producing cells (IPCs) from human iPS cells. In contrast to the conventional two dimensional (2D) cell culture systems used in many iPS derived IPC studies, the inner cell mass (ICM) from which various organs differentiate during embryogenesis is a cluster of cells that enables signaling crosstalk between cells of different types. Three dimensional (3D) cell culture systems allows cells to form cell clusters that promote cell - cell signaling. Hence, we hypothesized that 3D cell culture systems will yield better efficiency of differentiation to functional IPCs in vitro than 2D cultures.
Initially, the synthetic polymers sodium alginate and matrigel were analyzed for their ability to enable cell clustering to establish 3D cell culture systems. The 3D cell environment established using matrigel was used for the differentiation of human iPS cells to Insulin Producing Cells (IPC). The cells were first converted to endodermal cells. A mixture of growth factors then induced the differentiation of endodermal cells to pancreatic cells. The pancreatic cells were converted to IPCs that resemble pancreatic β-cells. Our 3D differentiated IPCs strongly expressed pancreatic endocrine transcription factors and pancreatic hormones. The IPCs also produced insulin when exposed to a high glucose environment. But the number of IPCs obtained at the end of the differentiation was low.
Hence, our results demonstrate that 3D differentiation generates functional IPCs in vitro unlike 2D differentiation. In the future we aim to improve the percentage of IPCs that we generate from the 3D differentiation. Our expectation is that these cells will be able to cure hyperglycemia in diabetic mice more rapidly compared to the 2D differentiated cells owing to their proven insulin production in the presence of a high glucose environment in vitro.
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Generation and function of glucose-responsive insulin producing cells derived from human induced pluripotent stem cellsManzar, Gohar Shahwar 01 August 2015 (has links)
Type I diabetes (T1D) is caused by autoimmune destruction of pancreatic β-cells. Immediate consequences of T1D are severe weight loss, ketoacidosis and death unless insulin is administered. The long-term consequences of T1D are dysregulation of metabolism leading to cardiovascular complications, neuropathy and kidney insufficiency. It is estimated that 3 million Americans have T1D, and its prevalence among young individuals is progressively rising. Islet transplantation is the most effective way to treat T1D. Unfortunately, there is a chronic shortage of cadaveric organ donors to treat all of the patients on the waiting list. Thus, an alternative source of insulin producing cells (IPCs) could significantly improve patient treatment. Our lab seeks to establish human induced pluripotent stem (iPS) cells as a novel source of IPCs that are patient tailored. The aim of this thesis was to 1) compare the differentiation of T1D and nondiabetic (ND) patient-derived iPS cells into IPCs, and 2) devise an effective protocol for differentiating skin fibroblast-derived T1D iPS cells into functional, glucose-responsive IPCs. Initially, T1D iPS cells were differentiated into IPCs. However, the yield was very poor. We hypothesized that epigenetic barriers were prevalent in T1D iPS cells, limiting their differentiation into IPCs. To address this problem, we utilized 5-aza-2’-deoxycytidine (5-aza-DC), a potent demethylating agent that inhibits the DNA methyltransferase (Dnmt). We reasoned that the use of a demethylation agent might induce a more labile, permissive state, allowing for greater cell responses to differentiation stimuli. Typically, after the differentiation of T1D iPS cells, several cell cluster types are obtained, namely compact cell clusters and hollow cysts. 5-aza-DC treatment appeared to convert all of the cell clusters into characteristic islet-like compact structures. In contrast, in untreated T1D IPC cultures, we observed the dominant presence of many hollow cysts with only a few tight spheroids. The hollow cysts stained negative for insulin whereas the rare solid spheroids highly expressed insulin. Flow cytometry analysis indicated a much greater percentage of Pdx1+ and insulin+ cells in 5-Aza-DC-treated cultures. These cells express markers typical of pancreatic β-cells, possessed insulin granules in similar quantities as islets, and were glucose-responsive. When transplanted in immunodeficient mice that had developed streptozotozin-induced diabetes, there was a dramatic decrease of hyperglycemia within 28 days. These mice effectively managed glucose challenge by recovering to normoglycemia, whereas nontransplanted mice did not. Altogether, our data for the first time reveal a very high yield of functional IPCs derived from human iPS cells derived from a patient with T1D, which presents a novel alternative source of IPCs that could be used to treat T1D.
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Diferenciação de células-tronco embrionárias murinas (mESCs) em células produtoras de insulina (IPCs) e caracterização funcional do gene Purkinje cell protein 4 (Pcp4) neste processo / Differentiation of murine embryonic stem cells (mESCs) into insulin-producing cells (IPCs) and functional characterization of the Purkinje Cell Protein 4 (Pcp4) gene in this processKossugue, Patricia Mayumi 28 May 2013 (has links)
Fontes alternativas de células β têm sido estudadas para o tratamento de Diabetes mellitus tipo 1, dentre as quais a mais promissora consiste das células-tronco diferenciadas em células produtoras de insulina (IPCs). Alguns trabalhos demonstram a capacidade de células-tronco embrionárias murinas (mESCs) de formarem estruturas semelhantes a ilhotas pancreáticas, porém, os níveis de produção de insulina são insuficientes para a reversão do diabetes em camundongos diabetizados. Este trabalho visa desenvolver um protocolo adequado para geração de IPCs e contribuir para a identificação e caracterização funcional de novos genes associados à organogênese pancreática. Logo no início da diferenciação das mESCs em IPCs, foi possível verificar o surgimento de células progenitoras, evidenciado pela expressão de marcadores importantes da diferenciação beta-pancreática. Ao final do processo de diferenciação in vitro, ocorreu a formação de agrupamentos (clusters) semelhantes a ilhotas, corando positivamente por ditizona, que é específica para células β-pancreáticas. Para avaliar seu potencial in vivo, estes clusters foram microencapsulados em Biodritina® e transplantados em camundongos diabetizados. Apesar dos níveis de insulina produzidos não serem suficientes para estabelecer a normoglicemia, os animais tratados com IPCs apresentaram melhores condições, quando comparados ao grupo controle, tendo melhor controle glicêmico, ganho de massa corpórea e melhor aparência da pelagem, na ausência de apatia. Além disso, análise dos clusters transplantados nestes animais indicou aumento da expressão de genes relacionados à maturação das células β. Porém, quando estes clusters foram microencapsuladas em Bioprotect® e submetidos à maturação in vivo em animais normais, ocorreu um aumento drástico na expressão de todos os genes analisados, indicando sua maturação completa em células beta. O transplante destas células completamente maturadas em animais diabetizados, tornou-os normoglicêmicos e capazes de responder ao teste de tolerância à glicose (OGTT) de forma semelhante aos animais normais. A segunda parte do trabalho visou analisar genes diferencialmente expressos identificados em estudo anterior do nosso grupo, comparando, através de DNA microarray, mESCs indiferenciadas e diferenciadas em IPCs. Um dos genes diferencialmente expressos é aquele que codifica para a Purkinge cell protein 4 (Pcp4), sendo 3.700 vezes mais expresso em IPCs. Para investigar o possível papel do gene Pcp4 em células β e no processo de diferenciação β-pancreática, adotou-se o enfoque de genômica funcional, superexpressando e inibindo sua expressão em células MIN-6 e mESCs. Apesar da alteração na expressão de Pcp4 em células MIN-6 não ter interferido de forma expressiva na expressão dos genes analisados, quando inibido, modificou o perfil da curva de crescimento celular, aumentando seu tempo de dobramento de forma significativa e diminuindo da viabilidade celular em ensaios de indução de apoptose. Já na diferenciação de mESCs em IPCs, a superexpressão de Pcp4 interferiu de forma positiva apresentando uma tendência a aumentar a expressão dos genes relacionado à diferenciaçãoβ-pancreática. Concluindo, desenvolvemos um novo protocolo de diferenciação de mESCs em IPCs as quais foram capazes de reverter o diabetes em camundongos diabetizados e descrevemos, pela primeira vez, o gene Pcp4 como sendo expresso em células β-pancreáticas, podendo estar relacionado à manutenção da viabilidade celular e maturação destas células. / New cellular sources for type 1 Diabetes mellitus treatment have been previously investigated, the most promising of which seems to be the insulin producing cells (IPCs), obtained by stem cells differentiation. Some reports show that murine embryonic stem cells (mESCs) are able to form islet-like structures, however, their insulin production is insufficient to render diabetic mice normoglycemic. This work aims at developing an adequate protocol for generation of IPCs and searching for new genes which could be involved in the pancreatic organogenesis process. Early on during mESCs differentiation into IPCs, we observed the presence of progenitor cells, which were able to express pancreatic β-cell markers. At the end of the differentiation process, the islet-like clusters positively stained for the insulin-specific dithizone. These clusters were microencapsulated in Biodritin® microcapsules, and then transplanted into diabetized mice. Although the levels of insulin production were insufficient for the animals to achieve normoglycemia, those which received IPCs displayed improved conditions, when compared to the control group, as judged by a better glycemic control, body weight gain and healthy fur appearance, in the absence of apathy. In addition, when these transplantated clusters were retrieved, high levels of expression of the genes related to β-cell maturation were detected. IPCs were also microencapsulated in Bioprotect® and subjected to in vivo maturation in normal animals. A dramatic increase of the analyzed genes expression was observed, indicating complete maturation of the differentiated cells. When these cells were transplanted into diabetized mice, these animals achieved normoglycemia and were able to display glucose tolerance test (OGTT) response very similar to that of normal mice. In the second part of this work, we analyzed upregulated genes described in previous work from our group, comparing undifferentiated mESCs to IPCs using a microarray platform. One of these genes is that coding for the Purkinje cell protein 4 (Pcp4), which is 3,700 more expressed than in undifferentiated mESC cells. We adopted a functional genomics approach to investigate the role played by the Pcp4 gene in β-cells and in β-cell differentiation, by inducing overexpression and knocking down this gene in MIN-6 and mESC cells. Although the differential expression of Pcp4 in MIN-6 was not able to interfere with the expression of the genes analyzed, we observed different cell growth rates, with increased doubling time and decreased cell viability when its expression was knocked down. In addition, overexpression of Pcp4 in mESCs subjected to differentiation into IPCs apparently increases the expression of genes related to β-cell differentiation. In conclusion, we developed a new protocol for ESCs differentiation into IPCs, which is able to revert diabetes in diabetized mice, and we also describe here, for the first time, the Pcp4 gene as being expressed in pancreatic β-cells and possibly being related to maintenance of cell viability and β-cell maturation.
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Diferenciação de células-tronco embrionárias murinas (mESCs) em células produtoras de insulina (IPCs) e caracterização funcional do gene Purkinje cell protein 4 (Pcp4) neste processo / Differentiation of murine embryonic stem cells (mESCs) into insulin-producing cells (IPCs) and functional characterization of the Purkinje Cell Protein 4 (Pcp4) gene in this processPatricia Mayumi Kossugue 28 May 2013 (has links)
Fontes alternativas de células β têm sido estudadas para o tratamento de Diabetes mellitus tipo 1, dentre as quais a mais promissora consiste das células-tronco diferenciadas em células produtoras de insulina (IPCs). Alguns trabalhos demonstram a capacidade de células-tronco embrionárias murinas (mESCs) de formarem estruturas semelhantes a ilhotas pancreáticas, porém, os níveis de produção de insulina são insuficientes para a reversão do diabetes em camundongos diabetizados. Este trabalho visa desenvolver um protocolo adequado para geração de IPCs e contribuir para a identificação e caracterização funcional de novos genes associados à organogênese pancreática. Logo no início da diferenciação das mESCs em IPCs, foi possível verificar o surgimento de células progenitoras, evidenciado pela expressão de marcadores importantes da diferenciação beta-pancreática. Ao final do processo de diferenciação in vitro, ocorreu a formação de agrupamentos (clusters) semelhantes a ilhotas, corando positivamente por ditizona, que é específica para células β-pancreáticas. Para avaliar seu potencial in vivo, estes clusters foram microencapsulados em Biodritina® e transplantados em camundongos diabetizados. Apesar dos níveis de insulina produzidos não serem suficientes para estabelecer a normoglicemia, os animais tratados com IPCs apresentaram melhores condições, quando comparados ao grupo controle, tendo melhor controle glicêmico, ganho de massa corpórea e melhor aparência da pelagem, na ausência de apatia. Além disso, análise dos clusters transplantados nestes animais indicou aumento da expressão de genes relacionados à maturação das células β. Porém, quando estes clusters foram microencapsuladas em Bioprotect® e submetidos à maturação in vivo em animais normais, ocorreu um aumento drástico na expressão de todos os genes analisados, indicando sua maturação completa em células beta. O transplante destas células completamente maturadas em animais diabetizados, tornou-os normoglicêmicos e capazes de responder ao teste de tolerância à glicose (OGTT) de forma semelhante aos animais normais. A segunda parte do trabalho visou analisar genes diferencialmente expressos identificados em estudo anterior do nosso grupo, comparando, através de DNA microarray, mESCs indiferenciadas e diferenciadas em IPCs. Um dos genes diferencialmente expressos é aquele que codifica para a Purkinge cell protein 4 (Pcp4), sendo 3.700 vezes mais expresso em IPCs. Para investigar o possível papel do gene Pcp4 em células β e no processo de diferenciação β-pancreática, adotou-se o enfoque de genômica funcional, superexpressando e inibindo sua expressão em células MIN-6 e mESCs. Apesar da alteração na expressão de Pcp4 em células MIN-6 não ter interferido de forma expressiva na expressão dos genes analisados, quando inibido, modificou o perfil da curva de crescimento celular, aumentando seu tempo de dobramento de forma significativa e diminuindo da viabilidade celular em ensaios de indução de apoptose. Já na diferenciação de mESCs em IPCs, a superexpressão de Pcp4 interferiu de forma positiva apresentando uma tendência a aumentar a expressão dos genes relacionado à diferenciaçãoβ-pancreática. Concluindo, desenvolvemos um novo protocolo de diferenciação de mESCs em IPCs as quais foram capazes de reverter o diabetes em camundongos diabetizados e descrevemos, pela primeira vez, o gene Pcp4 como sendo expresso em células β-pancreáticas, podendo estar relacionado à manutenção da viabilidade celular e maturação destas células. / New cellular sources for type 1 Diabetes mellitus treatment have been previously investigated, the most promising of which seems to be the insulin producing cells (IPCs), obtained by stem cells differentiation. Some reports show that murine embryonic stem cells (mESCs) are able to form islet-like structures, however, their insulin production is insufficient to render diabetic mice normoglycemic. This work aims at developing an adequate protocol for generation of IPCs and searching for new genes which could be involved in the pancreatic organogenesis process. Early on during mESCs differentiation into IPCs, we observed the presence of progenitor cells, which were able to express pancreatic β-cell markers. At the end of the differentiation process, the islet-like clusters positively stained for the insulin-specific dithizone. These clusters were microencapsulated in Biodritin® microcapsules, and then transplanted into diabetized mice. Although the levels of insulin production were insufficient for the animals to achieve normoglycemia, those which received IPCs displayed improved conditions, when compared to the control group, as judged by a better glycemic control, body weight gain and healthy fur appearance, in the absence of apathy. In addition, when these transplantated clusters were retrieved, high levels of expression of the genes related to β-cell maturation were detected. IPCs were also microencapsulated in Bioprotect® and subjected to in vivo maturation in normal animals. A dramatic increase of the analyzed genes expression was observed, indicating complete maturation of the differentiated cells. When these cells were transplanted into diabetized mice, these animals achieved normoglycemia and were able to display glucose tolerance test (OGTT) response very similar to that of normal mice. In the second part of this work, we analyzed upregulated genes described in previous work from our group, comparing undifferentiated mESCs to IPCs using a microarray platform. One of these genes is that coding for the Purkinje cell protein 4 (Pcp4), which is 3,700 more expressed than in undifferentiated mESC cells. We adopted a functional genomics approach to investigate the role played by the Pcp4 gene in β-cells and in β-cell differentiation, by inducing overexpression and knocking down this gene in MIN-6 and mESC cells. Although the differential expression of Pcp4 in MIN-6 was not able to interfere with the expression of the genes analyzed, we observed different cell growth rates, with increased doubling time and decreased cell viability when its expression was knocked down. In addition, overexpression of Pcp4 in mESCs subjected to differentiation into IPCs apparently increases the expression of genes related to β-cell differentiation. In conclusion, we developed a new protocol for ESCs differentiation into IPCs, which is able to revert diabetes in diabetized mice, and we also describe here, for the first time, the Pcp4 gene as being expressed in pancreatic β-cells and possibly being related to maintenance of cell viability and β-cell maturation.
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