A demanda por transfusões sanguíneas tem aumentado no Brasil e o número de doações de sangue permanecem insuficientes. Há escassez de componentes de sangue para transfusão, principalmente de concentrados de células vermelhas do sangue. As células-tronco pluripotentes induzidas humanas (hiPSCs) possuem um grande potencial para se tornar uma fonte de CÉLULAS VERMELHAS DO SANGUE, pois podem se diferenciar em qualquer tipo celular, incluindo CÉLULAS VERMELHAS DO SANGUE de fenótipo específico. O objetivo deste trabalho é a geração de hiPSCs para partir de células mononucleares de sangue periférico (PBMCs) de candidatos a doação de sangue que possuem fenótipo eritrocitário de baixa imunogenicidade, bem como a diferenciação eritroide das hiPSCs geradas. As amostras de sangue periférico (PB) de 11 indivíduos foram coletadas e caracterizadas quanto ao genótipo para os seguintes antígenos eritrocitários: Sistema Rh (RHCE*01/RHCE*02/RHCE*03/RHCE*04/RHCE*05), Kell (KEL*01/KEL*02), Duffy (FY*01/FY*02 and FY*02N.01), Kidd (JK*01/JK*02) e MNS (GYPB*03/GYPB*04). Outros antígenos de grupos sanguíneos distintos foram determinados por meio de fenotipagem. Duas amostras (PBMCs PB02 e PB12) foram selecionadas para a reprogramação devido ausência de múltiplos antígenos eritrocitários e, portanto, considerados de baixa imunogenicidade. Os PBMCs foram enriquecidos em eritroblastos e em seguida, as células foram transfectadas com os vetores episomais pEB-C5 e pEB-Tg e então, co-cultivados sobre fibroblastos de embriões murinos (MEFs) até o surgimento de colônias semelhantes a hiPSCs (hiPSC PB02 e hiPSC PB12). Estas colônias foram transferidas para condições de cultivo próprias e posteriormente caracterizadas quanto à sua pluripotência. A expressão dos genes de pluripotência OCT4, SOX2 e NANOG demonstrou níveis de expressão maior em comparação às linhagens não pluripotentes. As análises de imunofenotipagem por citometria de fluxo revelaram que em torno de 86% das células expressaram Nanog, 88% Oct4 e 88% Sox2. Os níveis de expressão de genes de pluripotência e marcadores foram consistentes com o estado indiferenciado encontrado em células pluripotentes conhecidas. A análise funcional para avaliação da pluripotência foi realizado pela injeção das hiPScs em camundongos imunodeficientes, demonstrando a formação de teratoma nas linhagens geradas. A metodologia para diferenciação hematopoética das hiPSCs geradas a partir dos corpos embrioides estão em progresso. O potencial de diferenciação foi confirmado durante a padronização deste processo, utilizando ensaio de formação de colônias em metilcelulose. Uma média de 10,5 colônias de precursores eritroide foram obtidas a partir de 50x103 hiPSC PB02 em diferenciação e uma colônia mista (mieloide e linfoide) a partir de 15x103 hiPSC PB12 foram obtidas. Neste trabalho foi possível gerar duas linhagens de hiPSCs com fenótipos de antígenos eritrocitários de interesse que podem ser mantidas em cultura por um longo período (26 passagens) e demonstram um potencial de diferenciação hematopoética. / The demand for blood transfusion has increased in Brazil and the number of blood donations remains insufficient. Therefore, there is a shortage of blood components for transfusion, mainly concentrates of red blood cells (RBCs). Human induced pluripotent stem cells (hiPSCs) have great potential to become a source of RBCs, because they can differentiate into every cellular type, including RBCs of a particular phenotype. The objective of this work was to generate hiPSC from mononuclear cells of peripheral blood (PBMCs) from blood donors who presented low immunogenic phenotype for transfusion, and erythroid differentiation of the generated hiPSCs. Peripheral blood samples from 11 individuals were collected and characterized for the following erythrocyte antigens: Rh system (RHCE*01/RHCE*02/RHCE*03/RHCE*04/RHCE*05), Kell (KEL*01/KEL*02), Duffy (FY*01/FY*02 and FY*02N.01), Kidd (JK*01/JK*02), MNS (GYPB*03/GYPB*04). Additionally, other antigens of different blood groups were determined by phenotyping. The samples PBMC PB02 and PBMC PB12 were chosen for iPS generation due to their multiple negative erythrocyte antigens. They were isolated, expanded into erythroblasts, and transfected using the reprogramming episomal vectors PEB-C5 and PEB-Tg. This population was co-cultured on mouse embryonic fibroblasts (MEFs) until the appearance of hiPSC like colonies (hiPSC PB02 and hiPSC PB12). These colonies were transferred to human embryonic stem cells (hESCs) culture conditions and characterized regarding their pluripotency. The expression of OCT4, SOX2 and NANOG pluripotency genes demonstrated that the expression of both lineages was higher in comparison with non-pluripotent lineages. Immunophenotyping performed by flow cytometry revealed that 86% of cells expressed Nanog, 88% Oct4 and 88% Sox2. Expression levels of pluripotency genes and markers were consistent with undifferentiated state found in known pluripotent cells. Functional analysis for pluripotency was achieved by the hiPSC injection in immunodeficient mice showing that both hiPSC cell lines were able to induce teratoma tumor. The hematopoietic differentiation potential was confirmed using methylcellulose assay, with an average of 10.5 erythroid colonies from 50x103 single cells and a mixed colonies of myeloid and lymphoid cells) and finally a colony composed of white cells from 15x103 PB12 hiPSC. In conclusion, it was possible to generate a hiPSC from a red blood cell phenotype that are negative for multiple antigens, and this cell line can be maintained for a long period in culture (26 passages) and show potential for hematopoietic differentiation.

Microsatellites, or simple tandem repeat sequences, occur naturally in the human genome and have important roles in genome evolution and function. However, the expansion of microsatellites is associated with over two dozen neurological diseases. A common denominator among the majority of these disorders is the expression of expanded tandem repeat-containing RNA, referred to as xtrRNA, which can mediate molecular disease pathology in multiple ways. Frontotemporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) are two fatal neurodegenerative diseases with significant clinical, neurological and genetic overlap thus referred to as C9FTD/ALS. Currently, gaps in the study of the underlying disease mechanisms persist, which can aid in the identification of promising therapeutic approaches. Access to simple models of neurological repeat expansion disease is critical for investigating biochemical mechanisms and for early therapeutic discovery. To better understand the molecular pathology of C9FTD/ALS repeat expansion disorder, we cloned GGGGCC repeats, which are the leading genetic cause of C9FTD/ALS. We employed a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors and validated the cloning to facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level using these vectors. In this study, we also differentiated C9FTD/ALS patient-derived induced pluripotent stem cells (iPSCs) to neural stem cells (NSCs) to be used as model systems. The use of iPSCs and NSCs to reveal important insights into the pathogenic mechanisms and to generate multiple neural cell types presents an excellent opportunity for researchers to model neurodegenerative diseases for cell therapy and drug discovery. We further investigated potential nuclear export mechanisms for C9FTD/ALS xtrRNA. The nuclear export mechanisms of xtrRNA in C9FTD/ALS are not well studied. ASOs and siRNAs were employed to knockdown genes of interest to study their involvement in the nuclear export of xtrRNA. We saw promising results on knockdown of TorsinA involved in nuclear export of xtrRNAs, corroborated by a substantial increase in the average number of xtrRNA foci in the nucleus. Our initial study provides evidence that TOR1A may be involved in the nuclear export of aberrant C9FTD/ALS repeat-containing RNAs. Due to the lack of reliable and robust assays to detect RAN translation products, the effect of the knockdown of TorsinA in these cell lines still remains to be explored. But the current study lays the groundwork for a deeper understanding of the less-studied nuclear export mechanisms in C9FTD/ALS and could reveal new therapeutic approaches to selectively block the nuclear export of xtrRNA through the use of RNAi and ASOs. The insights gained from this study will help us understand future events in the xtrRNA life cycle such as repeat translation mechanisms.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22006号 / 医科博第104号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 井上 治久, 教授 伊佐 正, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22327号 / 医博第4568号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 江藤 浩之, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23413号 / 医博第4758号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 小柳 義夫, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医科学) / 乙第13443号 / 論医科博第7号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 長船 健二, 教授 木村 剛, 教授 湊谷 謙司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

BACKGROUND: Inflammation is a key regulator in skeletal homeostasis during normal growth and tissue repair. However, the role that inflammation plays in skeletal processes is not well understood. Previous studies showed that damage associated molecular pattern (DAMP) molecules released after injury may contribute to immune activation and subsequent fibrosis. OBJECTIVE: This project aims to elucidate the link between tissue damage caused by trauma and the subsequent inflammatory response in a genetic condition of bone morphogenetic protein (BMP) pathway over activation. METHODS: We investigated this potential link by examining immune cells from patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of endochondral heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1). Patients with FOP show sensitivity to trauma, elevated serum cytokines and abnormal cytokine/chemokine secretion from monocytes and macrophages when stimulated with lipopolysaccharide in vitro. This suggested that BMP pathway activation may alter immune responses in patients with FOP. We studied macrophages derived from peripheral blood monocytes or created from human induced pluripotent stem cells (iPSC) from FOP and control subjects. Macrophages were evaluated by gene expression and culture media by multiplex cytokine analysis after stimulation with key DAMPs that were previously identified to be released after tissue injury. These DAMPs act as endogenous activators of inflammation. RESULTS: Monocyte derived macrophages from control subjects showed increased expression of pro-inflammatory cytokines in response to stimulation with DAMPs, HMGB1 and S100A8/A9. FOP monocyte-derived macrophages treated with each DAMP showed elevated production of CCL22, IL-8, CCL3, and CCL8 when compared to control macrophages. However, both control and FOP macrophages showed increased production of pro-inflammatory cytokines in response to DAMPs compared to non-stimulated conditions. RNA expression profiles of FOP iPSC derived macrophages did not show significantly increased responsiveness to DAMPs compared to control. Surprisingly, control patient iPSC derived macrophages show elevated expression of TNF-a and IL-1B CONCLUSIONS: Macrophages derived from peripheral blood monocytes show that DAMPs may be responsible for macrophage activation and the development of inflammatory complications in patients with FOP. Control iPSC derived macrophages showed similarity to monocyte derived macrophages in their response to DAMPs, suggesting that our iPSC derived macrophages are an applicable model for investigating the human immune system. The dissimilarity in FOP macrophage responsiveness to endogenous activators of our two macrophage models, suggest that iPSC derived macrophages may be affected by the different differentiation and polarization methods, and needs to be characterized further. Similarly, RNA expression profiles may not reflect cytokine production patterns of stimulated iPSC macrophages and warrants further studies. / 2021-06-07T00:00:00Z

Understanding the regenerative capacity and the role of human AT2s in the distal lung is imperative for defining alveolar response to injury and disease. Additionally, due to human AT2 expression of COPD genome wide association study (GWAS) genes, they are an especially relevant cell type to study the disease. Here we apply CRISPR-interference (CRISPRi) to reduce the expression of COPD GWAS gene, ADGRG6, to interrogate its function in induced pluripotent stem cell-derived type 2 alveolar epithelial cells (iAT2s). We find that decreased expression of ADGRG6 in iAT2s caused disruption to iAT2 cell polarity, organization of the actin cytoskeleton, and establishment of tight junctions. In addition, ADGRG6 knockdown (kd) causes a hyperproliferative phenotype. Finally, we find that ADGRG6-kd may contribute to dysregulation of tight junction formation in the presence of cigarette smoke.