Conjugation of the Human Immunodeficiency Virus Transactivator of Transcription (TAT) to active proteins allows transport into the intracellular environment. This feature can be harnessed to deliver combinations of reprogramming factors (RFs) such as c-Myc, Oct4, Klf4 and Sox2 into somatic cells to derive induced pluripotent stem cells (iPSCs). For this project, TAT-fusion proteins including four TAT-conjugated RFs (TAT-RFs) have been produced and purified. All four TAT-RFs can bind specific DNA sequences. Bioactivity was tested in live cells using a novel assay based on an engineered fibroblast cell line that can be induced to express RFs by doxycycline and subsequently generate iPSCs. To test each TAT-RF, reprogramming was blocked by transient silencing of a single RF by siRNA and rescued by the corresponding TAT-RF. The results of this assay suggested that TAT-Klf4 was bioactive in cells; however, definitive evidence could not be obtained for other RFs.

No description available.

Derivation of lung epithelial cells from stem cells remains a challenging task, due in part to a lack of understanding of the molecular mediators driving commitment of endoderm to an early lung lineage. Reciprocal signalling between the lung mesenchyme and epithelium is crucial for proper differentiation and branching morphogenesis to occur. We hypothesized that the combination of signalling pathways comprising early epithelial-mesenchymal interactions and the 3-D spatial environment are required for induction of embryonic and induced pluripotent stem cells (ESC and iPSC, respectively) into a lung cell phenotype with the hallmarks of the distal niche. Aggregating early lung mesenchyme with endoderm-induced ESC and iPSC resulted in differentiation to an NKX2.1 and pro-SFTPC positive lineage. The differentiating cells organized into tubular structures and became polarized epithelial cells. Ultrastructure analysis revealed precursors of lamellar bodies, and Sftpb mRNA expression was detected. Quantification of the differentiation using an Nkx2.1-reporter ESC line revealed that 80% were committed to an early lung lineage, a vast improvement over what has previously been published. The FGF growth factor family comprises well-known mediators of growth and differentiation during the development of many organs, including the lung. We found that FGF2 signalling through the FGFR2iiic receptor isoform was mediating the commitment of the stem cells to an early lung epithelial phenotype, as defined by NKX2.1/proSFTPC expression. FGF7 signalling through the FGFR2iiib receptor was found to be important for the maturation and morphogenesis of the NKX2.1/proSFTPC positive lineage, but did not play a role in the initial commitment. The addition of FGF2 to endoderm-induced ESC or iPSC in the absence of mesenchyme was able to commit the cells to an NKX2.1-positive lineage, but no proSFTPC was detected. Furthermore,the cells did not become polarized and no longer organized into tubular structures. These findings suggest that while FGF2 is important for initial commitment, additional mesenchyme components including matrix proteins, supporting cell lineages and other growth factors are crucial for an efficient differentiation to an early lung epithelial cell lineage.

Parkinson's disease (PD) primarily manifests as loss of motor control through the degeneration of nigrostriatal dopaminergic neurons. The microtubule-associated protein tau (MAPT) locus is highly genetically associated with PD, wherein the H1 haplotype confers disease risk and the H2 haplotype is protective. As this haplotype variation does not alter the amino acid sequence, disease risk may be conferred by altered gene expression, either of total MAPT or of specific isoforms, of which there are six in adult human brain. To investigate haplotype-specific control of MAPT expression in the neurons that die in PD, induced pluripotent stem cells (iPSCs) from H1/H2 heterozygous individuals were differentiated into dopaminergic neuronal cultures that expressed all six mature isoforms of MAPT after six months' maturation. A reporter construct using the human tyrosine hydroxylase locus was also generated to identify human dopaminergic neurons in mixed cultures. Haplotype-specific differences in the inclusion of exon 3 and total MAPT were observed in iPSC-derived dopaminergic neuronal cultures and a novel variant in MAPT intron 10 increased the inclusion of exon 10 by two-fold. RNA interference tools were generated to knockdown total MAPT or specific isoforms, wherein knockdown of the 4-repeat isoform of tau protein increased the velocity of axonal transport in iPSC-derived neurons. MAPT knockdown also reduced p62 levels, suggesting an impact of tau on macroautophagy, likely through altered axonal transport. These results demonstrate how variation at a disease susceptibility locus can alter gene expression, thereby impacting on neuronal function.

Immunotherapy traditionally made use of biological agents such as cytokines and monoclonal antibodies. Such first generation therapies lack antigen specificity and fail to induce immunological memory, suggesting that cell therapies may provide the next generation of treatments that are more discerning in their mode of action. Nevertheless, difficulties in obtaining sufficient immunologically-relevant cell types from patients has limited their success. Given that induced pluripotent stem cells (iPSC) may be generated from patients, we have investigated the feasibility of deriving two cell types whose availability is restricted in vivo: regulatory T cells (T_regs) and CD141⁺ cross-presenting dendritic cells (DCs). We describe the optimization of protocols for differentiation and purification of CD141⁺ DCs, focussing on their utility as a therapeutic vaccine for HIV-1. We investigate their phenotype, chemotactic capacity, phagocytic ability and propensity to harbour infectious virus. We also assess their immunostimulatory capacity and ability to cross-present exogenous antigen to MHC class I-restricted T cells. Our findings led us to speculate that iPSC-derived DCs (iPDCs) possess fetal phenotype, which is characterised by excessive secretion of IL-10 and failure to secrete IL-12, under all but the most stringent conditions. We hypothesised that constitutive secretion of IL-10 may be responsible for maintaining the fetal phenotype, a hypothesis we tested by developing an appropriate mouse model. iPSCs were derived from WT and IL-10^-/- mice and were shown to differentiate into iPDCs which recapitulate the fetal phenotype observed among human cells. However, loss of the endogenous Il-10 gene failed to restore full immunogenicity and IL-12 secretion. Finally, we developed protocols for differentiation of FoxP3+ T_regs from iPSCs, a feat that has not previously been achieved. These findings pave the way for the differentiation of T_regs from iPSCs reprogrammed from antigen-specific pathogenic T cells, thereby creating a source of T_regs with matched specificity for therapeutic intervention.

abstract: Alzheimer’s disease (AD), despite over a century of research, does not have a clearly defined pathogenesis for the sporadic form that makes up the majority of disease incidence. A variety of correlative risk factors have been identified, including the three isoforms of apolipoprotein E (ApoE), a cholesterol transport protein in the central nervous system. ApoE ε3 is the wild-type variant with no effect on risk. ApoE ε2, the protective and most rare variant, reduces risk of developing AD by 40%. ApoE ε4, the risk variant, increases risk by 3.2-fold and 14.9-fold for heterozygous and homozygous representation respectively. Study of these isoforms has been historically complex, but the advent of human induced pluripotent stem cells (hiPSC) provides the means for highly controlled, longitudinal in vitro study. The effect of ApoE variants can be further elucidated using this platform by generating isogenic hiPSC lines through precise genetic modification, the objective of this research. As the difference between alleles is determined by two cytosine-thymine polymorphisms, a specialized CRISPR/Cas9 system for direct base conversion was able to be successfully employed. The base conversion method for transitioning from the ε3 to ε2 allele was first verified using the HEK293 cell line as a model with delivery via electroporation. Following this verification, the transfection method was optimized using two hiPSC lines derived from ε4/ε4 patients, with a lipofection technique ultimately resulting in successful base conversion at the same site verified in the HEK293 model. Additional research performed included characterization of the pre-modification genotype with respect to likely off-target sites and methods of isolating clonal variants. / Dissertation/Thesis / Masters Thesis Bioengineering 2017

En 2006 et 2007, les équipes de Yamanaka et Thomson réalisent la reprogrammation de cellules somatiques murines et humaines en cellules souches pluripotentes à partir de deux cocktails de gènes : OCT4, SOX2, KLF4, cMYC (OSKM) et OCT4, NANOG, SOX2, LIN28 (ONSL). Les cellules souches à pluripotence induite générées (iPS) partagent les propriétés fondamentales des cellules souches embryonnaires : l’auto-renouvèlement, le maintien de la pluripotence et la capacité de différenciation. Ces cellules laissent entrevoir des applications considérables tant en recherche fondamentale (compréhension des mécanismes de développement et de pathologies, développement de modèles) qu’en recherche appliquée (médecine régénérative, toxicologie prédictive, criblage de médicaments). L’avantage majeur de l’utilisation des iPS réside dans leur origine non embryonnaire. Les contraintes d’ordre éthique sont écartées et une grande diversité de types cellulaires à partir de n’importe quel donneur a priori est disponible pour une reprogrammation. L’établissement d’une banque d’hiPS issus de donneurs sains ou de patients, serait d’une grande utilité pour la communauté scientifique se consacrant à l’étude des mécanismes physiopathologiques ou de développement et une source considérable pour la dérivation à des fins de thérapie cellulaire. Dans le but de mettre en place une telle banque, nous avons développé avec la société Vectalys des rétrovirus de reprogrammation contenant les cassettes polycistroniques ONSL et OSKM. Dans un premier temps, nous avons établi un protocole de reprogrammation robuste à l’aide des rétrovirus RV-ONSL. Nous avons ensuite mis en évidence la synergie entre les facteurs ONSL et OSKM, la combinaison équimolaire de RV-ONSL et RV-OSKM permettant d’atteindre 2% d’efficacité de reprogrammation. Nous avons également entrepris la reprogrammation propre par transfections d’ARNm polycistroniques ONSL et OKM mettant à profit cette incroyable synergie. / In 2006 and 2007, Yamanaka and Thomson teams achieved the reprogramming of mouse and human somatic cells into pluripotent stem cells through the transfection of two cocktails of genes: OCT4, SOX2, KLF4, cMYC (OSKM) and OCT4, NANOG, SOX2, LIN28 (ONSL). The generated cells, called induced Pluripotent Stem Cells (iPSC) share the same fundamental properties of ESC : self-renewing, pluripotency maintenance and capacity of differentiation into the three germ layers and suggest the same application potential in basic research (developmental and epigenetic biology) as well as in therapy (regenerative medicine, disease modeling for drug development). One of the major advantages of iPSC lies in their non-embryonic origin. Indeed, the use of iPSC resolves the ethical constraints and offers the possibility to work with extensive cell types directly from the patient to treat. Stéphane Viville’s research team aims to develop a hiPSC bank from patient suffering from genetic or other diseases which will be available for the scientific community. We are experienced in human primary fibroblasts reprogramming especially with the use of two polycistronic cassettes: ONSL encoding Thomson’s cocktail and OSKM encoding Yamanaka’s cocktail separated with 2A peptides. Thanks to the combination of RV-ONSL and RV-OSKM retroviral vectors (developed with Vectalys) we are yielding more than 2% of reprogramming efficiency in a highly reproducible way. Indeed, we demonstrated the reprogramming synergy of ONSL and OSKM combination. We are now focusing our effort on non-integrative strategies (ie mRNA) which are more appropriate for clinical usage.

A esclerose lateral amiotrófica (ELA) é uma doença neurodegenerativa, progressiva de início tardio que afeta principalmente os neurônios motores (NM). As causas que levam os NM à morte são variadas e ainda sendo investigadas. A descoberta de alterações genéticas como uma possível causa de ELA deu início à uma nova era na investigação desta afecção. Atualmente existem mais de 30 genes associados com a doença, entre eles o FUS, um gene que frequentemente aparece mutado em casos familiais da doença. A proteína FUS normalmente se localiza predominantemente no núcleo, mas na maioria dos casos de mutações na FUS relacionadas à ELA, ela aparece retida no citoplasma. O presente estudo traz um paciente de ELA (P) portando a mutação p.R521H no gene FUS e três de seus irmãos (dos quais um é portador da mutação e não apresnta sinais clínicos de ELA, e os outros dois não apresentam mutações no FUS) dos quais foram obtidas amostras de sangue e biópsia de pele. O DNA extraído das amostras de sangue, foi submetido ao sequenciamento do tipo Sanger para verificar a presença, ou ausência, da mutação R521H na FUS. A partir dos fibroblastos dos participantes, foram derivadas linhagens de células tronco pluripotentes induzidas (iPSC). As iPSC produzidas passaram por ensaios a fim de indicar o estado de pluripotência e de indiferenciação destas linhagens. Nós investigamos a posição da proteína FUS nas linhagens de iPSC e de fibroblastos e há evidências que, assim como descrito na literatura, a proteína FUS aparece retida no citoplasma das linhagens do paciente e de seu irmão portador da mutação. Desta forma, o presente estudo associa dois irmãos com quadros clínicos discordantes mas que apresentam a mesma mutação e sinais moleculares patológicos semelhantes. As linhagens de iPSC obtidas são um rico material para o uso em pesquisas futuras sobre a ELA / Amyotrophic lateral sclerosis (ALS) is a late onset, progressive, neurodegenerative disease that primarily affects motor neurons (MNs). The causes behind motor neuron death are diverse and still under investigation. The discovery of genetic alterations as possible causes of ALS initiated a new era for ALS research. There are currently over 30 genes associated with the disease, among which is FUS, one of the most frequently mutated in familial cases. The FUS protein is predominantly located in the nucleus, but in most of the ALS-related FUS mutations this protein is dislocated to the cytoplasm. The present work investigates the molecular aspects of a specific FUS mutation, p.R521H. An ALS patient (P) harboring the mutation and three siblings (of which one is a non-affected carrier and two present no mutations in FUS) were analyzed using blood samples and skin biopsies. We extracted DNA from blood samples and submitted it to Sanger sequencing for confirmation of the presence, or absence, of the R521H FUS mutation. The fibroblasts obtained from these biopsies were used for iPSC derivation. Assays were performed to confirm the undifferentiated state and pluripotency for the four strains obtained. We investigated the FUS location in these strains, and there is evidence for FUS retention in the cytoplasm of cells harboring the mutation (as seen in recent literature). Thus, this work associates two siblings with the same pathogenic mutation, showing the same molecular pathological signal but with discording clinical phenotypes. The iPSC strains obtained here are a valuable resource for further ALS investigation

A anemia aplástica (AA) é uma doença hematológica rara caracterizada pela hipocelularidade da medula óssea, o que provoca pancitopenia. Esta pode ser de origem genética (associada a encurtamento telomérico) ou adquirida (não-associada a desgaste excessivo dos telômeros). Na forma adquirida, a ativação anormal de linfócitos T provoca a destruição das células hematopoéticas. O mecanismo que leva a essa destruição ainda não foi elucidado. Um dos tratamentos mais eficazes para repovoar a medula óssea hipocelular é o transplante com célulastronco hematopoéticas (CTHs). Porém, uma grande porcentagem de pacientes não se beneficia de nenhum tratamento, fazendo-se necessário o desenvolvimento de novas alternativas para terapia. A geração de células-tronco pluripotentes induzidas (iPSCs) a partir de células somáticas (reprogramação) representa uma ferramenta promissora para o estudo de doenças e para o desenvolvimento de possíveis terapias paciente-especificas, como transplantes autólogos. Neste trabalho, avaliamos a capacidade de reprogramação de fibroblastos e eritroblastos de pacientes com AA adquirida. Metodologias de reprogramação utilizando lentivírus ou plasmídeos epissomais não integrativos foram testadas em células de quatro pacientes e de um controle saudável. Eritroblastos dos quatro pacientes e do controle foram reprogramados utilizando os plasmídeos não integrativos. As iPSCs geradas apresentaram-se similares a células-tronco embrionárias quanto à morfologia, expressão dos marcadores de pluripotência OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 e Tra-1-81, e capacidade de diferenciação in vitro em corpos embrioides (EBs). A dinâmica telomérica das células pré- e pós-reprogramação foi avaliada em diferentes passagens utilizando a técnica de flow-FISH. O comprimento telomérico foi aumentado nas iPSCs quando comparado às células parentais o que indica que a célula foi completamente reprogramada. No presente trabalho, células de pacientes com AA adquirida foram reprogramadas a um estado de pluripotência por meio de um método não integrativo. As iPSCs geradas serão essenciais para futuros ensaios de diferenciação hematopoética, o que poderá contribuir para o entendimento dos mecanismos envolvidos no desenvolvimento dessa doença. Além disso, a diferenciação dessas células livres de transgenes poderá servir como uma alternativa terapêutica para os pacientes com AA como, por exemplo, em transplantes autólogos / Aplastic anemia (AA) is a rare hematological disease characterized by bone marrow hypocellularity that leads to pancytopenia. Its origin can be genetic (associated with telomere shortening) or acquired (non-associated with telomere shortening). The acquired form exhibit T lymphocytes abnormal activation, which leads to hematopoietic cells destruction. The mechanisms behind this phenomenon are still unclear. One of the most effective treatments for hypocelullar bone marrow repopulation is hematopoietic stem cell (HSCs) transplantation. However, a large percentage of patients do not benefit from any of the available treatments. This highlights the need to develop new therapeutic strategies. The generation of induced pluripotent stem cells (iPSCs) from somatic cells (reprogramming) represents a powerful tool for disease modeling and for the development of patient-specific therapies such as autologous transplants. In this study, we evaluate the capacity of reprogramming acquired AA patients\' fibroblasts and erythroblasts. Reprogramming methods using lentivirus or non-integrative episomal plasmids were tested in four patients\' cells and in cells from one healthy donor. Erythroblasts from these four patients and healthy donor were reprogrammed using non-integrative plasmids. The iPSCs resembled human embryonic stem cells in morphology, in the expression of pluripotent markers such as OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 and Tra-1-81, and in in vitro differentiation (capacity to form embryoid bodies). The telomere dynamics of the cells before and after reprogramming was assessed along passaging using flow-FISH. The telomere length in the iPSCs was increased when compared to the parental cells. Thus, acquire AA patients\' cells could be reprogrammed to a pluripotent state by a nonintegrative method. The iPSCs will be essential for future hematopoietic differentiation assays that could contribute to the understanding of the mechanisms involved in the disease development. Furthermore, the differentiation of transgene-free cells may serve as an alternative therapy for patients with AA such as autologous transplants

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.