Tissues arising late in evolutionary time, such as lung alveoli that are unique to air breathing organisms, have been challenging to generate in vitro from pluripotent stem cells (PSCs), in part because there are limited lower organism model systems available to provide the necessary developmental roadmaps to guide in vitro differentiation. Furthermore, pulmonary alveolar epithelial type II cell (AEC2) dysfunction has been implicated as a primary cause of pathogenesis in many poorly understood lung diseases that lack effective therapies, including interstitial lung disease (ILD) and emphysema. Here we report the successful directed differentiation in vitro of human PSCs into AEC2s, the facultative progenitors of lung alveoli. Using gene editing to engineer multicolored fluorescent reporter PSC lines (NKX2-1GFP;SFTPCtdTomato), we track and purify human SFTPC+ alveolar progenitors as they emerge from NKX2-1+ endodermal developmental precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells are able to form monolayered epithelial spheres (“alveolospheres”) in 3D cultures without the need for mesenchymal co-culture support, exhibit extensive self-renewal capacity, and display additional canonical AEC2 functional capacities, including innate immune responsiveness, the production of lamellar bodies able to package surfactant, and the ability to undergo squamous cell differentiation while upregulating type 1 alveolar cell markers. Guided by time-series global transcriptomic profiling we find that AEC2 maturation involves downregulation of Wnt signaling activity, and the highest differentially expressed transcripts in the resulting SFTPC+ cells encode genes associated with lamellar body and surfactant biogenesis. Finally, we apply this novel model system to generate patient-specific AEC2s from induced PSCs (iPSCs) carrying homozygous surfactant mutations (SFTPB121ins2), and we employ footprint-free CRISPR-based gene editing to observe that correction of this genetic lesion restores surfactant processing in the cells responsible for their disease. Thus we provide an approach for disease modeling and future functional regeneration of a cell type unique to air-breathing organisms.

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.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25174号 / 医博第5060号 / 新制||医||1071(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 江藤 浩之, 教授 湊谷 謙司, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25197号 / 医博第5083号 / 新制||医||1072(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 齋藤 潤, 教授 斎藤 通紀, 教授 長船 健二 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25201号 / 医科博第157号 / 新制||医科||10(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 遊佐 宏介, 教授 近藤 玄, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

Stable porcine ES cell lines have not been succesfully established yet. Ability to selfrenew or to differentiate has been limited in different porcine ES-like cell lines so far. PiPSCs represent an alternative to pESCs. PiPSCs can be generated by reprogramming of somatic cells by introduction of several transcription factors on viral vectors and were established by several groups. However, the majority of piPS cell lines depend on transgene expression because of incomplete reprogramming and weak activation of endogenous pluripotency genes. Transgene expression can infuence differentiation potential of piPSCs. Therefore, we have used integrative and reexcisable PiggyBac transposons to generate viral free piPSCs. At the same time, small molecules (low-molecular inhibitors) with potential to increase reprogramming efficiency and to activate endogenous pluripotency genes were used in the reprogramming media. This strategy has a potential for generation of naive piPSCs. Successful excision of transgenes would generate transgene-free piPSCs with uncompromised differentiation potential. Pig (Sus Scrofa) is at the same time an important animal model in preclinical stage research of the diseases. Somatic cells used for generation of piPSCs were isolated from pigs carrying mutated huntingtin. Integration of the...

Em 2006, Takahashi e colaboradores demonstraram ser possível a obtenção de células-tronco pluripotentes por indução gênica (induced pluripotent stem cells ou iPSCs). Desde o surgimento desta tecnologia diversos modelos animais foram gerados, ampliando as possibilidades de seu uso na pesquisa, como por exemplo, na criação de modelos para doenças genéticas humanas como esclerose lateral amiotrófica, autismo, esquizofrenia, doença de Parkinson e Alzheimer, além do aprimoramento de características relevantes para produção animal. O modelo suíno é considerado vantajoso sobre os outros modelos animais principalmente pela criação já bem estabelecida e similaridades fisiológicas com os humanos. O intuito deste projeto foi reprogramar fibroblastos embrionários suínos através do sistema integrativo à iPSCs, para então diferenciá-las em células progenitoras neurais (neural progenitor cells, NPCs). Para isso, os fibroblastos foram transduzidos com vetores contendo sequencias humanas ou murinas dos genes OCT4, SOX2, c-Myc e KLF4 (hOSKM ou mOSKM) para formação das iPSCs. Estas foram caracterizadas quanto a morfologia, presença de fosfatase alcalina, a expressão dos genes exógenos e endógenos (OSKM, HS OCT4, OCT4, NANOG) através de imunofluorescência e RT-qPCR e formação de corpos embrióides. Então foram submetidas durante 14 dias ao meio de indução neural sob matriz extracelular comercial, gerando células potencialmente similares às NPCs. Estas foram caracterizadas morfologicamente, por imunofluorescência das proteínas NESTINA, BETA TUBULINA III e VIMENTINA, além da expressão de NESTINA e GFAP por RT-qPCR. Foram produzidas com sucesso 3 linhagens de iPSC em diferentes estágios de reprogramação e células positivas para todos os marcadores neurais testados. Os resultados apresentados deverão contribuir para a utilização do modelo suíno em futuros estudos voltados à medicina regenerativa e translacional. / In 2006, Takahashi and collaborators reported the induction into pluripotency of somatic cells (induced pluripotent stem cells, iPSCs). Since then, this technique has much been developed; many animal models have been created opening a new series of opportunities in research. They enable the creation of models for human genetic diseases, for example, amyotrophic lateral sclerosis, autism, schizophrenia, Parkinson´s disease, Alzheimer´s disease and the enhancement of relevant characteristics in agriculture. The swine model is considered to present many advantages over others, including the well-known production and maintenance and physiological similarities to humans. The aim of this project was to reprogram porcine embryonic fibroblasts (pEF) into iPSCs using the lentiviral integrative system, followed by its differentiation into neural progenitor cells (NPCs). The cells were reprogrammed using vector containing either the human sequences (hOSKM) or the mouse sequences (mOSKM) for the OCT4, SOX2, c-Myc and KLF4 genes to form the iPSCs. They were characterized regarding the presence of the Alkaline Phosphatase enzyme, expression of exogenous and endogenous genes (OSKM, HS OCT4, OCT4, NANOG) through immunofluorescence and RT-qPCR, and embryoid body formation. Then, the cells were cultured with neural induction media for 14 days in commercial extracellular matrix, generating cells potentially like NPCs. Those were characterized regarding their morphology, immunofluorescence for NESTINA, BETA TUBULIN III and VIMENTINA and gene expression of NESTINA and GFAP. iPSCs were successfully reprogramed, generating 3 cell lines at different stages of reprograming and cells positive for all the neural markers tested were produced. The results shown will contribute to the use of the porcine model in future regenerative and translational medicine research.

Atualmente, a única alternativa viável para pacientes que possuem um quadro de doença hepática em estágio final é o transplante total ou parcial de fígado. Devido à crescente defasagem entre doadores disponíveis e pacientes em fila de espera, o desenvolvimento de abordagens de engenharia tecidual hepática (ETH) se tornou uma necessidade crescente. As células pluripotentes induzidas (iPS) são uma atraente alternativa para servirem como fonte celular para aplicações de engenharia tecidual por serem capazes de produzir todos os fenótipos celulares. Dentre as principais abordagens de EHT podemos citar as técnicas de bioimpressão 3D, organóides hepáticos e descelularização/recelularização. Este trabalho buscou avaliar a utilização de células iPS no desenvolvimento das três tecnologias descritas. Visando avaliar como imprimir um tecido hepático funcional com células iPS, testamos a impressão com hepatócitos dispersos em células únicas em comparação com a impressão de esferóiedes hepáticos. Os esferóides hepáticos mostraram maior viabilidade e funcionalidade hepática por preservarem o fenótipo epitelial ao longo do tempo. A composição de células não parenquimáticas derivadas de iPS ou células primárias para a formação de organóides hepáticos foi testada neste trabalho. Os resultados indicam que, utilizando células mesenquimais primárias e endoteliais derivadas de iPS, obtém-se uma maturação hepatica mais eficiênte devido a inibição das vias de sinalização TGF-β? e modulação da via Wnt. A recelularização do tecido aórtico descelularizado de ratos com células derivadas de iPS mostrou ser capaz de prover função hepática em cultura assistida por biorreator, porém os resultados indicam a necessidade de aprimoramento do protocolo de recelularização. Este trabalho comprovou a viabilidade da aplicação de células iPS nas abordagens EHT testadas e contribuiu para o desenvolvimento de alternativas terapêuticas viáveis para pacientes em fila de espera de transplante hepático / Currently, the only feasible alternative for patients with end-stage liver disease is total or partial liver transplantation. Due to the growing gap between available donors and patients in waiting list, the development new tissue engineering technologies have become a growing need. Induced pluripotent cells (iPS) are an attractive alternative to serve as cell source for tissue engineering applications due to their ability to differentiate into all cellular phenotypes. Among the main liver tissue engineering technologies, 3D bioprinting, hepatic organoids and decellularization/recellularization of biological matrixes have generated much expectation. Thus, this work aimed to evaluate the use of iPS cells in the development of the aforementioned technologies. In order to evaluate how to bioprint a functional liver tissue using iPS-derived cells, we tested the effect of printing a single cell dispersion of hepatocytes versus printing hepatic spheroids. Hepatic spheroids showed greater viability and liver function, due to preserved epithelial phenotype over time. The composition of non-parenchymal cells using iPS-derived cells or primary adult cells for hepatic organoid formation was tested. The results indicated that, using primary mesenchymal cells and iPS-derived endothelial cells, we obtained a more efficient hepatic maturation due to the inhibition of TGF-β? and modulation Wnt signaling pathway. Recellularization of rat aortic decellularized scaffold with iPS-derived cells displayed hepatic function over time in a bioreactor-assisted culture, but the results indicate the need for improvements in the recellularization protocol. In conclusion, this work demonstrated the feasibility of use of iPS-derived cells for liver tissue engineering approaches and contributed to the development of the investigated technologies in order to generate future therapeutic alternatives for patients in waiting list for liver transplantation