京都大学 / 新制・課程博士 / 博士(医学) / 甲第23471号 / 医博第4778号 / 新制||医||1053(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 山下 潤, 教授 木村 剛, 教授 湊谷 謙司 / 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13518号 / 論医科博第10号 / 新制||医科||10(附属図書館) / (主査)教授 金子 新, 教授 松田 秀一, 教授 山中 伸弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Doctor of Philosophy / The liver is the largest internal organ in the body. It is responsible for performing an array of vital functions, including the filtration of blood, synthesis of different molecules, and metabolism of drugs and toxicants. Hepatocytes, or the main liver cell type, perform most of these functions. Typically, primary human hepatocytes (PHHs) are ideal for in vitro liver studies since they are obtained directly from tissues and exhibit adult characteristics and functions. However, sourcing these adult liver cells is extremely difficult since they are usually obtained through biopsies and are limited in quantity. Induced pluripotent stem cell (iPSC)-hepatocyte-like cells (iHLCs) hold tremendous potential to be used as a substitute since they can be obtained non-invasively. However, iHLCs require further maturation before they can be substituted for PHHs due to their lower liver-related functions and immature characteristics. Existing maturation approaches require the administration of chemical mixtures that can be up to a million times higher than the concentrations of these same molecules inside the body. The lack of a systematic approach to mature iHLCs currently limits their widespread use. During liver development, neighboring cells secrete different proteins that regulate and induce multiple pathways that aid in the maturation of cells into adult liver cells. We report the assembly of a multicellular 3D human liver organoid with iHLCs that recapitulates the in vivo hepatic microenvironment. Intra- and intercellular signaling between human hepatic cells in the organoid result in mature iHLCs that exhibit several markers and functions of PHHs within one to two weeks in culture. When two other hepatic cell types, Kupffer cells and liver sinusoidal endothelial cells, are present in the organoids, they secrete signaling molecules that synergistically mature iHLCs. Relying solely on intercellular secretion from the cells provides a systematic and reproducible approach to generate mature iHLCs for drug, disease, and patient-specific in vitro studies.

Stem cells have the ability to self-renew and to differentiate into a variety of cell types. Stem cells can be found naturally in the body, can be derived from the inner cell mass of blastocysts, or can be made by dedifferentiation of adult cells. Regenerative medicine aims to utilize the potential of stem cells to treat disease and injury. The ability to create stem cell lines from a patient's own tissues allows for transplantation without immunosuppressive therapy as well as patient-specific disease modeling and drug testing. The objective of this study was to use cellular dedifferentiation to create in vitro cell lines with which to study regenerative medicine. First, we used siRNA targeted against myogenin to induce the dedifferentiation of murine C2C12 myotubes into myoblasts. Timelapse photography, immunofluorescence, and western blot analysis support successful dedifferentiation into myoblasts. However, the inability to separate the myotubes and myoblasts prior to siRNA treatment confounded the results. This system has the potential to be used to study mechanisms behind muscle cell regeneration and wound healing, but a better method for separating out the myoblasts needs to be developed before this will be achievable. Second, we used a doxycycline-inducible lentiviral vector encoding the transcription factors Oct4, Sox2, cMyc, and Klf4 to create a line of naive-like porcine induced pluripotent stem cells (iPSCs). This reprogramming vector was verified first in murine cells, the system in which it was developed. Successful production of both murine and porcine iPSC lines was achieved. Both showed alkaline phosphatase activity, immunofluorescence for pluripotency marker (Oct4, Sox2, and Nanog) expression, PCR for upregulation of endogenous pluripotency factors (Oct4, Sox2, cMyc, Klf4, and Nanog), and the ability to form embryoid bodies that expressed markers of all three germ layers. Additionally, we were able to create secondary porcine iPSC lines by exposing cellular outgrowths from embryoid bodies to doxycycline to initiate more efficient production of porcine iPSCs. The secondary porcine iPSCs were similar to the primary porcine iPSCs in their morphology, behavior, alkaline phosphatase expression, and Nanog expression with immunofluorescence. The porcine iPSCs were dependent on doxycycline to maintain pluripotency, indicating that they are not fully reprogrammed. Despite this dependence on doxycyline, this system can be used in the future to study the process of reprogramming, to develop directed differentiation protocols, and to model diseases. / Master of Science / Stem cells have the ability to self-renew and to differentiate into a variety of cell types. Stem cells can be found naturally in the body, can be derived from the inner cell mass of blastocysts (the stage of development just prior to implantation), or can be made by dedifferentiating, or reprogramming, adult cells into stem cells. Regenerative medicine aims to utilize the potential of stem cells to treat disease and injury. The ability to create stem cell lines from a patient’s own tissues allows for transplantation without immunosuppressive therapy as well as patient-specific disease modeling and drug testing. The objective of this study was to use cellular dedifferentiation to create cell lines in the laboratory with which to study regenerative medicine. First, we knocked down the expression of myogenin, a key factor in muscle cell development, to induce the dedifferentiation of mouse myotubes (adult muscle cells) into myoblasts (progenitor cells). Various methods of analysis supported successful dedifferentiation into myoblasts, but the inability to completely separate myotubes and myoblasts prior to myogenin knockdown confounded the results. With better separation of the cells, this system has the potential to be used to study mechanisms behind muscle cell regeneration and wound healing. Second, we used a viral vector encoding reprogramming factors to create both mouse and pig induced pluripotent stem cells (iPSCs) from skin cells. Pluripotent cells have the ability to differentiate into any cell type in the body, except for the placenta. Multiple pluripotency assays indicated that both the mouse and pig iPSCs were truly pluripotent. Additionally, we were able to differentiate the iPSCs into adult cells, then reprogram those back into “secondary” iPSCs. The production of secondary iPSCs is much more efficient compared to the initial creation of the primary iPSCs, which increases the usefulness of these cells for future experiments. Unfortunately, the porcine iPSCs were dependent on the reprogramming vector to maintain pluripotency. This indicates that these cells are not fully reprogrammed. Despite this, the system can still be used in the future to study the process of reprogramming, to develop cellular differentiation protocols, and to model diseases.

Amyotrophic Lateral Sclerosis (ALS) is an incurable late onset neurodegenerative disorder characterised by the specific loss of motor neurones (MNs). It has been recently demonstrated that Transactive response DNA-binding protein (TDP-43) is the dominant disease protein in both ALS and a sub-group of frontotemporal lobar degeneration (FTLDTDP). Moreover, the identification of TARDBP mutations in familial ALS confirms a mechanistic link between the observed mis-accumulation of TDP-43 and neurodegeneration but also provides an opportunity to establish an in vitro platform to model these diseases, based on patient-derived induced pluripotent stem cells (iPSCs). This study presents the optimization of an iPSC-based platform to study the consequences of TDP-43 M337V mutation in human functional populations of MNs and astrocytes in isolation as well as in co-culture. To develop this platform, two protocols to differentiate patient-derived iPSCs into functional MNs and astrocytes were first optimized, and the obtained cellular populations were then used to characterize the behaviour of mutant TDP-43 and its effect on the different cell types. This study show that it is possible to use iPSC-based platforms to recapitulate in vitro key aspects of TDP-43 proteinopathies such as MN cell autonomous toxicity and TDP-43 accumulation, but they can also be used to highlight previously unrecognised disease specific mechanisms and to test novel therapeutic approaches. Moreover, by performing co-culture experiments it was possible to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of co-cultured neurons. This iPSC-based platform represents an in vitro model to study both the effect of somatic mutations on isolated patient-specific cultures, but also to investigate cellular autonomy and neurodegeneration in the context of TDP-43 proteinopathies.

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.

A great need exists for an abundant, easily accessible source of patient-specific cells that will function for use in regenerative medicine. One promising source is the adult stem cell derived from adipose tissue (ASCs). Isolated from waste lipoaspiration, these cells could serve as a readily available source for the regeneration of damaged tissues. To further define the biology of ASCs, we have isolated multiple cell strains from different adipose tissue sources, indicating wide-spread distribution in the body. We find that a widely used set of cell surface markers fail to distinguish ASCs from normal fibroblasts. However, our ASC isolations are multipotent while fibroblasts show no differentiation potential. In further contrast to fibroblasts, these cells also show expression of genes associated with pluripotent cells, Oct-4, SOX2, and NANOG. Together, our data suggest that while the cell surface profile of ASCs do not distinguish them from normal fibroblasts and their lack of telomerase shows their limited proliferation capacity, the expression of genes closely linked to pluripotency and their differentiation capacity clearly define ASCs as multipotent stem cells. iPS cells are another promising cell type for tissue regeneration, due to their expression of hTERT and their capacity to differentiate into all three germ layers. Interestingly, telomerase is activated during the induction process, accomplished by the exogenous expression of four genes in normal, non-hTERT-expressing fibroblasts. To elucidate the mechanisms behind this activation, we examined the overexpression of these four factors in BJ fibroblasts and ASCs, which resulted in undetectable hTERT expression. We then demonstrated a lack of an acetylated histone H3K9 with the opposing di-methylation, indicative of a closed chromatin state at the hTERT promoter. Subsequent treatment of cells with TSA alone showed an upregulation of hTERT mRNA without telomerase activity. However, telomerase activity was found when ASCs, but not BJs were treated with TSA and all four factors, indicating differential regulation of hTERT in cells of similar mesenchymal origins. Our data suggest that while hTERT’s expression is universally dependent on the presence of a relaxed chromatin state and sufficient transactivating factors, other cell to cell differences can prevent its expression.

There is a growing epidemic of chronic heart failure (CHF) in the developed world. The costs associated with providing care is profound and despite our best efforts, new, more effective treatments for CHF are needed; 50% of patients diagnosed with CHF are dead within 5 years. Current paradigms rely heavily on pharmacologic interventions, which merely help manage the disease. Surgical interventions may also be considered for late stage CHF patients such as heart transplant or left ventricular assist device (LVAD) but require burdensome and invasive surgical procedures. In addition they are costly, and require the need for life long immunosuppressive and anticoagulant therapies respectively. Despite our best intentions, the long-term prognosis for CHF patients remains poor. With over a decade of clinical investigation taken place, data from cell-based therapy trials remains inconsistent. While demonstrating safety, limited efficacy has been reported and to date, no stem cell therapy has been approved by the FDA. Despite these shortcomings important lessons have been learned that can be applied to future developments. Retrospective analysis of early cell-based clinical trial data has suggested that variations in isolated cell number, viability, and potency from donor to donor in autologous preparations yielded wide discrepancies in functional outcomes. In addition, sub culturing adult stem cells, even for short periods of time in 2D polystyrene environments void of complementary cell populations and extra cellular matrix protein interactions, may alter the therapeutic potential of a given cell. As a solution, allogeneic approaches where donor cell quality and potency can be assessed and optimized may help achieve functional benefits. Furthermore, co-dosing with multiple cell populations or developing 3D sub-culture environments that more closely mimic the in vivo milieu may ultimately yield more potent therapeutic cell populations. While these alterations may improve cell-based therapy outcomes, other solutions have been proposed such as tissue engineering. While the concept of tissue engineering is not new, advancements in biomaterials, bioreactor design and cell sources have greatly enhanced the reality of these preparations. Previously, one of the greatest limitations to tissue engineering is overcoming the cell requirements for developing and testing where millions if not billions of cells are required. Cell sourcing limitations appear to have been solved with the discovery and development of induced pluripotent stem cell (iPSC) derived cell populations. First reported in 2007, they have the ability to generate embryonic like pluripotent stem cells without the ethical concerns of embryonic stem cells. These iPSCs hold tremendous potential for drug toxicology / screening, personalized medicine and cell therapies. The body of work described in this dissertation looks at developing and testing a tissue engineered cardiac patch to treat heart failure. For which, an emphasis has been to provide 1) structural support for engrafted cells and 2) a rapidly inducible vascular supply once implanted in vivo. Biomaterials were sourced that facilitate infill by multiple cell populations in 3D culture and the establishment of extra cellular matrix deposits. Together, these patches enhanced cellular development in vitro and result in long term functional improvements in small animal models for CHF. Additional feasibility work was performed in large animal models to permit upscaling and development of surgical implantation techniques to demonstrate clinical applicability

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.

Schizophrenia is a major psychiatric disorder that affects 1% of the world population and is among the 10 leading worldwide causes of disability. Disrupted-In- Schizophrenia (DISC1) is one of the most studied risk genes for mental illness and is disrupted by a balanced translocation between chromosomes 1 and 11 that co-segregates with major mental illness in a single large Scottish family. DISC1 is a scaffold protein with numerous interactors and has been shown to hold key roles in neuronal progenitor proliferation, migration, cells signalling and synapse formation and maintenance. The studies herein provide the platform in order to investigate the molecular and cellular consequences of the t(1;11) translocation using induced pluripotent stem cells (iPSCs)-derived neural precursor cells and neurons from within-family carriers and controls. Towards this end, several iPSC lines have been converted into neural progenitor cells (NPCs) and differentiated into physiologically active forebrain neurons following well-characterised protocols. These cells were characterised in terms of basic marker expression at each developmental stage. Inter-line variation was observed in all subsequent experiments but overall t(1;11) lines did not generate less neuronal or less proliferating cells compared to control lines. Furthermore, the expression pattern of genes disrupted by the t(1;11) translocation was investigated by RT-qPCR. DISC1 was reduced by ~50% in the translocation lines, both neural precursors and neurons. This observation corresponds to previous findings in lymphoblastoid cell lines (LBCs) derived from members of the same family. Moreover, DISC1 expression was found to increase as neural precursors differentiation to neurons. Two other genes are disrupted by the t(1;11) translocation;DISC2 and DISC1FP1. Their expression was detectable, but below the threshold of quantification. Similarly, DISC1/DISC1FP1 chimeric transcripts corresponding to such transcripts previously identifies in LBCs from the family were detectable, but not quantifiable. A fourth gene, TSNAX, was also investigated because it is located in close proximity to, and undergoes intergenic splicing with, DISC1. Interestingly, TSNAX was found to be altered in some but not all time points studied, in the translocation carriers compared to control lines. In addition to breakpoint gene expression profiling, iPSC-derived material was used to investigate neuronal differentiation. There seemed to be attenuation in BIII-TUBULIN expression at two weeks post-differentiation, while NESTIN, MAP2 and GFAP expression was similar between translocation carrier and control lines at all time points studied. I also had access to targeted mice designed to mimic the derived chromosome 1 of the t(1;11) balanced translocation. Using RT-qPCR Disc1 expression was found to be 50% lower in heterozygous mice compared to wild types, and I detected a similar profile of chimeric transcript expression as detected in translocation carrier-derived LBCs. These observations support my gene expression studies of the human cells and indicate that the iPSC-derived neural precursors and neurons can be studied in parallel with the genome edited mice to obtain meaningful insights into the mechanism by which the t(1;11) translocation confers substantially elevated risk of major mental illness. In conclusion, the studies described in this thesis provide an experimental platform for investigation of the effects of the t(1;11) translocation upon function and gene and protein expression in material derived from translocation carriers and in brain tissue from a corresponding mouse model.