The basis of developmental biology lies in the idea of when and how cells decide to divide or to differentiate. Previous studies have established several signaling pathways that determine cell fate decisions, including Notch, Wingless, Hedgehog, Bone morphogenetic protein, and Fibroblast growth factor. Signaling converges on transcriptional factors that regulate gene expression. In mouse embryonic stem cells, I explored how pluripotency and differentiation are regulated through opposing actions of beta-catenin-mediated canonical Wnt signaling, and the mechanisms underlying Sonic hedgehog signaling in generating progenitor cells in the ventral neural tube.

Those who are very wealthy may also be extremely free. Independently wealthy philanthropists epitomize this type of freedom. They seem to be able to act in whichever way they please, as long as they respect the limits of the law. Their freedom also implies that they do not experience as much accountability as other funders. Considering philanthropists’ ambitions as policymakers, and given their imposition of performance demands on their grantees, their accountability is relevant to investigate. However, there are no comprehensive comparative studies of philanthropists’ accountability, and there is mainly anecdotal evidence of a lack of accountability being derived from their independent wealth. This dissertation is a study of philanthropists’ accountability. I compare their experienced and exhibited accountability to that of other funders within societies, and I also compare philanthropists’ accountability across societies. I investigate whether philanthropists’ independent wealth influences to whom they are accountable, for what they are accountable, and how they are accountable. To learn about these topics, I examine their accountability relationships, their accountability mechanisms, and how they justify their potentially controversial funding of human embryonic stem cell research. Across these dimensions, I study their legal, financial, hierarchical, peer, professional, political, and fiduciary/social accountability. Empirically, I make a cross-sectional comparison of philanthropists to other funders of human embryonic stem cell research within and across three welfare regimes - liberal California, social democratic Sweden, and statist South Korea. I compare the accountability of independently wealthy philanthropists to that of public agencies, corporations, and fundraising dependent nonprofits. The empirical materials include 101 structured interviews with open-ended questions covering 51 funding organizations, as well as questionnaires explored in ANOVA and social network analysis. The study indicates that philanthropists experience and exhibit less accountability than other funders in some ways, in some contexts. By developing and using a framework to analyze their accountability, I show that philanthropists’ accountability is patterned within the societies in which they fund, and it differs greatly across societies. In California, philanthropists enact themselves as free actors, whereas in Sweden they enact a moral identity as funders of science. In South Korea, there is no clear boundary between philanthropic and corporate accountability. My results point to the contextual limits of philanthropists’ accountability. By enacting their moral identity in a way that conforms to local norms, philanthropists simultaneously retain and enable their continued freedom. In terms of their accountability, philanthropists are free to conform, and they become free by conforming.

Em fêmeas de mamíferos, um dos cromossomos X é inativado proporcionando compensação de dose entre os produtos gênicos de machos e fêmeas. A inativação do cromossomo X (ICX) ocorre no embrião em desenvolvimento, e se caracteriza pela aquisição de marcas heterocromáticas no cromossomo X inativado (Xi), que são mantidas nas células somáticas ao longo das divisões celulares. O melhor modelo para estudo do início da ICX são as células-tronco embrionárias femininas. Provenientes da massa celular interna de blastocistos, elas representam um embrião em desenvolvimento e possuem os dois X ativos; a diferenciação das células promove a ICX in vitro, o que permite a identificação dos fatores e mecanismos moleculares envolvidos. A derivação de linhagens de célulastronco embrionárias humanas (human embryonic stem cells - hESCs) em 1998 permitiu novas possibilidades de estudo da ICX, pois a maioria dos trabalhos procurou esclarecer o mecanismo da ICX no modelo murino. Tradicionalmente, a manutenção da ICX em humanos tem sido investigada em células somáticas híbridas ou transformadas; porém, sabe-se que estas não representam um contexto celular natural. Assim, o presente trabalho teve como objetivos principais explorar a potencialidade de hESCs no estudo do início da ICX, e ainda investigar a função de três fatores na manutenção da ICX em células humanas imortalizadas: DNMT1 (enzima responsável pela manutenção da metilação do DNA), SMCHD1 (proteína da família de coesinas/condensinas), e XIST (um RNA não-codificador que inicia o processo de heterocromatinização do futuro Xi) foram selecionados para este estudo, uma vez que todos participam da manutenção da ICX em camundongos. Até o momento foram derivadas em nosso laboratório quatro linhagens de hESCs, as primeiras da América Latina. A caracterização das linhagens mostrou que, apesar de se manterem indiferenciadas, as hESCs femininas encontram-se em estágio pós-ICX, pois mesmo indiferenciadas já apresentam um dos X inativado. Nossos dados indicam que, submetidas às atuais condições de cultivo, as hESCs não são bons modelos para o estudo do início da ICX, e é possível que a inativação de um cromossomo X durante o cultivo confira alguma vantagem seletiva às células. A estratégia utilizada no estudo da manutenção da ICX foi o silenciamento dos três genes por interferência de RNA (RNAi). Não foi possível diminuir significativamente a expressão dos genes XIST e SMCHD1. Porém, o silenciamento de DNMT1 foi expressivo, e em resposta foi observada reativação do gene MAOA, localizado no cromossomo X e submetido à inativação. Apesar de nossas análises mostrarem que os efeitos da diminuição de DNMT1 foram restritos ao gene MAOA, estes resultados sugerem a existência de diferentes hierarquias de controle epigenético dos genes submetidos à ICX em células humanas / In female mammals, one of the X chromosomes is inactivated to achieve dosage compensation between males and females. The X chromosome inactivation (XCI) occurs early during embryogenesis and is characterized by the acquisition of heterochromatic features on the inactive X (Xi), which are maintained during all the subsequent cell divisions. Embryonic stem cells are the most suitable cells to study the establishment of XCI. They are obtained from the inner cell mass (ICM) of blastocysts, and can represent a developing female embryo, possessing two active X-chromosomes; when differentiated, these cells recapitulate XCI in vitro, and thus one can identify XCI regulators and factors involved. The derivation of human embryonic stem cells (hESCs) in 1998 offered new possibilities to study XCI, since most of the mechanistic studies of XCI have so far been investigated in the mouse model system. Traditionally, maintenance of XCI in humans has been addressed in somatic cell hybrids or transformed cells; however, they do not represent a natural cellular context. The main goals of the present work were to verify the potential of hESCs as models of XCI, and also to study the function of three important factors in XCI maintenance in immortalized human cells. DNMT1 (DNA-methyltransferase 1), SMCHD1 (a cohesin/condensin protein family member) and the XIST gene (a non-coding RNA which triggers XCI and promotes X heterochromatin formation on the future Xi) were selected, as they are key factors in XCI maintenance in the mouse. Until now four hESCs lines were derived in our lab. Their characterization showed that, in spite of been undifferentiated, the female hESCs have already undergone XCI. Our data suggest that, under the actual culture conditions, hESCs are not good models to study XCI, and it is also possible that X inactivation confers selective advantage to hESCs. Knockdown by RNA interference was used to study the roles of three genes in XCI maintenance. We could not efficiently knockdown XIST or SMCHD1. However, the DNMT1 silencing was substantial, and led to the reactivation of MAOA, an X-linked gene subjected to XCI. Although the effect of DNMT1 silencing was restricted to MAOA, our data suggest that there are different epigenetic hierarchies to control the expression of the genes subjected to XCI in human cells.

The Hedgehog (HH) signalling pathway and its primary transducer, GLI2, regulate cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells. To further assess the role of HH signalling during mouse embryonic stem (mES) cell differentiation, we studied the effects of GLI2 overexpression during mES cell differentiation. GLI2 overexpression resulted in temporal enhancement of cardiac progenitor genes, Mef2c and Nkx2-5, along with enhancement of Tbx5, Myhc6, and Myhc7 in day 6 differentiating mES cells. Mass spectrometric analysis of proteins that immunoprecipitate with GLI2 determined that GLI2 forms a complex with BRG1 during mES cell differentiation. Furthermore, modulation of HH signalling during P19 EC cell differentiation followed by chromatin immunoprecipitation with an anti-BRG1 antibody determined that HH signalling regulates BRG1 enrichment on Mef2c. Therefore, HH signalling accelerates cardiac progenitor gene expression during mES cell differentiation potentially by recruiting a chromatin remodelling factor to at least one cardiac progenitor gene.

Embryonic stem (ES) cells possess the remarkable ability to form cells and tissues from all three germ layers, a characteristic known as pluripotency. In particular, the generation of ES cell-derived hematopoietic cells could serve as an alternate source of hematopoietic stem cells for transplantation in place of bone marrow cells, which are limited by donor availability and high immunogenicity. The advantages of ES cell-derived hematopoietic cells over bone marrow cells include a greater proliferative capacity, which alleviates the problems of donor shortage, and low level expression of MHC antigens, which suggests immune privilege. However, it is unclear whether the immune system is capable of recognizing and rejecting ES cell-derived hematopoietic cells following transplantation. The observation that ES cell-derivatives express low levels of MHC class I, the predominant inhibitory ligand for NK cells, led us to hypothesize that ES cell-derived hematopoietic progenitor cells (HPC) are susceptible to NK cell-mediated killing. To test this hypothesis, we first generated HPCs from murine ES cells ectopically expressing HOXB4, a homeobox transcription factor that confers hematopoietic self-renewal, and confirmed that HPCs expressed low levels of MHC class I antigens. To specifically investigate the role of NK cells in regulating the in vivo engraftment of HPCs, we transplanted NK-replete Rag2-/- or NK-deficient Rag2-/-γc-/- mice with HPCs. We observed permanent HPC engraftment in Rag2-/-γc-/- mice; however, HPC engraftment was significantly reduced in Rag2-/- mice and was eventually eliminated over time. Bone marrow harvested from these animals showed that HPC-derived Lin-c-kit+ and Lin-Sca-1+ progenitor cells, critical progenitor cells for long-term hematopoietic engraftment, were deleted in Rag2-/- but not in Rag2-/-γc-/- mice. Next, we focused on the mechanism of NK cell activation by HPCs. Increased expression of the cytotoxic proteins Granzyme B and Perforin in the NK cells of HPC-transplanted Rag2-/- mice confirmed in vivo NK cell activation. Phenotypic analysis of HPCs revealed high level expression of H60, a ligand of the NK activating receptor NKG2D, and neutralization of H60 rescued HPCs from NK cell-mediated killing. Altogether, our results demonstrate that NK cells are a major barrier to the successful engraftment of ES cell-derived hematopoietic cells, underlining an important role of the innate immune system in regulating the long-term engraftment of ES cell derivatives.

In eukaryotic interphase cells, heterochromatin mostly localizes either at the nucleolar periphery or at the nuclear lamina. Genome localization studies are crucial due to evidence that spatial organization of the genome affects gene function. Nucleolus-associated domains (NADs) are mainly heterochromatic regions that have been mapped only in a handful of mouse and human somatic cells, and in plants. The extent to which changes in NAD localization occur during cellular differentiation remains unknown. In this thesis, we characterize a map of genome-wide NADs in F121-9 mouse embryonic stem cells (mESCs). We identified NADs by deep sequencing chromatin associated with biochemically purified nucleoli and using NADfinder software to call NAD peaks. F121-9 NADs are mostly comprised of genomic regions with inactive or lowly transcribed genes and overlap extensively with lamina-associated domains (LADs) and regions with late replication timing. Similar to somatic mouse embryonic fibroblasts (MEFs), where NADs have been previously characterized by our laboratory, F121-9 mESCs display abundant “Type I” NADs. This subset of NADs frequently associates with nuclear lamina and nucleolar periphery and resembles constitutive heterochromatin. Compared to MEFs, F121-9 mESCs have fewer “Type II” NADs; this subset of NADs is frequently found at the nucleolar periphery but not at the nuclear lamina. mESC NADs are also less enriched in H3K27me3 modified regions compared to MEF NADs. This suggests that Polycomb complex-mediated facultative vii heterochromatin expansion is part of NAD maturation during cellular differentiation. Comparison of MEF and mESC NADs also revealed enrichment of developmentally regulated genes in NADs specific to these cell types. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin. Our F121-9 mESC NAD studies identified distinct features of stem cell NADs and will facilitate future studies of genome organization changes during mammalian development.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19568号 / 医博第4075号 / 新制||医||1013(附属図書館) / 32604 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊佐 正, 教授 渡邉 大, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Final publication is available at http://www.sciencedirect.com/science/article/pii/S1873506115001191 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19579号 / 医博第4086号 / 新制||医||1013(附属図書館) / 32615 / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 髙橋 良輔, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Embryonic stem (ES) cells have the ability to maintain pluripotency and self-renewal during in vitro maintenance, which is a key to their clinical applications. ES cells are a model in developmental biology studies due to their potential to differentiate in vitro. Understanding critical pathways of pluripotency, self-renewal, and differentiation during early embryonic development is important for the evaluation of the therapeutic potential of ES cells because of their ability for tumor transformation due to genetic and epigenetic instability acquired during in vitro culture maintenance. Single tandem repeats are sequences of DNA that have been implicated in the deregulation of gene expression in different human conditions. Understanding the origin of repetitive sequence instability and functions in the genome allow characterization of early genomic instability signals in ES cell pluripotency, differentiation, and tumor transformation pathways. The hypothesis of this study was that genetic stability, in repetitive sequences, located near embryonic developmental genes is responsible for pluripotency, self-renewal, differentiation, and chromatin assembly and could be a signal for adaptation, differentiation, or transformation of ES cells in vitro. Our result showed instability in specific repetitive sequences which increased during ES cell passages and embryoid body differentiation in vitro. ES cells displayed significant mean frequencies of genomic instability in repetitive regions that lead to ES cells pluripotency, self-renewal maintenance, or cell lineage specialization. The present study reports potentially biomarkers for identifying accumulation of genomic instability in specific genes that may contributes to adaptation of ES cells and could be the switch that initiates early ES cell lineage commitment in vitro. Determining genetic and epigenetic modifications, including single tandem repeat instability, gene expression changes, and chromatin modifications, is essential for elucidating possible molecular mechanisms of genomic instability and determining novel molecular characterization for diagnostic purposes to ensure ES cell stability and integrity that could potentially lead to use of ES cell derivatives that could then be a safe source needed for regenerative medicine applications