Pluripotency is the ability of a cell to differentiate into derivatives of all three somatic lineages and germ cells. In vivo, pluripotent cells exist transiently in the epiblast of the developing embryo and in rare tumour cells. In vitro, pluripotent cells have been isolated and propagated from teratocarcinomas (EC cells), preimplantation epiblast (ES cells) and post-implantation epiblast (EpiSCs). Pluripotency is governed by a gene regulatory network centred on the triumvirate of transcription factors Oct4, Sox2 and Nanog. Interestingly, transcription factors that are important to direct pluripotent cell identity are not all equally distributed throughout the pluripotent cell population. While Oct4 levels are relatively homogeneous, other transcription factors, such as Nanog, are more heterogeneously expressed. Additionally, an increasing body of evidence indicates that extrinsic cues also play a critical role in the establishment and maintenance of pluripotency. Using biochemical and genetic tools in mouse ES cells, the role of FGF signaling and Sox2 levels on heterogeneous Nanog expression was examined. Interference with FGF or ERK activity by genetic ablation or signal inhibition, promoted high, homogenous Nanog expression and enhanced self-renewal. This is consistent with reports showing that similar manipulations reduced the ability of ES cells to commit to differentiation. Moreover, ES cells with reduced Sox2 levels displayed greater heterogeneity for Nanog expression than wild-type ES cells. Pluripotency is lost in the mouse embryo around E8.5, however, the precise timing and mechanism involved in this process has not yet been defined. Here it is shown that pluripotency is extinguished at the onset of somitogenesis, coincident with reduced expression and chromatin accessibility of Oct4 and Nanog regulatory regions. Prior to somitogenesis, the expression of both Nanog and Oct4 is regionalized. Interestingly, pluripotency tracks the in vivo level of Oct4, this correlation does not hold true for Nanog. However, Nanog expression reports on pluripotent cells. Indeed, ectopic Oct4 expression in somitogenesis-stage tissue provokes rapid reopening of Oct4 and Nanog chromatin, Nanog re-expression and resuscitation of moribund pluripotency. Competence to re-activate the pluripotency network upon enforced Oct4 expression is gradually lost with the progression of embryonic development. ES cells and EpiSCs are two distinct pluripotent populations as they show differences in their ability to undergo clonal propagation, re-colonize embryos, growth factor responsiveness, morphology and gene regulatory networks. It is possible to harness this differential growth factor responsiveness to convert ES cells into EpiSCs. Conversely, EpiSC can be reverted back to ES cell pluripotency through the overexpression of a small number of transcription factors. The inter-conversion of ES cells and EpiSCs has been documented, but detailed analyses of the changes that occur during such transitions had not been performed. The current work shows that Nanog levels are critical for the specification of the pluripotent state of the cells. Furthermore, it is shown that orphan nuclear receptor Esrrb is a potent inducer of ES cell pluripotency in EpiSC. Interestingly, Esrrb was able to restore naïve pluripotency in cells genetically depleted of Nanog.

No description available.

The Oct-4 transcription factor is believed to co-regulate early embryonic development of mammals due to the correlation of its presence with the maintenance of pluripotency. It is commonly used as a marker for the identification of embryonic stem (ES) cells for this reason. Until 1999, Oct-4 studies were limited to in vivo-produced embryos; equine embryos have not been studied for their Oct-4 expression patterns. In addition, equine stem-like cells (defined by marker expression, induced differentiation, passage survival, and morphology) have recently been isolated from in vivo-produced embryos, but no work has been performed in horses to isolate ES cells from in vitroproduced embryos. This study investigated the expression of Oct-4 transcription factor using immunocytochemistry in 42 in vitro-produced embryos aged 1-10 days and in 5 in vivoproduced blastocysts aged 7-10 days. Effective conditions for rapid establishment of a feeder layer of equine fetal fibroblasts were established, and this feeder layer was used to grow isolated equine inner cell mass (ICM) cells from in vitro-produced embryos. The expression of Oct-4 was examined in resultant cell growths. In vitro-produced embryos less than 6 days of age showed variable staining within blastomeres of the same embryo, and the peak of variability correlated with maternal-zygotic transition. After Oct-4 staining of in vitro-produced blastocysts, no cells could be identified as an ICM based on a difference in fluorescent intensity from the other cells of the blasyocysts. However, in vitro-produced blastocysts that were subsequently cultured in vivo contained a presumptive ICM, visible based on greater fluorescent intensity of Oct-4 stain. The trophoblast of all blastocysts also stained positively for Oct-4 protein. Fibroblasts were successfully isolated from equine feti. Treatment with 20 µg/ml of Mitomycin C arrested cell growth without causing excessive death. Fibroblasts were inactivated and frozen, then thawed as needed to establish a confluent monolayer for ICM isolation overnight. ICMs from in vitro-produced embryos formed outgrowths, but none that could be identified morphologically as ES cells. Outgrowth cells contained about 20% Oct-4 expressing cells in sporadic groupings. Assuming appropriate binding of the Oct-4 antibody, Oct-4 expressing cells (potentially indicating pluripotency) are found throughout the embryo in early development and in the feeder layer after co-culture.

The rat has been an established experimental animal model within many areas of biological investigation for over one hundred years due to its size, breeding characteristics, and knowledge of its physiology and behaviour. In recent years its status as a leading biomedical model has been somewhat surpassed by the mouse. This is largely the result of the isolation and application of mouse embryonic stem (ES) cells. Mouse ES cells have the capacity for unlimited self-renew in vitro whilst maintaining pluripotency and germline competence, and most importantly are amenable to sophisticated reverse genetics strategies such as gene targeting, which have provided a route to germ line modification. Thus far, the derivation of rat ES cells has proved elusive. The generation of rat ES cells would therefore facilitate equivalent applications to rat genetics and significantly strengthen the rat as an experimental model system. Previous attempts to derive rat ES cells led to the isolation of rat ES-like cells. However, whilst these cells exhibit extensive self-renew in vitro, it was known that they fail to maintain significant levels of the key functional ES cell marker Oct4 and do not contribute to chimeras. Rather, these cells express the trophectoderm markers Cdx2 and CyclinD3, and have been termed ExS cells due to their probable extra-embryonic nature. In the work described in this thesis, further investigation of ExS cells revealed the absence of expression of the key pluripotency gene Nanog, although the expression pattern of Nanog in the rat embryo was shown to be similar to that of mouse. It was hypothesised that expression of exogenous Oct4 and Nanog or Sox2 genes could facilitate reprogramming of ExS cells into a 'true' ES cell state. Initial work described in this thesis demonstrated that it was possible to introduce transgenes into rat ExS cells and obtain stable transformants with long term transgene expression. On this basis Oct 4, Nanog and Sox2 transgene expression vectors were constructed and stably integrated into ExS cells, and transgene expression verified. However, no reactivation of an endogenous gene expression profile, characteristic of a true ES cell-like state, was observed in any of the transgenic lines produced. Concurrent with work on ExS cells, investigations by others using chemically defined, serum-free medium containing small molecule inhibitors of MEK and GSK3 (called 3i/2i medium) had demonstrated that it was possible to readily isolate mouse ES cells, even from strains known to be refractory to ES cell isolation. Therefore, the ability of this culture system to facilitate rat ES cell derivation was investigated. Rat 3i/2i cell lines were established from ICM outgrowths of Fischer, DA and Sprague Dawley E4.5 rat embryos. These cells maintained expression of Oct4 and Nanog and could generate complex teratomas consisting of all three germ layers. They were distinct from epiblast stem cells (EpiSC) in that they expressed Klf4, Rex1 and Stella and most importantly, they could contribute to the formation of adult chimaeras and demonstrated germline competency. Isolation of these authentic rat ES cells paves the way for gene targeting in the rat, a development that should greatly facilitate new biomedical discoveries.

CpG islands (CGIs) are short GC rich sequences with a high frequency of CpGs that are associated with the active chromatin mark H3K4me3. Most occur at gene promoters and are often free of cytosine methylation. Recent work has begun to clarify the functional significance of CGIs with respect to chromatin structure and transcription. In particular, proteins associated with histone-modifying activities, such as Cfp1 and Kdm2a, bind specifically to non-methylated CGIs via their CxxC domains. For example, artificial promoterless CpG-rich sequences integrated at the 3’ UTR of genes recruit Cfp1 and generate novel peaks of H3K4me3 in mouse ES cells without apparent RNA polymerase recruitment. There is also evidence that G+C-rich DNA recruits H3K27me3, a gene silencing mark. In this thesis I am exploring the constraints on DNA sequence and genomic location that are required to impose both H3K4me3 and H3K27me3 at CGI sequences. Showing that the generation of novel peaks of H3K4me3 and H3K27me3 over a promoter-less CpG rich sequence in a gene desert region is independent of it’s location in the genome extends earlier findings. These findings suggest that shared features of the primary DNA sequence at CGIs directly influence chromatin modification. Thus CGIs are not passive footprints of other cellular mechanisms, but play an active role in setting up local chromatin structure. However, the relative contribution of CpG frequency versus G+C content remains unclear. Therefore a sequence was generated that contains low levels of CpGs, comparable to the bulk genome, but has a G+C content similar to that of CGIs (Low CpG / High G+C). When this sequence was inserted into a gene desert neither marks of H3K4me3 or H3K27me3 were formed, indicating the importance of CpGs. Surprisingly, the reverse sequence with a high CpG frequency similar to that of CGIs and a low G+C content similar to that of the bulk genome (High CpG / Low G+C) did not establish H3K4me3 or H3K27me3 either. However, it was found that this sequence becomes heavily methylated in contrast to CGI-like sequences that remained unmethylated when introduced into a gene desert. This finding suggests that a high G+C content is important for keeping CGI-like sequences methylation free. Upon insertion of this High CpG / Low G+C sequence into mouse ES cells that were devoid of the de-novo DNA methyltransferases 3a and 3b (Dnmt3a/3b -/-) both H3K4me3 and H3K27me3 marks were established at the inserted sequence. This discovery confirms the importance of CpGs for setting up local chromatin structure.

Specification of a germ cell lineage and appropriate maturation are essential for the transfer of genetic information from one generation to the next. Germ cells form from pluripotent precursor cells that migrate into the gonadal ridge and undergo commitment to either the female or male lineage. In the fetal ovary, germ cells enter meiotic prophase I, then arrest at the diplotene stage; in the testis germ cells do not begin meiosis until puberty. Abnormal differentiation of germ cells can result in malignant transformation. Somatic cells play a key role in modulating the developmental fate of the germ cells. Research into germ cell development during fetal life has almost exclusively focused on studies in rodents, but we, and others, have reported several fundamental differences in the expression of germ cell specific markers in the human compared with the mouse. The studies described in this thesis have investigated germ cell-specific gene expression and the possible impact of the somatic cells during development. This was achieved by studying human fetal gonads obtained during the 1st and 2nd trimesters of pregnancy and through the use of both wild-type and mutant mouse ES cell lines. Studies on germ cells in the human fetal testis have extended the findings of others, and confirmed that germ cell populations at different stages of maturation co-exist in the human fetal testis, a situation that is in contrast to that in rodents. For example expression of M2A and AP2γ was restricted to the OCT4-positive gonocyte population, while VASA and NANOS1 were localised exclusively to the to the OCT4-negative prespermatogonia. DAZL was expressed in both populations. Analysis also revealed that both the gonocyte and prespermatogonial populations proliferate throughout the 2nd trimester. Recent studies have implicated retinoic acid (RA) in the control of meiotic entry in germ cells of the fetal mouse ovary. In this study we demonstrated for the first time that two genes implicated in the action of RA in mouse gonad, STRA8 and NANOS2, are also expressed in a similar sexspecific- manner in the human fetal gonads, and that the RA receptors are present in both somatic and germ cells suggesting that RA may regulate germ cell function in the human as well as the mouse. However, whilst the mesonephros appears to be the primary site of RA synthesis in the mouse our initial studies indicate that in the human the gonad itself may be a more likely site of RA biosynthesis. In the fetal mouse testis, RA is degraded by the enzyme Cyp26b1 present in the somatic cells and germ cells do not enter meiosis, our novel findings suggest that CYP26B1 is more abundant in the human fetal ovary than the testis, suggesting that meiotic entry may be controlled by an alternative signalling pathway in the human. One of the methods that can aid our understanding of somatic cell gene expression in the gonad is in vitro culture. To date, there have been no published reports of the successful in vitro culture of somatic cells from the human fetal testis. In the current study, populations of human somatic cells were dissociated and maintained in vitro and characterised. Analysis demonstrated that cells expressing mRNAs characteristic of Sertoli cells, Leydig cells and peritubular myoid (PTM) cells were present initially, but long-term culture resulted in downregulation in expression of mRNAs specific for Sertoli cells and Leydig cells, suggesting that these cells either failed to survive or underwent alterations to their phenotype. In contrast PTM/fibroblast cells proliferated in vitro and initially maintained androgen receptor expression. These cultures therefore hold promise for studies into the signalling or cell-cell interactions in testicular somatic cells especially those relevant to the PTM population. Several studies have claimed differentiation of putative germ cells from ES cells. In the current study, analysis of mouse ES cell lines has expanded on results showing that ES cells and early germ cells express a number of genes in common. Kit signalling was shown to be important for ES cell survival as they differentiate although expression of Kit was heterogeneous. We also demonstrated that ES cells that did not express Kit displayed a decreased expression of the early germ cell genes Blimp1, Fragilis and Stella, implicating Kit signalling in the control of germ cell-associated gene expression in ES cells. This may be important to future studies optimising germ cell derivation from ES cells. In conclusion, this study has demonstrated important differences in protein expression patterns in germ cells of the human fetal testis compared to the mouse, and has raised questions about whether the proposed mechanism controlling meiotic entry of germ cells in the mouse can be applied to the human. The establishment of a system for culturing human fetal gonadal somatic cells may lead to further understanding of gene expression and development in the human fetal testis, and data suggest that the Kit/Kitl signalling system may influence germ cell gene expression in mouse ES cells.

Nanog is a transcription factor that plays a central part in the gene regulatory network that maintains and induces pluripotency of embryonic stem cells (ESCs). However, the molecular basis by which Nanog achieves its functions is not fully understood. At the centre of C-terminal domain of Nanog a tryptophan repeat (WR) is located, comprising 10 penta-peptide repeats each starting with a tryptophan. A mutant form of Nanog (Nanog-W10A) in which all 10 tryptophan residues have been substituted by alanine has an impaired capacity to drive LIF-independent self-renewal and a reduced efficiency in reprogramming primed epiblast stem cells to naïve pluripotency. To understand how the WR contributes to Nanog function, Nanog-W10A-ERT2 was introduced into Nanog null cells. Upon hydroxytamoxifen addition, the Nanog-ERT2 fusion proteins were detected on chromatin within 1 hour, allowing a comparison of genome-wide transcriptional responses to Nanog and Nanog-W10A by microarray. When treated with LIF, Nanog-W10A can activate most of Nanog targets as efficiently as Nanog. In contrast, Nanog-W10A did not efficiently repress most Nanog targets, including Otx2 and Tcf15 that were previously suggested to prime ESCs for differentiation. The microarray experiments performed in the absence of LIF signalling showed that Nanog and LIF co-regulate an extensive list of targets, including Klf4 and Mras. When LIF is absent, wildtype Nanog can still activate pro-self-renewal factors, including Esrrb and repress differentiation-priming factor, such as Tcf15 and Otx2. In contrast, in the absence of LIF, the activation of pro-self-renewal factors Klf4 and Mras is reduced. In addition, activation of Esrrb by Nanog-W10A induction delays but does not prevent differentiation. These effects allow the de-repression of Otx2 and Tcf15 by Nanog-W10A to dominate. Therefore, the function of Nanog is not only mediated by the activation of pro-self-renewal genes, but also repression of pro-differentiation signals. The functional significance of the repression of Nanog targets was further exemplified by the robust capacity of Otx2 to dominate over the self-renewal signals and to drive differentiation. The Otx2 protein is a direct interacting partner of Nanog that binds the Nanog WR tryptophan residues. The previously identified Otx2 “tail domain” comprises two imperfectly aligned repeats and aromatic residues of each repeat align with aromatic residues of the Sox2 “SXS/TY” motif previously identified to mediate the interaction between Sox2 and Nanog. Aromatic residues of Otx2 were demonstrated to directly interact with both Nanog and Sox2. The interactions between Otx2, Nanog and Sox2 are essential for Otx2 functions in driving ESCs differentiation, as Otx2 mutants with alanine substitutions of the aromatic residues in both or either of the repeats have reduced efficiency to drive differentiation. As Nanog and Sox2 may co-occupy many loci important in maintaining ESC self-renewal, Otx2 may be able to “read” the Nanog/Sox2 co-binding sites to dissolve the pluripotent networks. In summary, the repression function of Nanog is located within the Nanog WR region and represents an important module of Nanog in fine-tuning the balance between self-renewal and differentiation. This module involving Nanog WR can also be recognised by differentiation-priming factor Otx2 and may represent an initial step during the exit of differentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20790号 / 医博第4290号 / 新制||医||1025(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 高橋 淳, 教授 井上 治久, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

The thymus is the major site for T-cell generation and thus is important for the adaptive immune system. Development of a properly selected, functional T-cell repertoire relies on interactions between developing T cells and a series of functionally distinct thymic stroma cell types including the cortical and medullary thymic epithelial cells (TECs). The thymus is one of the first organs to degenerate in normal healthy ageing. Related to this, there is strong interest in developing protocols for improving thymus function in patients by cell replacement or regenerative therapies. Thymic epithelial progenitor cells (TEPCs) represent a potential source of cells for thymus transplantation. However, the only source of these cells for transplantation is currently fetal thymus tissue. If TEPCs could be generated from pluripotent cells, this could provide an alternative source of cells for transplantation. The work described in this thesis therefore had two central aims (i) to test the stability of thymic epithelial progenitor cells in vivo and (ii) to investigate the possibility of generating TEPCs or TECs from mouse embryonic stem (ES) cells. The forkhead transcription factor, Foxn1, is essential for the development of a functionally mature thymic epithelium, but is not necessary for formation of the thymic primordium or for medullary thymic epithelial sub-lineage specification. By reactivating Foxn1 expression postnatally in mice carrying a revertible hypomorphic allele of Foxn1, Foxn1R, I herein demonstrate that TEPCs that can express only low levels of Foxn1 mRNA can persist postnatally in the thymic rudiment in mice until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression. In parallel with this work, I confirmed the possibility of generating Foxn1-expressing cells from mouse ES cells by using a Foxn1 reporter cell line. As the thymic epithelium has a single origin in the third pharyngeal pouch (3pp) endoderm, I then tested whether or not TEPCs and /or TECs were generated during ES cell differentiation via existing protocols for generating anterior definitive endoderm differentiation cells from mouse ES cells. From this work, I showed that genes expressed in the 3pp and/or TEPC,-including Plet-1, Tbx1, Hoxa3 and Pax9, were induced by differentiation of ES cells using these protocols. I further showed that cells expressing both Plet-1, a marker of foregut endoderm and 3pp, and EpCAM, a marker of proliferating epithelial cells, were induced using a novel protocol (2i ADE) for generating ES cells from ADE. However, gene expression analysis and functional testing suggested that the majority of these cells were non-thymus lineage. I subsequently developed a novel protocol which combined this 2i ADE protocol with co-culturing of the differentiating ES cells with fetal thymic lobes, and demonstrated that this further induced 3pp and TEPC related genes. Finally, I modified the culture conditions in this protocol to conditions predicted to better support TEPC/TEC, and showed that in these conditions, the TEPC-specific markers Foxn1 and IL-7 were induced more strongly than in any other conditions tested. The data presented in this thesis therefore represent an advance towards an optimized protocol for successfully generating TEPCs from ES cells in vitro.