Indiana University-Purdue University Indianapolis (IUPUI) / This dissertation describes structure-function studies of CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, in order to determine the functional significance of Cfp1 protein domains and properties. Cfp1 is an important regulator of chromatin structure and is essential for mammalian development. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1-/-) are viable but demonstrate a variety of defects, including hypersensitivity to DNA damaging agents, reduced plating efficiency and growth, decreased global and gene-specific cytosine methylation, failure to achieve in vitro differentiation, aberrant histone methylation, and subnuclear mis-localization of Setd1A, the catalytic component of a histone H3K4 methyltransferase complex, and tri-methylated histone H3K4 (H3K4me3) with regions of heterochromatin. Expression of wild-type Cfp1 in CXXC1-/- ES cells rescues the observed defects, thereby providing a convenient method to assess structure-function relationships of Cfp1. Cfp1 cDNA expression constructs were stably transfected into CXXC1-/- ES cells to evaluate the ability of various Cfp1 fragments and mutations to rescue the CXXC1-/- ES cell phenotype. These experiments revealed that expression of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) is sufficient to rescue the hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and differentiation defects. These results reveal that Cfp1 contains redundant functional domains for appropriate regulation of cytosine methylation, histone methylation, and in vitro differentiation. Additional studies revealed that a point mutation (C169A) that abolishes DNA-binding activity of Cfp1 ablates the rescue activity of the 1-367 fragment, and a point mutation (C375A) that abolishes the interaction of Cfp1 with the Setd1A and Setd1B histone H3K4 methyltransferase complexes ablates the rescue activity of the 361-656 Cfp1 fragment. In addition, introduction of both point mutations (C169A and C375A) ablates the rescue activity of the full-length Cfp1 protein. These results indicate that retention of either DNA-binding or Setd1 association of Cfp1 is required to rescue hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and in vitro differentiation. In contrast, confocal immunofluorescence analysis revealed that full-length Cfp1 is required to restrict Setd1A and histone H3K4me3 to euchromatic regions.

Mouse embryonic stem cells (ESCs) are capable of differentiating into any somatic cell type and are known to display fragile immune privilege in vivo and in vitro. The extent to which the depletion of essential amino acids (EAAs) by ESCs contributes to this phenomenon was investigated. ESCs were found to express various enzymes capable of catabolising EAAs within the culture medium. In particular, depletion of threonine, valine and lysine was found to have significant impact on T cell proliferation and differentiation, biasing their polarisation towards a FoxP3⁺ T regulatory (T_reg) phenotype. Supplementing ESC conditioned medium with these three EAAs alone rescued normal T cell proliferation, whereas artificially limiting their availability was sufficient to induce T_reg cell differentiation to a level equivalent to general EAA depletion. The pattern of EAA catabolism by mouse ESC was shared by induced pluripotent stem cells, while mouse melanoma cell lines and human ESCs displayed distinct patterns of EAA depletion. The cytosolic branched chain aminotransferase enzyme, Bcat1, catalyses the first step of branched chain amino acid catabolism (isoleucine, leucine and valine), and is highly expressed by both mouse and human ESCs. The contribution of this enzyme to the establishment of acquired immune privilege by ESC-derived tissues was, therefore, investigated. ESC lines were derived from mice lacking Bcat1 activity and were characterised. Bcat1^−/− ESC lines displayed no difference to their wildtype counterparts (Bcat1^LoxP) in terms of in vitro proliferation and their capacity to form teratomas in vivo. Furthermore, the loss of Bcat1 function had little impact on the inhibition of T cell proliferation in culture, ability to induce T_reg cell commitment or their ability to prevent rejection by T cell receptor transgenic recipients, suggesting the minimal contribution of Bcat1 to the depletion of EAAs by ESCs. In conclusion, EAA depletion by mouse ESC may provide a mechanistic explanation for the previously described immune-suppressive capacity of ESC.

Murine embryonic stem (ES) cells are derived from the inner cell mass of the developing embryonic blastocyst. These cells can self renew which allows them to be propagated indefinitely in the laboratory and they can differentiate into cell types derived from all three germ layers. Manipulation of the mouse genome using gene targeting techniques in conjunction with ES cell technology has provided valuable insights into embryonic development and cell lineage specification. KIT is a trans-membrane receptor tyrosine kinase (RTK) that dimerises upon binding to its ligand, stem cell factor (SCF) resulting in the auto-phosphorylation of intracellular kinase domains. This activity is crucial for the transmission of signals from the cell surface to the nucleus. KIT is expressed on stem and progenitor cells of many lineages and defects in the SCF/KIT signaling pathway causes detrimental effects at both the cellular and physiological level. This project aimed to investigate the role of the SCF/KIT signalling pathway during murine ES cell differentiation and survival. To assess the role of SCF/KIT signalling in ES cell proliferation and survival, we knocked out the c-kit gene in mouse ES cells to produce heterozygous (KitW-lacZ/+) and KIT Null (KitW-lacZ/W-lacZ) cell lines. The self renewal and differentiation profile of these cell lines revealed an auxiliary role for SCF/KIT during ES cell self renewal and an absolute role in survival upon in vitro differentiation. This phenotype of apoptosis upon differentiation was recapitulated in wild type E14 ES cells treated with a KIT neutralising antibody (ACK2). Wild type cells that were treated with the JNK inhibitor, SP600125 had a comparable phenotype to KIT null cells indicating that this could be one of the mediators of KIT signalling that has a protective role in the survival of differentiating ES cells. We hypothesised that blocking classical apoptotic pathways might prevent the death on differentiation observed in KIT null cells. However, neither blocking the pro-apoptotic P38 pathway with the chemical inhibitor PD169316 nor over-expressing the pro-survival protein BCL2 in KIT Null cells could prevent their apoptosis upon differentiation phenotype. This strongly suggests that these pathways are not involved in KIT mediated survival of differentiating ES cells. Although compensatory mechanisms are thought to exist for defective KIT signaling in vivo, an absolute role is assigned to KIT during ES cell differentiation. Further analysis of micro array data comparing gene expression from wild type E14 and KIT Null cell lines may reveal the specific mechanisms of KIT mediated survival during differentiation onset.

During development of the neocortex, many diverse projection neuron subtypes are generated under regulation of cell-extrinsic and cell-intrinsic controls. One broad projection neuron class, corticofugal projection neurons (CFuPN), is the primary output neuron population of the neocortex. CFuPN axons innervate sub-cortical targets including thalamus, striatum, brainstem, and spinal cord.

Current feeder-free culture systems employing undefined Matrigel are still more effective in maintaining human embryonic stem (ES) cells than defined surfaces using extracellular matrix (ECM) proteins. While the role of substrate stiffness in stem cell fate is becoming increasingly evident, all previous culture systems use ECM proteins on rigid polystyrene surfaces. Here, we used factorial designs to screen and evaluate combinations ECM proteins and substrate stiffness for their effect on short-term pluripotency and self-renewal. Using optimal conditions determined from our screening experiments, defined and near xeno-free culture systems maintained CA1 human ES cells for over 10 passages in Essential 8 (E8) medium. Under these conditions, we found that human ES cell self-renewal was greater on soft polydimethylsiloxane (PDMS) substrates than on rigid polystyrene dishes. The culture systems and screening tools developed in this project will help develop robust and defined xeno-free culture systems that incorporate both biochemical and biomechanical factors.

Current feeder-free culture systems employing undefined Matrigel are still more effective in maintaining human embryonic stem (ES) cells than defined surfaces using extracellular matrix (ECM) proteins. While the role of substrate stiffness in stem cell fate is becoming increasingly evident, all previous culture systems use ECM proteins on rigid polystyrene surfaces. Here, we used factorial designs to screen and evaluate combinations ECM proteins and substrate stiffness for their effect on short-term pluripotency and self-renewal. Using optimal conditions determined from our screening experiments, defined and near xeno-free culture systems maintained CA1 human ES cells for over 10 passages in Essential 8 (E8) medium. Under these conditions, we found that human ES cell self-renewal was greater on soft polydimethylsiloxane (PDMS) substrates than on rigid polystyrene dishes. The culture systems and screening tools developed in this project will help develop robust and defined xeno-free culture systems that incorporate both biochemical and biomechanical factors.

Tumours are not autonomous transformed cell populations, but rather a society composed of both malignant and normal, including immune, cells that together foster tumour growth and development. Tumour-associated macrophages have been reported to enhance tumour growth, progression and metastasis. In high-grade non-Hodgkin’s lymphomas, prototypically the B-cell neoplasm, Burkitt’s lymphoma (BL), infiltrating macrophages engulf large numbers of apoptotic tumour cells. Evidence suggests that apoptotic BL cells can condition the tumour microenvironment to promote lymphoma development by selectively attracting macrophages while inhibiting neutrophil infiltration and by stimulating macrophages to produce the B-cell growth and survival factor. Tumour cells grow in a hypoxic and nutrient-deficient environment and the resultant cellular stress can induce apoptosis. It is therefore possible that hostile environmental conditions in the tumour also contribute to the generation of a pro-tumour microenvironment. This thesis describes investigations which examined this hypothesis. BL cells were cultured at high density to mimic conditions of metabolic stress existing in the tumour environment. Cell-free supernatants from such stressed BL cells demonstrated potent chemoattractive activity for mononuclear phagocytes. Supernatants from BL cells that were protected from apoptosis by over-expression of bcl-2 had similar ability, confirming that chemoattractant release was apoptosis-independent. The observation that apyrase and suramin could inhibit the chemotactic activity of these supernatants suggested that nucleotides might be the apoptosis-independent chemoattractant. Detection of ATP in stress supernatants by bioluminescence assay was consistent with this proposal. Significantly, supernatants from BL cells and those transfected with bcl-2 were both found to inhibit neutrophil migration, suggesting the occurrence of a neutrophil migration inhibitory factor whose release was apoptosis-independent. Furthermore, stress supernatants could promote BL cell proliferation in vitro, which was apoptosis and cell line-independent. In order to study the role of TAM in the tumour microenvironment, a novel macrophage model was devised using mouse embryonic stem cells (ES cells). Cells derived from ES cells generated in vitro expressed macrophage-specific markers and were free of dendritic cells and undifferentiated ES cells. ES cell-derived macrophages (ESDM) could migrate towards apoptotic BL cells and engulf them. However, ESDM migrated to stress supernatants with decreasing efficiency as they matured. Preliminary data indicated that the phagocytic ability of ESDM to engulf apoptotic cells increased as they matured, consistent with distinct roles for circulating monocytes and tissue macrophages with regard to this function. Considering the high yields and purities of ESDM described here, together with their non-malignant nature and genetic versatility these cells should provide a superior source of undifferentiated mononuclear phagocytes with which to elucidate the molecular mechanisms underlying tumour infiltration and microenvironmental conditioning by TAM. In conclusion, this work suggests that under conditions of pre-apoptotic stress, BL cells have the capacity to regulate their micro-environment upstream of their apoptosis programme to promote net tumour growth through paracrine signals that attract supportive macrophages and inhibit destructive neutrophils and through release of autocrine/juxtacrine tumour growth factors.

Transwell inserts with microporous membranes, available from multiple commercial sources, have been widely used for various mammalian cell culture applications, including the reduction of cell culture mixing. In this study, we examined the feasibility and functionality of using this technology for separating human embryonic stem cells (hESCs) from their respective feeder cells. We found that when hESCs were propagated on transwell inserts positioned directly above feeder cells grown in a separate dish, the hESCs could be maintained in an undifferentiated state for over 10 passages with no change in their basic pluripotent characteristics. In parallel with our transwell insert experiments, we also evaluated the ability of a new defined, xeno-free medium, HEScGRO™, to enhance the animal-free characteristics of the transwell insert-based culture system. Results from our studies demonstrate that HEScGRO™ medium assists in maintaining the pluripotent characteristics of hESCs propagated in the transwell insert- based culture system. These combined results represent a significant development in properly segregating stem cells from their feeders, thus eliminating cell mixing, contamination, and providing the cells with a superior environment for nourishment and controlled self-renewal. Overall, this development in hESC propagation could have wide-reaching applications for self-renewal and differentiation studies within the field of stem cell biology.

Les cellules souches ont attiré l’attention du public ces dernières années, grâce non-seulement à leur utilisation comme thérapies visant à s’attaquer à certains types de cancers, mais aussi en relation avec leur potentiel dans le domaine de la médecine regénérative. Il est établi que le destin cellulaire des cellules souches embryonnaires (ESC) est régulé de façon intensive par un groupe de facteur clés agissant sur leur pluripotence. Il est néanmoins envisageable que certains déterminants influençant l’auto-renouvellement et la différenciation de ces cellules soient toujours inconnus. Afin de tester cette hypothèse, nous avons généré, en utilisant une méthode par infections virales, une collection de ESC contenant des délétions chromosomales chevauchantes que nous avons baptisée DelES (Deletion in ES cells). Cette librairie contient plus de 1000 clones indépendants dont les régions délétées couvrent environ 25% du génome murin. À l’aide de cette ressource, nous avons conduit un criblage de formation de corps embryoïdes (EB), démontrant que plusieurs clones délétés avaient un phénotype de différenciation anormal. Nos études de complémentation sur un groupe de clones ont par la suite permis l’identification de Rps14 - un gène codant pour une protéine ribosomale (RP) comme étant haploinsuffisant pour la formation de EB. Dans un deuxième temps, l’analyse approfondie des résultats de notre crible a permis d’identifier un groupe de gènes codants pour des RP qui semblent essentiels pour la différenciation des ESC, mais dispensables pour leur auto-renouvellement. De manière intéressante, les phénotypes anormaux de formation en EB les plus marqués sont associés à des délétions de RP qui se retrouvent au site de sortie des ARN messagers (ARNm) du ribosome, soit Rps5, Rps14 et Rps28. Étonnament, alors qu’un débalancement des RP conduit généralement à une réponse de type p53, l’haploinsuffisance de ces trois gènes ne peut être renversée par une simple réduction des niveaux d’expression de ce gène suppresseur de tumeurs. Finalement, nos études de profilage polysomal et de séquençage à haut-débit montrent une signature spécifique de gènes liés au mésoderme chez un clone hétérozygote pour Rps5, suggérant ainsi une explication au phénotype de différenciation p53-indépendant identifié chez ces ESC. Nos travaux rapportent donc la création d’une ressource intéressante de génomique fonctionnelle qui a permis de mettre à jour le rôle essentiel que jouent les RP dans le processus de formation de EB. Nos résultats permettent aussi de documenter une réponse p53-indépendante suite à un débalancement de RP dans un contexte opposant l’auto-renouvellement et la différenciation des ESC. / Stem cells have captured public’s attention in the last years, thanks to their involvement in cancer therapies and also their huge theoretical potential in the regenerative medicine field. In order to translate this new technology to the clinic, a better understanding of their regulatory mechanisms is still needed. It is well established that mouse embryonic stem cell (ESC) fate is highly regulated by core pluripotency factors. However, it is conceivable that novel self-renewal or differentiation regulators are not yet described. To investigate this possibility, we used a viral-based approach to generate a collection of ESC with nested chromosomal deletions called DelES (Deletion in ES cells). This library contains more than a thousand independent ESC clones highly enriched in chromosomal deletions which together cover ~25% of the mouse genome. Using this resource, we conducted an embryoid body (EB) differentiation screen and showed that several clones were having an abnormal EB formation phenotype. Complementation studies later identified Rps14-a ribosomal protein (RP) coding gene- as a novel haploinsufficient gene in EB formation from undifferentiated ESC. Further analyses of our screen results showed a strong bias for a subset of small subunit ribosomal protein genes which are critical for ESC differentiation but not for their self-renewal activity. Interestingly, the most severe differentiation phenotypes were found with ribosomal proteins associated to the ribosome’s mRNA exit site, namely Rps5, Rps14 and Rps28. While RP gene imbalance often leads to a p53 response that can be corrected by p53 suppression, ESC clones with decreased expression of mRNA exit site RP genes were surprisingly insensitive to p53 reduction, but were rescued by BAC or cDNA complementation, thus confirming the causative nature of these genes in the ESC phenotype. Finally, polysomal profiling and RNA-Seq studies showed that Rps5 deleted ESC exhibit an abnormal mesodermal gene signature. Together, our work presents a highly valuable resource for functional genomic studies in ESC and also highlights a novel p53-independent role linked to RP gene imbalance. Our results shed light on the relevance of these subunits for the developmental transition of ESC from a pluripotent to a differentiated state.

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