Mesenchymal stem cells (MSCs) are promising candidates for treating diverse inflammatory disorders due to their capacity to be immunosuppressive. This phenotype is not present at baseline but develops in response to instructive cues. To date, clinical trials use cells grown in basic culture conditions, anticipating the cells will acquire a useful phenotype in response to in vivo cues. This strategy has failed to produce any FDA approved therapies, based on inconsistent efficacy. This thesis explores whether priming MSCs prior to administration can lead to a more uniformly therapeutic phenotype, and it details the design of an optimal in vitro priming regimen. Because interferon gamma (IFN-γ) is known to induce an anti-inflammatory state in MSCs, hypoxia can confer survival benefits, and both cues coexist in known situations of immune tolerance, we hypothesized dual IFN-γ/hypoxia priming would yield a superior immunosuppressive MSC therapy. We show that priming MSCs with hypoxia or IFN-γ alone improves their ability to inhibit T-cells in vitro, but combining these cues results in additive improvements. We next characterize the proteomic and metabolomic changes MSCs undergo when exposed to single or dual IFN-γ/hypoxia priming. While IFN-γ induces MSCs to suppress inflammation and fibrosis, hypoxia leads to cell adaptations to low oxygen, including upregulation of proteins involved in anaerobic metabolism, autophagy, angiogenesis, and cell migration. Dual priming results in additive effects, with many instances of synergy. Finally, we show initial evidence that dual primed MSCs are better able to inhibit disease progression in a mouse model of acute graft-vs-host disease (GvHD).

Hematopoietic stem cells facilitate lifelong production of a diverse repertoire of functional mature blood cells. They are a critical biological reservoir that enable organisms to endure physiological challenges such as inflammation, disease, and age. The functional maintenance of hematopoietic stem cells depends not only on intrinsic cell pathways, but also on extrinsic cues that guide core behaviors like homing and self-renewal. Careful study of these extrinsic regulatory networks can deepen our appreciation of fundamental stem cell biology and motivate therapeutic approaches to treat hematologic disease. Here I show how derangement of the bone marrow regulatory environment perturbs normal hematopoiesis, and demonstrate the dependence of hematopoietic stem cells on a circulating endocrine factor.

Cellular quiescence, a reversible state of cell-cycle exit, and developmental potential, the ability to differentiate into appropriate cell types, are properties essential for normal development and stem cell function (reviewed in (Cheung and Rando, 2013; Fiore et al., 2018; Mihaylova et al., 2014). Understanding the mechanisms by which cells maintain quiescence has important implications for developmental biology, as this reversible state of cell-cycle exit is a key attribute of stem cells, as well as for cancer biology, as quiescence plays a key role in tumor dormancy and metastasis. Environmental conditions are key in regulating whether stem cells maintain quiescence or exit to resume divisions and developmentally progress. I aim to investigate how the properties of quiescence and developmental potential are retained over long periods of time and how they are appropriately regulated by external environmental inputs. The nematode Caenorhabditis elegans is an excellent model for investigating both of these questions because it is capable of entering and maintaining a developmentally arrested state for an unusually long time compared to the normal lifetime of the worm, and because the decision to enter this arrest is regulated entirely by external environmental inputs (Cassada and Russell, 1975). Upon encountering conditions unfavorable for growth, C. elegans enters an alternative, developmentally arrested state called dauer diapause in which precursor cells remain quiescent for months – a period many times the lifespan of a worm grown under favorable conditions (Cassada and Russell, 1975). Maintaining precursors in this arrested state is important in order for the worms to develop normally once conditions improve and requires components of the conserved Insulin/Insulin-like (IIS) signaling pathway (Karp and Greenwald 2013 and this work); of note, the IIS pathway also regulates mammalian quiescence (Eijkelenboom and Burgering, 2013). Canonical regulation of dauer diapause includes IIS, TGFß, and dafachronic acid (DA)/nuclear hormone receptor (NHR) signaling (reviewed in (Murphy and Hu, 2013a)). Here, I investigate how DAF-18, the sole C. elegans ortholog of the tumor suppressor PTEN (Phosphatase and tensin homolog) (Gil et al., 1999; Mihaylova et al., 1999; Ogg and Ruvkun, 1998; Rouault et al., 1999), maintains quiescence in dauer through regulation of these conserved signaling pathways using the C. elegans gonad as a model. The gonad is composed of somatic cells and the germline. Both the somatic gonad and germline develop post-embryonically from precursor cells present when dauer arrest occurs, and these precursor cells remain quiescent for the duration of dauer diapause (Cassada and Russell, 1975; Hong et al., 1998; Narbonne and Roy, 2006). After exit from dauer, division and differentiation resume. DAF-18/PTEN is required for germline quiescence during dauer diapause (Narbonne and Roy, 2006), and my results implicate DAF-18/PTEN in the control of quiescence of the somatic tissues as well, including the somatic gonad. In this role, DAF-18/PTEN activity in the somatic gonad non-autonomously coordinates both germline stem cell (GSC) and somatic gonad blast (SGB) quiescence. I have demonstrated this somatic gonad focus through mosaic analysis, tissue-specific rescue, and tissue-specific excision mosaics. We propose that DAF-18/PTEN mediates production of a signal promoting quiescence from the somatic gonad to the SGBs and GSCs and that this signal does not absolutely require or solely target the IIS, TGFß, or DA/NHR signaling pathways normally implicated in regulation of dauer diapause.

La crête neurale (CN) dérive de la partie dorsale du tube neural des Vertébrés. Pendant le développement, ces cellules migrent et contribuent à la formation de différents tissus et organes. Le long de l'axe antéro-postérieur, la CN donne naissance aux neurones et cellules gliales du système nerveux périphérique, et aux mélanocytes. Par ailleurs, la CN céphalique est aussi à l’origine de tissus mésenchymateux qui constituent tous les os et cartilages de la face, la plus grande partie du crâne, le derme facial, et les adipocytes et cellules de muscles lisses dans la tête. Dans le tronc des Vertébrés amniotes, ces tissus dérivent du mésoderme. Chez les Vertébrés inférieurs, la CN troncale génère cependant des tissus mésenchymateux, comme les rayons des nageoires du poisson-zèbre. La question qui se pose est de savoir si la capacité de la CN à produire des cellules mésenchymateuses a été totalement perdue dans le tronc au cours de l’évolution, ou bien si elle peut encore se manifester chez les Amniotes dans des conditions spécifiques. Ce travail s’est intéressé à dévoiler le potentiel mésenchymateux de la CN troncale.Notre approche expérimentale a été d'examiner le potentiel de différenciation squelettogénique et adipogénique des cellules de la CN troncale de caille en culture in vitro, par hybridation in situ (HIS), immunocytochimie et RT-PCR. L’ostéogenèse a été initialement caractérisée par l'expression de Runx2, premier facteur de transcription des ostéoprogéniteurs, qui a été détectée par HIS à partir 5 jours de culture. Plus tard, nous avons observé la maturation des ostéoblastes, avec l’expression de la protéine collagen1, des ARNm de l'ostéopontine et de la phosphatase alcaline, jusqu’à l'étape de minéralisation de la matrice osseuse. Les cellules de CN troncale ont effectué également un processus de chondrogenèse, mis en évidence par l'expression des ARNm de Sox9, aggrecan et collagène10, et par la coloration au bleu Alcian. L'observation des zones minéralisées et des régions chondrogéniques suggère que les cellules de la CN troncale in vitro effectuent une ossification de types endochondral et intramembranaire. Dans les mêmes conditions de culture, les cellules se sont aussi différenciées en adipocytes, identifiés à partir de 10 jours de culture par le colorant Oil Red O. Les ARNm des facteurs de transcription CEBP et PPAR, essentiels pour l'adipogenèse, et de la protéine FABP4, ont été détectés par RT-PCR dès 3 jours de culture. Afin de caractériser les précurseurs des cellules osseuses et adipocytaires, nous avons examiné le potentiel de différenciation des cellules individuelles de la CN troncale. L'analyse des types cellulaires dans les cultures clonales a montré que 76% des clones contiennent des ostéoblastes Runx2-positifs. De plus, les cellules de CN troncale comprennent des progéniteurs multipotents dotés à la fois de potentiels neuraux et ostéogénique. Dans une autre condition de culture clonale, les adipocytes ont été trouvés dans la descendance de 35,3% des cellules, et environ la moitié de ces cellules possédaient aussi un potentiel glial et/ou mélanogénique. Ces résultats montrent que, in vitro, les cellules de la CN troncale sont capables de se différencier non seulement dans ses dérivés traditionnels trouvés in vivo (mélanocytes, neurones et cellules gliales), mais aussi dans des phénotypes mésenchymateux, y compris adipocytes et ostéoblastes. Comme dans les cellules de la CN céphalique, ces phénotypes mésenchymateux se différencient à partir de progéniteurs multipotents. Ceci suggère que, au cours de l’évolution, les cellules souches de la CN, dotées de potentiels à la fois mésenchymateux et neuraux, ont eu l'expression de leur potentiel mésenchymateux inhibée dans le tronc. Ainsi, chez les Vertébrés amniotes, même s’il ne se manifeste pas et reste dormant in vivo, un potentiel de différenciation mésenchymateuse est bien présent dans les cellules de la CN troncale et peut être révélé in vitro. / The neural crest (NC) derives from the dorsal borders of the vertebrate neural tube. During development, the NC cells migrate and contribute to the formation of different tissues and organs. Along the anteroposterior axis, the NC gives rise to neurons and glia of the peripheral nervous system and to melanocytes. Furthermore, the cephalic NC yields mesenchymal tissues, which form all facial cartilages and bones, the large part of skull, facial dermis, fat cells and smooth muscle cells in the head. In the trunk of amniotes Vertebrates, these tissues are derived from the mesoderm, not from the NC. In lower Vertebrates, however, the trunk NC generates some mesenchymal tissues, such as in the dorsal fins of zebrafish. The question therefore is raised whether the ability of the NC to produce mesenchymal cells was totally lost in the trunk of amniote Vertebrates during evolution, or if it can still be achieved under specific conditions. This work is interested in uncovering the mesenchymal potential of the avian trunk NC, with special interest in the differentiation into osteoblasts and adipocytes.Our experimental approach was to examine the skeletogenic and adipogenic differentiation potentials of quail trunk NC cells after in vitro culture. Cell differentiation was evidenced by the analysis of lineage-specific genes and markers using in situ hybridization (ISH), immunocytochemistry and RT-PCR. The established culture conditions allowed observation of both skeletogenesis and adipogenesis. Osteogenesis was initially characterized by expression of Runx2, the first transcription factor specific of the osteoprogenitors, which was detected by ISH from 5 days of culture. Later, we observed osteoblast maturation, with the expression of collagen1 protein, osteopontin mRNA and alkaline phosphatase mRNA, until the bone matrix mineralization stage. The trunk NC cells also underwent chondrogenesis, as demonstrated by Sox9, aggrecan and collagen10 mRNA expression, and Alcian blue staining. The observation of the mineralized areas and chondrogenesis suggested that the trunk NC cells in vitro are able to perform endochondral and membranous ossifications. In same culture conditions, the cells differentiated also into adipocytes, identified from 10 days of culture by Oil Red O staining. The mRNAs of the CEBP, PPAR and FABP4 adipogenic markers were detected by RT-PCR from 3 days of culture. For the characterization of bone and adipocyte progenitors, we evaluated the differentiation potential of individual trunk NC cells. The phenotypic analysis of these clonal cultures showed that 76% of the cells generated Runx2-positive osteoblasts. Moreover, most of the clone-forming trunk NC cells were multipotent progenitors endowed with both neural and osteogenic potentials. Furthermore, in another clonal culture condition, adipocytes were found in 35.3% of the clones, and approximately half of them also contained glial and/or melanogenic cells.These results show that the trunk NC cells in vitro are able to differentiate not only in their classical derivatives found in vivo (melanocytes, neurons and glial cells), but also in mesenchymal phenotypes, including adipocytes and osteoblasts. Importantly, as in cephalic NC cells, mesenchymal phenotypes differentiated from multipotent progenitor cells, suggesting that, during evolution, the NC stem cells intended for both mesenchymal and neural fates, had the expression of their mesenchymal potential inhibited in the trunk. Thus, although at the dormant state and not expressed in vivo, a significant mesenchymal potential is present in the trunk NC cells of amniotes Vertebrates and can be disclosed in vitro

No description available.

Gene expression is regulated at various levels, such as transcription, RNA transport and translation. Additionally, it has been shown that chromatin structure, location and dynamics also have an important role in gene expression control. While active gene regions are strongly associated with an open chromatin structure at the surface of the chromosome territory (CT) and a location in the nuclear interior, inactive gene regions seem to be related with a closed structure within the CT and a position at the nuclear periphery. However, it is still unclear how these features are regulated. Importantly, malfunction of gene regulation can impact on health and longevity. Therefore, the aim of this project was to investigate the correlation of gene expression and chromatin organisation both in single gene loci and the MHC gene cluster. The MHC locus has the highest gene density in mammalian cells and contains genes that can be reprogrammed by pro-inflammatory cytokines. The original goal of this project was to label the MHC locus by the Lac operator/repressor (LacO/LacI) approach in order to study chromatin dynamics in living cells using labelled CTs as reference for genome mobility. The thymidine analogue EdU, that can be used to label CTs, was analysed for its effects on cell cycle progression and survival, and revealed to have a strong negative impact on the cells' well-being. In the end, the LacO/LacI-recognition system for live-cell imaging did not succeed, thus FISH analyses were carried out to study chromatin dynamics in snap-shots. The location and structure of the hybridised gene regions were analysed in response to gene activation and inactivation during ESC differentiation to neuroepithelial progenitors (NPs). Single-gene focused experiments were performed using the cell line specific genes, Oct4 and Sox1, together with Gapdh as a housekeeping gene. Even though, the results showed less changes between the days of differentiation on the Gapdh locus, the gene expression profiles for the cell line specific genes did not match with the hypothesised chromatin organisation (see above). However, investigations on the gene-dense MHC locus showed structural chromatin changes that correlated with the activation of genes in this region. Interestingly, ESC treated with TNFalpha were unable to activate NF-kappaB signalling, probably due to the lack of a functional IKK complex. In summary, this project was focussing on the regulation of gene expression by the chromatin architecture and revealed complex chromatin dynamics that are likely to be affected by the sum of genes in a genome region, rather than a single gene.

Human umbilical cord perivascular cells (HUCPVCs) derived from regions surrounding the umbilical cord vessels represent an attractive cell source for cellular therapies, given their proliferative potential and the accessibility of donor material compared with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). However, these cells remain poorly characterised. Using flow cytometry, HUCPVCs were shown to express conventional MSC markers CD29, CD44, CD73, CD90, CD105, CD146, CD166 and integrins alpha1 to -5, alphaV, alphaVβ3, alphaVβ5, β1 and β3, but not CD14, CD34, CD45, STRO-1 or integrin alphaVβ6. HUCPVC marker profiles were consistent between three donors and at different passage numbers. Immunostaining for smooth muscle cell (SMC) markers; alpha-SMA, SM22alpha and smoothelin revealed that HUCPVCs shared expression of these markers with SMCs. However, in comparison with SMCs, HUCPVCs deposited more extensive fibronectin-rich matrices. When compared with hBM-MSCs, HUCPVCs differentiated along adipogenic and osteogenic lineages more slowly and did not progress to terminal phenotypes. mRNA expression of recently identified mesenchymal progenitor cell markers, ROR2, EPHA2, PLXNA2, CDH13 and CD9, was confirmed in HUCPVCs from two donors. In addition, all these markers (except EPHA2) were detected in the umbilical cord vessel wall cells of three donors, confirming their expression in both cultured HUCPVCs and cells of the primary tissue. To determine the roles of these markers in HUCPVCs, they were depleted individually using siRNA. Knockdown (KD) efficiencies of 90-97% were achieved. CD9 KD cells appeared elongated compared to cells treated with control siRNA, and these cells along with ROR2, EPHA2 and PLXNA2 KD cells exhibited larger cell areas than controls. All KD cells also showed decreased proliferative potential by day 6 compared with control siRNA or lipofectamine treated cells. A decrease in total β1 integrins was detected in the CD9 KD cells. Up-regulation of ROR2 and PLXNA2 mRNA expression was detected in HUCPVCs from two donors, when they underwent osteogenic differentiation. ROR2 and PLXNA2 knockdown resulted in increases in PLXNA2 and ROR2 mRNAs respectively, when cells were cultured in osteogenic medium compared with basal conditions. In addition, each individual knockdown revealed that the KD cells showed trends in increasing RUNX2 mRNA expression after 13-16 days in osteogenic medium. These data suggest that ROR2 and PLXNA2 may co-operate in promoting an osteogenic phenotype. Culturing HUCPVCs on non-mineralised BVSMC-derived matrices had very little impact on their differentiation status. In contrast, when HUCPVCs were cultured on mineralised BVSMC-derived matrices in osteogenic medium, their ability to further deposit mineralised matrix was enhanced by 7 days; no accompanying changes in RUNX2, ROR2 or PLXNA2 mRNA expression were detected. Taken together, early up-regulation of RUNX2, ROR2 and PLXNA2 appears to be important in driving osteogenic differentiation in HUPCVCs, whilst subsequent down-regulation of these markers may be required for mineralisation to occur. HUCPVCs express ROR2, PLXNA2, CDH13 and CD9 in vitro and in situ; these markers have distinct roles in regulating cell proliferation, shape and differentiation which may be regulated via changes in β1 integrins. It is not known why HUCPVCs might differentiate along adipogenic and osteogenic lineages more incompletely than hBM-MSCs. Further comparative characterisation of HUCPVCs and hBM-MSCs is a prerequisite for exploiting their vast clinical potential.

"February 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 171-209). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Pluripotency is defined as the capacity to differentiate into cells from each of the three primary germ layers, the ectoderm, mesoderm and endoderm. This is a property of cells located in the inner cell mass (ICM) of preimplantation blastocysts and in the epiblast layer of postimplantation, presomite embryos. Preimplantation and postimplantation pluripotency can be captured indefinitely in cultured embryonic stem (ES) cells and epiblast stem cells (EpiSCs) respectively. Preimplantation pluripotency in ES cells is regulated by a network of genes centred on three transcription factors (TFs) Oct4, Sox2 and Nanog. Oct4 and Sox2 form a mutually-reinforcing circuit and cooperatively stimulate transcription of downstream genes, including Nanog. All three TFs are expressed in EpiSCs and in the postimplantation epiblast. Functional studies established a role for Oct4 and Nanog in the specification of ICM cell identity, and a role for Oct4 in the maintenance of postimplantation pluripotency. Although the role of Sox2 in preimplantation ICM cells is unclear, it is critical for the establishment of egg cylinder following implantation and indispensable for ES cell pluripotency. However, despite the presence of Sox2 in postimplantation pluripotent cells the role of Sox2 in postimplantation pluripotency is unknown. In this thesis the role of Sox2 in the regulation of postimplantation pluripotency was examined. In contrast to the situation in the preimplantation ICM, Sox2 and Nanog are expressed in opposing gradients in the gastrulation-stage postimplantation epiblast, with Sox2 highest anteriorly and Nanog highest posteriorly. Interestingly the posterior epiblast of neural-plate (NP)-staged embryos was shown not to be pluripotent. Furthermore, forced expression of Sox2 but not Oct4 in this region rescued pluripotency. The ability of Oct4 to reinstate pluripotency in the somitogenesis-stage embryo is limited to Sox2-positive tissues. This strongly suggests that coexpression of Sox2 and Oct4 is important for establishing postimplantation pluripotent identity. Sox2HIGH cultured EpiSCs were not positively correlated with NanogHIGH cells. This reciprocal relationship emerged during the transition from ES cells to EpiSCs in culture. Using mutant cells with reduced levels of Sox2 or Nanog, Sox2 positively influences Nanog but Nanog negatively influences Sox2 expression post-transcriptionally. The negative influence of Nanog on Sox2 protein level was confirmed using doxycycline-inducible Nanog overexpressing EpiSCs. This negative relationship indicates that the regulation of Sox2 expression is different in postimplantation pluripotency and that Nanog may negatively regulate Sox2 on the protein level in the posterior epiblast. Sox2 is expressed at a lower level in EpiSCs than ES cells and the significance of this was further investigated by microarray transcription profiling using cells in which a fluorescent reporter (tdTomato) was knocked in to the Sox2 gene. Sox2- tdTomatoHIGH cells cultured in LIF/FCS/GMEMβ correlate with an undifferentiated cell identity and Sox2-tdTomatoLOW cells are associated with non-neural differentiation. Interestingly the global profile of ES cells and EpiSCs that share similar Sox2-tdTomato signal are non-identical. This suggests that Sox2 has different roles in different pluripotent states. ES cells with enforced Sox2 expression were unable to enter the EpiSC state, while ES cells with lowered Sox2 levels were inefficient in neural differentiation. Therefore, levels of Sox2 are critical for cell fate decisions. Strikingly, given the apparent requirement for Sox2 during Oct4-induced reinstatement of post-implantation pluripotency, deletion of Sox2 had no effect on the maintenance of EpiSC pluripotency. This is likely due to the presence of redundant Sox factors and indeed Sox3 is able to rescue the Sox2-null phenotype in ES cells. Taken together, these results suggest the hypothesis that postimplantation pluripotency is maintained by multiple Sox factors, while Nanog negatively regulates Sox2 post-transcriptionally to repress neural specification in the posterior epbilast. The positive influence of Sox2 on Nanog protein level suggests a possible negative feedback loop to balance the proneural and pluripotent properties of Sox2 in postimplantation pluripotency.

Embryonic stem (ES) cells are maintained in an undifferentiated state by a gene regulatory network centred on the triumvirate of transcription factors Nanog, Oct4 and Sox2. Genome-wide chromatin immunoprecipitation studies indicate that in many cases target genes contain closely localised binding sites for each of these proteins, as well as additional members of the extended pluripotency transcription factor network. However, the biochemical basis of the interactions between these proteins is largely unknown, as are the mechanisms by which these interactions control ES cell identity. By purifying Nanog from ES cells and identifying co-purified proteins, we determined a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members and RNA processing factors. Validation of interactions was obtained by co-immunoprecipitation of Nanog with putative partners. Sox2 was identified as a robust interacting partner of Nanog and the interaction was investigated further. We show that the interaction is independent of DNA binding and that a region of Nanog known as tryptophan repeat, in which tryptophan is present every 5th residue is necessary and sufficient for the binding of Sox2. Furthermore, mutation of tryptophan residues within the Nanog tryptophan repeat (WR) abolishes the interaction with Sox2. A region of Sox2 known as serine rich region, a triple-repeat motif (S X T/S Y) within a stretch of 21 residues is required for the interaction with Nanog. Mutation of tyrosines to alanine within the three motifs (S X T/S Y) abrogates the Nanog–Sox2 interaction. The disruption of the Nanog-Sox2 interaction results in the alteration of expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines of the motif with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal. Together these data shed light on the extent of the interactions of Nanog with protein partners as well as the biochemical nature of the interaction between Nanog and one of the most important partners Sox2, an interaction crucial for maintaining optimal mouse ES cell self-renewal efficiency.