Hematopoietic stem cells (HSCs) transplantations are widely used for the treatment of hematological and non-hematological disorders in clinic. Successful transplantation requires sufficient number and efficient homing of HSCs. Many studies have focused on developing an effective strategy to expand functional HSC population. Some regulatory molecules have been recently shown great promise for controlling the amplification of HSCs. In these dissertation studies, I first aim to identify gene(s) and their allelic variants contributing to strain-specific difference in HSC numbers between C57BL/6 (B6, low) and DBA/2 (D2, high) mice by using a classic forward genetic approach. Firstly, 3 quantitative trait loci (QTL) on chromosome (Chr) 3,5 and 18 were mapped by linkage analyses and confirmed in congenic mice. Secondly, Chr.3 QTL affected several HSC number-related biological processes. The D2 allele increased cycling and self-renewal whereas it decreased apoptotic rates of HSCs. Both actions conspired to increase HSC population size. Lastly, a small number of differentially-expressed genes was identified in Chr.3 congenic HSCs by a microarray-based candidate gene method, and the differential expression of one candidate, latexin, was found to relate to HSC number variations. Our studies report the strong evidence for the potential functions of latexin in HSC number regulation, and they are important for understanding molecular mechanisms of stem cell regulation and developing effective stem cell expansion strategies for clinical applications. In the second part of my studies, I studied homing and engraftment capabilities of HSCs. By using functional assays for progenitor and stem cells, I first reported the absolute homing efficiencies of murine young or old donor cells into young or old recipient mice. The results indicated that homing of primitive hematopoietic cells was not efficient and significantly decreased by aging of donors and recipients. The proliferation and differentiation states of HSCs were also impaired by homing itself, as well as by donors' and recipients age. Moreover, the hematopoietic reconstitution dynamics following transplantation were also affected by aging. Together, these findings will provide useful information for clinical applications especially when older individuals increasing serve as stem cell donors for elderly patients.

It is well documented that both quantitative and qualitative changes in the murine hematopoietic stem cell (HSC) population occur with age. In mice, the effect of aging on stem cells is highly strain-specific, thus suggesting genetic regulation plays a role in HSC aging. In C57BL/6 (B6) mice, the HSC population steadily increases with age, whereas in DBA/2 (D2) mice, this population declines. Our lab has previously mapped a quantitative trait locus (QTL) to murine chromosome 2 that is associated with the variation in frequency of HSCs between aged B6 and D2 mice. In these dissertation studies, I first aim to characterize the congenic mouse model which was generated by introgressing D2 alleles in the QTL onto a B6 background. Using a surrogate assay to mimic aging, I analyzed the cell cycle, apoptotic and self-renewal capabilities of congenic and B6 HSCs and show that D2 alleles in the QTL affect the apoptotic and selfrenewal capabilities of HSCs. In the second aim of these studies, I used oligonucleotide arrays to compare the differential expression of B6 and congenic cells using a population enriched for primitive stem and progenitor cells. Extensive analysis of the expression arrays pointed to two strong candidates, the genes encoding Retinoblastoma like protein 1 (p107) and Sorting nexin 5 (Snx5). B6 alleles were associated with increased p107 and Snx5 expression in old HSCs therefore both genes were hypothesized to be positive regulators of stem cell number in aged mice. Finally, in the third aim of these studies, I show that the individual overexpression of p107 and Snx5 in congeic HSCs increases day35 cobblestone area forming cell (CAFC) numbers, therefore confirming their roles as positive regulators of HSC number in vitro. These studies uncover novel roles for p107 and Snx5 in the regulation of HSC numbers and provide additional clues in the complex regulation of HSC aging.

The adaptor protein Shb has been implicated in the signaling of several tyrosine kinase receptors and previous studies have suggested a role for Shb in the signal transduction of T cells. Shb associates with the T cell receptor (TCR) and partakes in the signal propagation of activated T lymphocytes. In order to explore Shb’s influence on TCR signaling in vivo, T cell development and function was studied in a Shb knockout mouse. The loss of Shb led to aberrant TCR signaling in both thymocytes and peripheral CD4+ TH cells, with elevated basal phosphorylation of key components in the signal cascade. Shb was found to be dispensable for thymocyte development, but its absence resulted in a TH2 bias in in vitro stimulated peripheral CD4+ TH cells. As imbalances in TH2 responses are linked to allergic diseases, we further explored Shb’s role in immune regulation in a mouse model of atopic dermatitis. Shb knockout mice exhibit more aggravated signs of atopic dermatitis, including increased immune cell recruitment to the affected areas and elevated mRNA levels of typical TH2 cytokines. The effect of Shb on hematopoiesis in general was determined by examining populations of long-term hematopoietic stem cells (LT-HSCs) and hematopoietic progenitor cells in bone marrow of Shb knockout and wild type mice. Shb deficient bone marrow was found to contain significantly fewer relative numbers of LT-HSCs due to a proliferative defect. The reduced cell cycle activity of Shb LT-HSCs could further be linked to an abnormal regulation of the focal adhesion kinase/Rac1/p21-activated kinase pathway. Since alterations in LT-HSC proliferative abilities may have implications for leukemia development, BCR-Abl induced myeloid neoplasia was investigated in the absence of Shb. Shb deficiency confers a more aggressive progression of BCR-Abl induced myeloid neoplasia characterized by an increased peripheral blood neutrophilia and a deregulated cytokine profile. In addition, focal adhesion kinase and STAT3 signaling is hyperactivated in Shb knockout leukemic cells. In conclusion, Shb appears to be a multifunctional signaling mediator that controls several responses in hematopoietic cells, under homeostatic as well as disease conditions.

Homeostasis of hematopoietic stem cells (HSC) in the mammalian bone marrow stem cell niche is regulated by signals of the local microenvironment. Besides juxtacrine, endocrine and metabolic cues, paracrine and autocrine signals are involved in controlling quiescence, proliferation and differentiation of HSC with strong implications on expansion and differentiation ex vivo as well as in vivo transplantation. Towards this aim, a cell culture analysis on a polymer microcavity carrier platform was combined with a partial least square analysis of a mechanistic model of cell proliferation. We could demonstrate the discrimination of specific autocrine and paracrine signals from soluble factors as stimulating and inhibitory effectors in hematopoietic stem and progenitor cell culture. From that we hypothesize autocrine signals to be predominantly involved in maintaining the quiescent state of HSC in single-cell niches and advocate our analysis platform as an unprecedented option for untangling convoluted signaling mechanisms in complex cell systems being it of juxtacrine, paracrine or autocrine origin.

O transplante de células-tronco hematopoéticas (CTHs) é o tipo de terapia celular mais usada atualmente. As células-tronco da medula óssea humana, do sangue periférico mobilizado e do sangue do cordão umbilical (SCU) são as únicas fontes de células utilizadas clinicamente para a recuperação hematopoética, mas elas têm uma disponibilidade limitada e apenas um terço dos pacientes apresentam um doador compatível. Uma fonte alternativa para suprir esta demanda seriam as células hematopoéticas derivadas a partir de células-tronco pluripotentes. Célulastronco embrionárias (CTE) são um tipo de células pulripotentes caracterizadas por sua capacidade ilimitada de autorrenovação e diferenciação em todas as células especializadas do indivíduo adulto. A alta capacidade de diferenciação dessas células em linhagens específicas por métodos de indução in vitro faz delas grandes promessas para o desenvolvimento de novas tecnologias aplicáveis à medicina regenerativa e terapia celular. Entretanto, ainda é necessário determinar se células-tronco hematopoéticas (CTH) geradas a partir de células pluripotentes são funcionais in vivo. Assim, o objetivo desse estudo consistiu no desenvolvimento de um modelo animal para o transplante de células hematopoéticas humanas provenientes de células pluripotentes e utilizamos CTH de sangue de cordão umbilical como controle. Para tanto, realizamos a padronização da dose de irradiação subletal nos camundongos NOD / SCID IL2R ? null (NSG) e utilizamos duas concentrações de células, 1x106 e 0,75x106 de células hematopoéticas diferenciadas a partir de CTE e células do cordão umbilical. Os animais que receberam as células do SCU apresentaram uma taxa mais elevada de enxertia em comparação aos que receberam ás células diferenciadas a partir de CTE humanas. Foi confirmado que após quinze dias do transplante, a hematopoese é restabelecida e que células CD45+ humanas estavam presentes, porém em baixas quantidades. Sobretudo, os resultados aqui apresentados enfatizaram o descrito na literatura, porém não ultrapassando a 1,5% de enxertia na medula óssea. Estes dados indicaram que os camundongos NSG proporcionaram um microambiente hematopoético favorável para as CTE humanas porém, essas células precisam ser investigadas no que tange aos fatores que aumentem de sua duração in vivo, otimizando a enxertia. / Hematopoietic stem cell transplantation (HSC) is the most widely used type of cell therapy nowadays. Human bone marrow, mobilized peripheral blood, and stem cells in the umbilical cord (UC) are the only sources of cells used clinically for hematopoietic recovery, but they have limited availability and only a third of patients have a matching donor. An alternative source to supply this demand would be hematopoietic cells derived from pluripotent stem cells. Embryonic stem cells (ESC) are a type of pulp-like cells characterized by their unlimited capacity for self-renewal and differentiation in all specialized cells of the adult individual. The high differentiation capacity of these cells in specific strains by in vitro induction methods makes great promises for the development of new technologies applicable to regenerative medicine and cell therapy. However, it remains to be determined whether hematopoietic stem cells (HTC) generated from pluripotent cells are functional in vivo. Thus, the objective of this study was to develop an animal model for the transplantation of human hematopoietic cells from pluripotent cells and to use HTC in the umbilical cord blood as a control. To do so, we performed standardization of the sublethal irradiation dose on the NOD / SCID IL2R ? null (NSG) mice and used two concentrations of cells, 1x106 and 0.75x106 hematopoietic cells differentiated from ESC and umbilical cord cells. The animals that received UC cells had a higher rate of grafting compared to those that received the differentiated cells from the human ESC. It was confirmed that after fifteen days of transplantation, hematopoiesis restored and that human CD45+ cells were present, but in low amounts. Above all, the results presented here emphasize the described in the literature, but not exceeding 1.5% of grafting in bone marrow. These data indicated that the NSG mice provided a hematopoietic microenvironment favorable to the human ESC, however, these cells need to be investigated with regard to factors that increase their duration in vivo, optimizing grafting.

Yau Fung-wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 147-183). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Publications --- p.iv / Abbreviations --- p.vii / Appendix Some cell surface antigens expressed on hematopoietic cells --- p.ix / Abstract --- p.x / Chapter Chapter One --- Introduction --- p.1 / Chapter Section A --- Sources of blood stem cells for transplantation --- p.1 / Chapter Section B --- Hematopoiesis --- p.7 / Chapter Section C --- Human CD34+ blood cells --- p.15 / Chapter Section D --- Human stem and progenitor cells in neonates --- p.19 / Chapter Section E --- Methods of CD34 detection --- p.23 / Chapter Section F --- Adhesion molecule: migratory properties of hematopoietic stem and progenitor cells --- p.33 / Chapter Section G --- Project objectives --- p.37 / Chapter Chapter Two --- Materials and Methods --- p.38 / Chapter Section A --- Quality and quantity of CD34+ cells in neonatal blood --- p.38 / Chapter Section B --- Kinetics of hematopoietic stem and progenitor cellsin human neonatal blood after birth --- p.48 / Chapter Section C --- Enumeration of long term culture initiating cells by limiting dilution assay --- p.56 / Chapter Chapter Three --- Results & Discussion --- p.61 / Chapter Section A --- Characterization of hematopoietic stem and progenitor cells in neonatal blood --- p.61 / Results --- p.61 / Discussion --- p.78 / Chapter Section B --- Kinetics of hematopoietic stem and progenitor cellsin neonatal blood --- p.88 / Results --- p.88 / Discussion --- p.119 / Chapter Section C --- Comparison of CD34+ cell enumeration by flow cytometry using two antibodies and two protocols --- p.125 / Results --- p.125 / Discussion --- p.129 / Conclusion --- p.131 / Future prospective --- p.133 / References --- p.134

L’obtention d’un nombre élevé de cellules souches hématopoïétiques (CSH) représente un enjeu majeur pour le développement de protocoles de thérapies cellulaires d’hémopathies ou de tumeurs solides. L’expansion ex vivo de ces cellules met en jeu différents acteurs (cytokiniques, environnementaux) et notamment les basses concentrations d’oxygène (O2), qui reflètent des conditions physiologiques retrouvées au sein de structures spécifiques de la moelle osseuse où résident les CSH et auxquelles notre équipe s’intéresse depuis plusieurs années. Les effets bénéfiques de ces basses concentrations d’O2 sur le maintien des CSH sont actuellement bien établis lors de courtes cultures de cellules de moelle osseuse, de sang placentaire ou mobilisées dans le sang. Nous avons cherché à confirmer et à étendre ces résultats à des cellules peu étudiées, les cellules souches de sang périphérique en situation d’homéostasie (CSSP-H). Ces cellules représentent en effet une source possible de CSH à usage thérapeutique, du fait de leur disponibilité et de leur facilité d’accès. Nos travaux ont permis d’établir et d’optimiser un protocole, rapide et simple, de purification de cellules CD34+ à partir de filtres de leucoréduction (LRF). La quantité et la pureté de ces cellules adaptées à la poursuite de nos travaux, ainsi que leur validation fonctionnelle, nous ont permis de les utiliser comme modèle pour l’étude des effets de cultures de 7 jours très faiblement oxygénées (0,1% d’O2). La détermination de combinaisons cytokiniques assurant le maintien et l’expansion des cellules primitives a révélé un rôle bénéfique de l’IL-3 et du SCF couplé à la TPO. Ces conditions de culture ont permis de révéler, comparativement à des cultures réalisées à 20% d’O2, le rôle majeur des faibles concentrations d’O2 dans le maintien de cellules quiescentes, indifférenciées, ne se divisant pas ou très peu et capables de reconstituer une hématopoïèse, suite à leur injection dans des souris NOG. Les mécanismes moléculaires et métaboliques intervenant dans ces processus restent, cependant, encore à établir. / Obtaining a high number of hematopoietic stem cells (HSCs) is a major challenge for developing cell therapies for blood diseases. Ex vivo expansion of HSCs involves various factors (cytokines, environment), including low oxygen (O2) concentrations, that reflect the physiological conditions found in specific structures of the bone marrow where HSCs reside. Our team is interested with the study of these low O2 levels for several years and their beneficial effects are currently well established during short-term cultures of cells from bone marrow, cord blood or mobilized in the blood. We sought to confirm and extend these results to poorly studied cells: stem cells from steady state peripheral blood (SSPB). Indeed, these cells represent a possible HSCs source devoted to the therapeutic use, because of their availability and their easy access. Our work has led to the establishment and the optimisation of a procedure, rapid and easy to set up, for CD34+ cells purification from leukoreduction filters (LRFs). The cell quantities and purities, adapted to our further work, together with their functional validation, led us to use these cells as a model for 7-days in vitro cultures performed at very low O2 concentration (0.1%). Cytokine combination assays, allowing the maintenance and the expansion of primitive cells, have revealed a beneficial influence of IL-3 or SCF + TPO additions. These cultures have revealed, comparatively to those performed at 20% O2, a major role of the very low O2 concentrations in the maintenance of quiescent and undifferentiated cells, showing an un- or low-cycling status and able to reconstitute hematopoiesis, consecutively to their injection into NOG mice. However, the molecular and metabolic mechanisms involved in these processes remain unknown.

In this study, we show that removal of a quorum sensing subtype of stromal macrophage expands the support capacity of ex vivo bone marrow culture. Notably, this system maintains much of the remaining paracrine signaling of the organ, unlike traditional macrophage ablation or cytokine supplemented media and does not place undue stress on the HSPC itself. Recent studies have independently identified alternatively activated macrophages that suppress hematopoiesis in in vitro culture. We have identified for the first time, a small molecule capable of preferentially killing those cells, thus providing a method to both culture unaltered HSPC ex vivo for long periods of time and significantly expand transient progenitor cells to assist transplantation efficiency. Our culture system in unique in its ability to maintain cultured HSPC in the physiological micro-environment of the bone marrow We found the small molecule “999” capable of expanding hematopoietic capacity of stroma culture by selectively eliminating an MHCII-Hi subpopulation of stromal macrophages that suppress HSPC growth. Removal of these macrophages enables long-term HSC ex vivo stability and massive expansion of the MPP and its progeny. Cultures expanded in this manner have increased engraftment potential and behave physiologically normal upon transplantation. This investigation has also helped to uncover the role of TGFB in bone marrow quiescence signaling. The MHCII-HI target cells express TGFB and through it, signal quiescence to the HSPC, likely as a form of quorum sensing. Targeted acute elimination of that signal leads to unabashed expansion of MPP. Furthermore, macrophage polarization in the tumor microenvironment has also been show to promote tumor formation and often leads to poor prognosis. Molecular tools such as 999 that have the ability to alter macrophage polarization ratios may prove to be valuable synergistic tools for oncologists in conjunction with current therapies.

Opportunistic infection is always a potentially life threatening complication in pediatric oncology patients and hematopoietic stem cell transplant recipients. With the advances in various disease treatment protocols, the overall and event-free survivals of this high risk population improve significantly. In this thesis, the author reported a number of original studies to discuss different strategies in prevention of this serious complication. Firstly, the author demonstrates that pediatric oncology patients are still vulnerable to various vaccine-preventable infectious diseases up to 18 months after stopping chemotherapy. For those vaccine-preventable infectious diseases, pediatric oncology patients can mount a significant and persistent immune response to common inactivated vaccine (namely diphtheria-tetanus-pertussis vaccine). For non-vaccine preventable infectious diseases, regular monitoring of plasma viral load and strategic use of antiviral agents as pre-emptive or prophylactic agent is an effective approach to prevent infection. In hematopoietic stem cell transplant setting, adoptive transfer of acquired immunity from donor to recipient and incorporation of this parameter in donor selection process can be considered. The findings of the studies can be applied to clinical setting. The future direction of our studies includes the immune responses of other common vaccines namely pneumococcal vaccine and pandemic influenza vaccine in high risk population. The role of transfer of donor's varicella zoster immunity in prevention of herpes zoster infection in transplant recipient can be further explored. With the advances in supportive care of our vulnerable patients, the survival rate is expected to be further improved in the future. / by Frankie Wai Tsoi, Cheng. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 193-208). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

Les cellules souches hématopoïétiques (CSH) sont à l’origine de l’ensemble des cellules hématopoïétiques, mais les mécanismes responsables de leurs réponses au stress ne sont que partiellement compris. J’ai étudié le rôle du facteur de transcription Helios dans les CSH, dans lesquelles il est fortement exprimé. J’ai trouvé que les CSH de souris nulles pour Helios (He-/-) possèdent un plus fort potentiel de reconstitution que les CSH WT dans des expériences de transplantations en série. De manière frappante, les souris âgées de 2 ans possèdent 8 fois plus de CSH fonctionnelles par rapport aux souris vieilles WT. De plus, le pool de CSH de long terme de souris He-/- vieilles ressemblent aux CSH WT jeunes en termes de phénotype et de fréquences. Les CSH He-/- vieilles présentent une dérégulation de gènes impliqués dans le vieillissement. De plus,les CSH He-/- jeunes sous-expriment des gènes codant des protéines impliquées dans la réparationde l’ADN ainsi que dans la voie p53. Quand les CSH He-/- et WT sont traitées avec différents agents radiomimétiques qui induisent des cassures simple et double brins à l’ADN, tels que la néocarzinostatine, la camptothécine ou l’étoposide, l’entrée en apoptose, en sénescence et l’arrêt de la prolifération des CSH He-/- sont altérées. Ce phénotype est accompagné d’une faible induction des gènes cibles de p53 et d’une altération du dégagement des foyers gammaH2AX. De plus, j’ai montré que Helios agit en synergie avec p53 pour réguler les réponses aux dommages à l’ADN des CSH. Mes résultats suggèrent qu’en synergie avec p53, Helios contrôle le vieillissement des CSH en prévenant l’accumulation des dommages de l’ADN des CSH. / Hematopoietic stem cells (HSCs) give rise to all blood cell lineages but the mechanisms responsible of HSCs responses to stress remain partially understood. I studied the role of the transcription factor Helios in HSCs, where Helios is highly and specifically expressed. I found that HSCs from young Helios null mice (He-/-) reconstitute the hematopoietic system of irradiated recipient mice similarly to HSCs from WT mice in primary transplantations, but out-perform WT cells in secondary and tertiary transplantations. Strikingly, HSCs from 2-year-old He-/- mice had 8-fold higher reconstitution potential than old WT HSCs in primary transplantations. Moreover, the pool of long-term HSCs in old He-/- mice resembles that of young WT animals in both phenotype and frequency. HSCs from old He-/- mice present a down regulation of genes involved in aging. Further, young He-/- HSCs express reduced mRNA levels of genes encoding DNA repair proteins as well as those associated with thep53 pathway. When He-/- and WT HSCs were subjected to DNA damage by different agents like neocarzinostatin, camptothecin, or etoposide, DNA damage-induced apoptosis, senescence and cell cycle arrest were significantly impaired in He-/- HSCs. This phenotype was accompanied by a poor induction of p53 target genes and impaired clearance of gammaH2AX foci. Furthermore, I found that Helios synergies with p53 to regulate the DNA damage responses of HSCs. My results suggest that,in synergy with p53, Helios controls HSC aging by preventing the accumulation of DNA damage in these cells.