Human umbilical cord blood, an alternate source of haematopoietic stem cells (HSC), has been successfully used to reconstitute haematopoiesis in both related and unrelated transplant recipients. However, because CB has fewer total cells (and as a consequence fewer HSC and progenitor cells) CB transplant recipients often experience delayed engraftment as compared with that seen in bone marrow or mobilized peripheral blood transplant recipients. Delayed engraftment exposes patients to an increased risk of infection and bleeding. Cytokine-mediated expansion has been investigated to improve engraftment after CB HSC transplantation as a means to expand the total cell number and both the HSC and progenitors populations. However, its effect on HSC function remains controversial. We hypothesise that if cytokine-mediated expansion promotes divisional recruitment and multilineage differentiation it causes changes in phenotype and cell cycle related gene expression which may be detrimental to the engraftment capacity of haematopoietic cells. Therefore we investigated the relationship between cell division, phenotype and engraftment potential of CB CD34+ cells following cytokine-mediated expansion. High resolution cell division tracking using the fluorescent dye CFSE was used to monitor changes as a consequence of cytokine-mediated expansion in phenotype and function in CB CD34+ cells. Cytokine-mediated expansion caused upregulation of lineage and proliferation markers and adhesion molecules and downregulation of putative stem cell markers with concomitant cell division. However, these changes in phenotype as a consequence of cytokine-mediated expansion may not reflect or be predictive of a functional change in the expanded population. Cytokine-mediated expansion of CB CD34+ also caused changes in cell cycle related gene expression of G1 phase regulators. CB CD34+ cells exhibited expression of all D cyclins, albeit at different levels and p21WAF1 was differentially expressed across CB samples. The effect of cell division on the engraftment potential as a consequence of cytokine-mediated expansion was examined in CB CD34+. Cytokine-mediated expansion of CB CD34+ cells reduced, but did not completely eliminate engraftment potential, as a proportion of the expanded and divided cell populations retained their ability to engraft the NOD-SCID mouse. Overall, this study confirms reports in the literature that cytokine-mediated expansion induces changes in the phenotype of HSC and compromises their in vivo function.

Umbilical cord blood (UCB) is a source of hematopoietic stem and progenitor cells (HSPC) used for allogeneic transplantation. Ex vivo expansion of HSPC can improve the slow platelet and neutrophil engraftment associated with UCB transplants. HSPCs reside in niches, some of which are near the endosteal bone surface, where they can associate with immature osteoblasts. Interestingly, osteoblasts can enhance the growth of HSPC in culture and their platelet engraftment activity. Using a proteomics approach, I identified 47 differentially expressed proteins between mesenchymal stem cells and immature osteoblasts. Several of these were previously implicated in HSPC maintenance such as IGF2, IGFBP2, DCN, GAS6 and VCAM1. Moreover, several other proteins belong to the alternative and classical complement pathways. Finally, I discovered that microvesicles found in osteoblast conditioned medium may also modulate the growth of HSPC, at least in ex vivo cultures.

Osteoblasts (OST) provide strong intrinsic growth modulatory activities on hematopoietic stem and progenitor cells via different mechanisms that include secretion of growth factors, and cellular interaction. Previously we showed that medium conditioned by mesenchymal stromal cell (MSC)-derived osteoblasts (M-OST) improve the expansion of cord blood (CB) CD34+ cells. I hypothesize that the hematopoietic supporting activity of M-OST would vary as a function of their maturation. This was tested by producing osteoblast conditioned media (OCM) from M-OST at distinct stages of maturation, and testing their growth regulatory activities in CB CD34+ cell cultures. My results showed that some of the growth promoting activity of OCM on CB cells are not dependent on the maturation status, while others are and those are largely independent of Notch signalling. In conclusion, these results provide further evidence that osteoblasts release factors that can promote the growth of immature CB progenitors in a Notch-independent way.

L'émergence des thérapies ciblées dans le traitement des cancers a rendu indispensable la mise au point de marqueurs plus spécifiques et sensibles pour la surveillance des patients. Dès le stade invasif, des cellules tumorales peuvent passer dans le sang où elles constituent les Cellules Tumorales Circulantes (CTC). Les CTC sont accessibles par une simple prise de sang, évitant les biopsies invasives. De plus, elles représentent le seul matériel tumoral résiduel après traitement. C'est la raison pour laquelle les CTC constituent un axe de recherche très actif avec plus de 400 essais cliniques incluant ces cellules comme biomarqueurs. Ces essais apportent des renseignements importants sur le risque de récidive ou de progression métastatique, et ont pour objectif de pouvoir gérer en temps réel la conduite thérapeutique. Cependant, les CTC potentiellement métastatiques ne représentent qu'une fraction très minoritaire de ces cellules circulantes. Les technologies existantes, essentiellement basées sur une simple numération, ne suffisent pas pour guider efficacement la stratégie thérapeutique. Ce projet a évalué un ensemble de critères pouvant être utile pour la prise de décisions thérapeutiques pertinentes, adaptées à chaque patient, et la mesure de l'efficacité des traitements. Ce projet sera centre sur le mélanome. Les stades d'évolution de ce cancer sont bien définis, et dans les stades avances, le risque de développer des métastases est très élevé et la détection précoce de celles-ci est un enjeu important. Par ailleurs, ce cancer bénéficie de rapides progrès thérapeutiques, les CTC constituent donc un outil intéressant pour tester l'efficacité de ces nouveaux traitements. / The emergence of targeted therapies in cancer treatment has made essential the development of more specific and sensitive markers for monitoring patients. At the invasive stage, tumor cells can pass to blood. These cells are called Circulating Tumor Cells (CTC). CTCs are accessible through a simple blood test, avoiding invasive biopsies. Moreover, they represent the only residual tumor after treatment. It is why CTCs are a very active center of research with more than 400 clinical trials involving these cells as biomarkers. These tests provide important information on the risk of recurrence or metastatic progression and aim to manage in real time the therapeutic conduct. But the CTC potentially metastatic represents only a fraction very minority of these circulating cells. Existing technologies, mainly based on simple enumeration, are not enough to effectively guide therapeutic strategy. This project has evaluated a set of criteria to make appropriate therapeutic decisions, adapted to each patient, and able to measure the effectiveness of treatments. This project will focus on melanoma. Evolution stages of this cancer are well defined, and in advanced stages, the risk of developing metastases is very high and the early detection is an important issue. Moreover, CTC could be is an interesting tool to test the effectiveness of these new treatments.

Ex vivo expansion of T lymphocytes is a central process in the generation of cellular therapies targeted at tumors and other disease-relevant structures,which currently cannot be reached by established pharmaceuticals. The influence of culture conditions on T cell functions is, however, incompletely understood. In clinical applications of ex vivo expanded T cells, so far, a relatively classical standard cell culture methodology has been established. The expanded cells have been characterized in both preclinical models and clinical studies mainly using a therapeutic endpoint, for example antitumor response and cytotoxic function against cellular targets, whereas the influence of manipulations of T cells ex vivo including transduction and culture expansion has been studied to a much lesser detail, or in many contexts remains unknown. This includes the circulation behavior of expanded T cells after intravenous application, their intracellular metabolism and signal transduction, and their cytoskeletal (re)organization or their adhesion, migration, and subsequent intra-tissue differentiation. This review aims to provide an overview of established T cell expansion methodologies and address unanswered questions relating in vivo interaction of ex vivo expanded T cells for cellular therapy.

In dieser Arbeit wurden Methoden entwickelt und verglichen, um aus Corneoskleralringen langzeitorgankonservierter Hornhäute und intakten, kryokonservierten Amnionmembranen Limbusepithel-Amnion-Transplantate herzustellen. Als erfolgreichste Kultivierungsmethode stellte sich hierbei signifikant die Explantat-Technik mit nach unten gerichtetem Limbusepithel heraus. Hier konnte eine Auswachsrate von 42 % erzielt werden. Es wurde weiterhin gezeigt, dass das ausgewachsene, mehrschichtige Limbusepithel proliferationsfähige TACs (Transient Amplifying Cells) enthält. Weiterhin konnten mittels Regressionsanalyse signifikante Zusammenhänge zwischen Spenderalter, Post-mortem-Zeit, Organkultur-Dauer und der Auswachsrate beschrieben werden. Kurzgefasst wurde die Vermutung bestätigt, dass jede Verlängerung der unterschiedlichen Zeiten eine Verringerung der Auswachsrate zur Folge hat. Die hergestellten Limbusepithel-Amnion-Transplantate könnten für Patienten mit Limbusstammzellinsuffizienz unterschiedlicher Genese verwendet werden.

Les cellules souches hématopoïétiques (CSH) sont des cellules souches adultes, responsables du maintien du système sanguin tout au long de la vie des vertébrés. Les CSH sont des cellules multipotentes spécialisées qui possèdent deux propriétés principales : leur capacité à se différencier en de multiples lignées et leur capacité à créer d'autres cellules souches (c'est-à-dire l'autorenouvellement). Grâce à ces caractéristiques, les CSH ont un énorme potentiel thérapeutique. En effet, la transplantation de CSH constitue à ce jour une option de choix pour le traitement de plusieurs maladies et troubles hématologiques. Les CSH ne se retrouvent que dans certains échantillons biologiques comme la moelle osseuse, les cellules mobilisées de la moelle osseuse dans le sang périphérique ou les cellules de sang de cordon ombilical. Les applications cliniques des CSH sont souvent limitées en raison de leur faible fréquence dans les échantillons biologiques, c’est pourquoi leur expansion ex vivo est un domaine de recherche en plein essor. Des approches basées sur des petites molécules pour amplifier le nombre les cellules couches ex vivo ont été testées avec succès pour permettre la prolifération des cellules et freiner leur différentiation. Notre groupe a contribué à ce domaine en identifiant la petite molécule UM171 qui peut amplifier les CSH ex vivo par reprogrammation épigénétique. Dans le cadre des efforts d’expansion ex vivo des CSH, un obstacle majeur est la caractérisation des cellules qui ont proliféré ex vivo afin d’évaluer de façon exhaustive le potentiel des greffons pour des applications ultérieures. La caractérisation phénotypique des CSH amplifiées ex vivo est une approche prometteuse pour aider à isoler et à purifier les cellules souches. Les travaux de cette thèse explorent l'association de l'immunophénotype à la fonctionnalité des cellules souches pour nous aider à définir l'hétérogénéité des cellules amplifiées. Au chapitre 2, en utilisant un profilage de cellules amplifiées basée sur le transcriptome, nous avons pu identifier CEACAM1 comme un nouveau marqueur fonctionnel des CSH. Concomitamment, au chapitre 3, nous appliquons une approche alternative basée sur le protéome de la surface cellulaire pour aider à caractériser le phénotype des cellules souches et progénitrices hématopoïétiques (CSPH) amplifiées ex vivo afin d'identifier GPA33 en comme marqueur probable de CSH. Les marqueurs de surface compatibles avec la culture constituent un excellent outil pour un isolement prospectif rapide et des manipulations in vitro et in vivo supplémentaires pour permettre une meilleure compréhension de la biologie des cellules souches. La caractérisation des HSPC expansées ex vivo est donc une tentative de combler le fossé et de permettre des stratégies thérapeutiques améliorées. / Hematopoietic stem cells (HSCs) are responsible for maintaining the blood system throughout the lifespan of vertebrates. HSCs are specialized multipotent cells that have two main properties – their ability to differentiate into multiple lineages and their ability to create more stem cells (i.e. self-renewal). Due to these special abilities, HSCs have tremendous therapeutic potential. HSCs thus to date are the best curative measure against most hematological malignancies and disorders. HSCs occur in limited frequency and can be found only from certain conserved sources like the bone marrow or mobilized cells from the bone marrow in the peripheral blood or umbilical cord blood cells. Clinical applications of HSCs are often restricted due to their low occurring frequencies, therefore ex vivo expansion is a growing research field. Small molecule-based approaches to expand stem cells ex vivo have been successfully tested to allow for proliferation of cells by curbing their differentiation. Our group has contributed to this field by the identification of the small molecule UM171 which can expand hematopoietic stem and progenitor cells (HSPCs) ex vivo via epigenetic reprogramming. To expand HSPCs ex vivo a major hurdle is the proper characterization of the ex vivo expanded cells to evaluate the full potential of grafts for further downstream applications. Phenotypic dissociation of ex vivo expanded HSPCs is a prospective tool to help isolate and purify stem cells. Identification of culture-compatible surface markers is therefore the first step to help characterize the ex vivo expanded cells. The work in this thesis explores the association of immunophenotype to the functionality of stem cells to help us delineate the heterogeneity of expanded cells. In Chapter 2, using transcriptome-based interrogation of expanded cells, we were able to identify CEACAM1 as a novel functional marker of HSCs. Whereas, in Chapter 3 we apply an alternative cell surface proteome-based approach to help characterize the phenotype of ex vivo expanded HSPCs to identify GPA33 as a probable HSC marker. Culture-compatible surface markers make for an excellent tool for rapid prospective isolation and additional in vitro and in vivo manipulations to allow a better understanding of stem cell biology. Characterization of ex vivo expanded HSPCs is thus an attempt to help bridge the gap and allow for enhanced therapeutic strategies.

In dieser Arbeit wurden Methoden entwickelt und verglichen, um aus Corneoskleralringen langzeitorgankonservierter Hornhäute und intakten, kryokonservierten Amnionmembranen Limbusepithel-Amnion-Transplantate herzustellen. Als erfolgreichste Kultivierungsmethode stellte sich hierbei signifikant die Explantat-Technik mit nach unten gerichtetem Limbusepithel heraus. Hier konnte eine Auswachsrate von 42 % erzielt werden. Es wurde weiterhin gezeigt, dass das ausgewachsene, mehrschichtige Limbusepithel proliferationsfähige TACs (Transient Amplifying Cells) enthält. Weiterhin konnten mittels Regressionsanalyse signifikante Zusammenhänge zwischen Spenderalter, Post-mortem-Zeit, Organkultur-Dauer und der Auswachsrate beschrieben werden. Kurzgefasst wurde die Vermutung bestätigt, dass jede Verlängerung der unterschiedlichen Zeiten eine Verringerung der Auswachsrate zur Folge hat. Die hergestellten Limbusepithel-Amnion-Transplantate könnten für Patienten mit Limbusstammzellinsuffizienz unterschiedlicher Genese verwendet werden.