published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

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.

Derivation of lung epithelial cells from stem cells remains a challenging task, due in part to a lack of understanding of the molecular mediators driving commitment of endoderm to an early lung lineage. Reciprocal signalling between the lung mesenchyme and epithelium is crucial for proper differentiation and branching morphogenesis to occur. We hypothesized that the combination of signalling pathways comprising early epithelial-mesenchymal interactions and the 3-D spatial environment are required for induction of embryonic and induced pluripotent stem cells (ESC and iPSC, respectively) into a lung cell phenotype with the hallmarks of the distal niche. Aggregating early lung mesenchyme with endoderm-induced ESC and iPSC resulted in differentiation to an NKX2.1 and pro-SFTPC positive lineage. The differentiating cells organized into tubular structures and became polarized epithelial cells. Ultrastructure analysis revealed precursors of lamellar bodies, and Sftpb mRNA expression was detected. Quantification of the differentiation using an Nkx2.1-reporter ESC line revealed that 80% were committed to an early lung lineage, a vast improvement over what has previously been published. The FGF growth factor family comprises well-known mediators of growth and differentiation during the development of many organs, including the lung. We found that FGF2 signalling through the FGFR2iiic receptor isoform was mediating the commitment of the stem cells to an early lung epithelial phenotype, as defined by NKX2.1/proSFTPC expression. FGF7 signalling through the FGFR2iiib receptor was found to be important for the maturation and morphogenesis of the NKX2.1/proSFTPC positive lineage, but did not play a role in the initial commitment. The addition of FGF2 to endoderm-induced ESC or iPSC in the absence of mesenchyme was able to commit the cells to an NKX2.1-positive lineage, but no proSFTPC was detected. Furthermore,the cells did not become polarized and no longer organized into tubular structures. These findings suggest that while FGF2 is important for initial commitment, additional mesenchyme components including matrix proteins, supporting cell lineages and other growth factors are crucial for an efficient differentiation to an early lung epithelial cell lineage.

The cancer stem cell hypothesis has recently re-emerged as a compelling paradigm for the development and progression of neoplastic disease. The hypothesis proposes that a specific subset of “cancer stem cells” (CSC), believed to share many features with normal stem cells, is exclusively responsible for maintaining tumour growth and driving progression. If the CSC hypothesis applies, it may require re-evaluation of the clinical approach to neoplasia. Spontaneous cancer in the domestic dog represents a significant welfare problem, with dogs developing many tumours strongly reminiscent of those affecting humans. This study sought to investigate whether cells with characteristics of CSC are identifiable in canine cancer. Assays to identify, isolate and characterise CSC were adapted to the canine system, and cancer cell lines and spontaneous tumours of diverse origin evaluated for the presence of candidate populations. Whilst analysis of surface expression patterns did not identify specific subpopulations within canine cancer cell lines, these were detectable in cells derived directly from primary tumours. Assays for stem cellassociated drug resistance mechanisms could also be used to identify subsets of putative canine CSC. Formation of “tumourspheres” by canine cancer cell lines was found to be highly density-dependent, so a potentially unreliable method of isolating CSC. Expression of the cell surface glycoprotein CD44 was associated with cellular proliferation status, although it may not represent a stable canine CSC marker. The NFκB survival pathway, associated with apoptosis resistance of some putative CSC, was constitutively active in canine cancer cell lines; suppression using specific inhibitors could reduce cell viability, indicating that this may represent a rational therapeutic target. Overall, these studies demonstrated that CSC assays may be adapted to the canine model system, although they require rigorous interrogation to distinguish apparent CSC attributes from basic biological properties. Cell lines have provided a stable background upon which to optimise assays, but appear less likely to demonstrate discrete CSC subpopulations. Putative CSC subsets may be more readily identifiable within heterogeneous primary tumour cells. The application of some of these adapted assays within a clinical setting may enable further characterisation of individual patients’ tumours, and inform therapeutic regimes for improved treatment outcomes.

Busulfan, cyclophosphamide, and etoposide (BuCyE) is a commonly used conditioning regimen for autologous stem cell transplantation (ASCT). This multicenter, phase II study examined the safety and efficacy of BuCyE with individually adjusted busulfan based on preconditioning pharmacokinetics. The study initially enrolled Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) patients ages 18 to 80 years but was amended due to high early treatment-related mortality (TRM) in patients > 65 years. BuCyE outcomes were compared with contemporaneous recipients of carmustine, etoposide, cytarabine, and melphalan (BEAM) from the Center for International Blood and Marrow Transplant Research. Two hundred seven subjects with HL (n = 66) or NHL (n = 141) were enrolled from 32 centers in North America, and 203 underwent ASCT. Day 100 TRM for all subjects (n = 203), patients > 65 years (n = 17), and patients ≤ 65 years (n = 186) were 4.5%, 23.5%, and 2.7%, respectively. The estimated rates of 2-year progression-free survival (PFS) were 33% for HL and 58%, 77%, and 43% for diffuse large B cell lymphoma (DLBCL; n = 63), mantle cell lymphoma (MCL; n = 29), and follicular lymphoma (FL; n = 23), respectively. The estimated rates of 2-year overall survival (OS) were 76% for HL and 65%, 89%, and 89% for DLBCL, MCL, and FL, respectively. In the matched analysis rates of 2-year TRM were 3.3% for BuCyE and 3.9% for BEAM, and there were no differences in outcomes for NHL. Patients with HL had lower rates of 2-year PFS with BuCyE, 33% (95% CI, 21% to 46%), than with BEAM, 59% (95% CI, 52% to 66%), with no differences in TRM or OS. BuCyE provided adequate disease control and safety in B cell NHL patients ≤ 65 years but produced worse PFS in HL patients when compared with BEAM.

Human haematopoiesis resembles a complex hierarchy, however most intermediate stages are only poorly defined. Efforts to characterise human progenitors have been inconsistent and failed to integrate previous knowledge. Furthermore, characterisation of normal progenitors has important implications in acute myeloid leukaemia (AML) biology. We previously established that leukaemic stem cells (LSCs) resemble the immunophenotypic progenitor compartments more closely than the stem cell fraction. Therefore, I set out to characterise human stem and progenitor cells (HSCPs) on phenotypic, molecular and functional level to complete the picture of human haematopoiesis. I purified HSPCs based on their immunophenotype from adult bone marrow (BM) and umbilical cord blood (CB) to investigate steady state and neonatal haematopoiesis. To define differentiation potentials, HSPCs were subjected to functional in vitro assays on bulk and clonal level. Limit dilution assays were used to determine the frequency of cells with multiple differentiation potentials. RNA sequencing revealed underlying lineage priming and specific gene expression signatures. I successfully characterized the incompletely defined Lin^-CD34⁺CD38^-CD45RA⁺ fraction in BM and CB, containing a CD10^lo lymphoid-primed multipotent progenitor (LMPP) with T cell, B cell, NK cell, granulocytic and monocytic differentiation potential, and succeeded in placing it in the haematopoietic hierarchy with relation to similar lympho-myeloid progenitors defined by other groups. This research lays the foundation to characterise early human progenitors with a comprehensive toolkit on a phenotypic, molecular and functional level. Findings from this thesis might provide knowledge about potential targets in LSCs.

Stem cell niches are specialized signalling microenvironments that allow maintenance of the stem cells. According to the traditional model of the stem cell niche, the niche signalling input is integrated by a cell towards a binary decision between stemness and differentiation. I have studied the regulation of somatic cyst stem cell (CyCS) proliferation in the testicular stem cell niche of Drosophila melanogaster by performing the DamID screen for targets of the transcriptional regulator Zfh1, a shared target of Jak/STAT and Hedgehog niche signalling. I have found that Zfh1 binds to the regulatory regions of kibra and salvador, tumour suppressors of the Hippo/Yorkie pathway, and downregulates them, restricting Yorkie activity to the Zfh1 positive CySCs. Clonal inactivation of the Hippo pathway is sufficient for CySC proliferation, but does not affect their differentiation ability. I therefore proposed a different stem cell niche model, whereby the niche signalling directly “micromanage” stem cell behavior, not involving the cell fate decision making.

The use of adult hepatic stem cells for the treatment of diabetes, based both on the close embryological association of the pancreas and liver, and on a putative shared tissue stem cell, has been proposed by a number of studies. This study investigated the capacity of hepatic oval cells to differentiate into pancreatic endocrine cells in the presence of pancreatic mesoderm. The GaIN model of hepatic injury was used to induce oval cell activation in Male Sprague-Dawley rats. A viable and significant oval cell population could not however, be isolated and propagated in culture. In order to continue experimentation, a PHeSC-A2 cell line, derived from normal adult porcine liver, was cultured with quail pancreatic mesoderm in the GFRM-Ham s F12.ITS culture system. Cells demonstrating positive immulocalization of the pancreatic markers, insulin and glucagon, were identified as PHeSC-A2-derived, by visual assessment of their nuclear morphology. Techniques used to confirm these results and preclude the derivation of the pancreatic endocrine cells from pancreatic endodermal contamination, proved ineffectual. The tentative results obtained in this study have lead to the following postulations: firstly, the PHeSC-A2 cell line may possess a higher level of potentiality than previously demonstrated; secondly, this potential may be due to the shared embryological origins of the pancreas and liver, and thirdly, permissive signaling from pancreatic mesoderm may have the capacity to induce the differentiation of hepatic oval cells into pancreatic endocrine cells. Further research is required to confirm the results obtained in this study and to substantiate the aforementioned propositions.

Dans les tumeurs hiérarchiques, les cellules souches du cancer (CSC), au sommet de la hiérarchie tumorale, peuvent s'auto-renouveler et se différencier en progéniteurs amplificateurs transitoires (TAP) avec un potentiel d'auto-renouvellement limité. Au cours du développement, les cellules souches neurales de Drosophile, appelées neuroblastes (NB), expriment en séquence deux protéines antagonistes se liant à l'ARN, Imp et Syncrip (Syp), qui respectivement favorise et réprime l'auto-renouvellement des NB. La perturbation de mécanismes de division asymétrique des NB peut générer leur amplification illimitée induisant de véritables tumeurs. À l’aide d’une analyse clonale et de modélisations mathématiques, nous avons démontré que les progéniteurs Imp+ dans les tumeurs agissent comme des cellules semblables aux CSC, capables de se renouveler indéfiniment et de se différencier en progéniteurs Syp+, qui, à l’instar des TAP, ont un potentiel d’auto-renouvellement limité et une forte tendance à entrer en quiescence. De plus, nous avons démontré que les tumeurs du NB suivent une organisation hiérarchique rigide dans laquelle la transition Imp-Syp est irréversible. Fait intéressant, en utilisant l’analyse transcriptomique, nous avons constaté que la transition Imp à Syp dans les NB de tumeurs induit une régulation négative des gènes glycolytiques et respiratoires, épuisant vraisemblablement la capacité de croissance et d’auto-renouvellement des progéniteurs Syp+. La conservation frappante de ces protéines de liaison à l'ARN ouvre la possibilité passionnante que des hiérarchies analogues puissent exister dans les cancers humain. / In hierarchical tumors, cancer stem cells (CSCs), at the top of the tumor hierarchy, can self-renew and differentiate in transient-amplifying progenitors (TAPs), with a limited self-renewal potential. Understanding the molecular mechanisms that drive tumor hierarchy and heterogeneity is crucial to develop effective therapies to eliminate CSCs. During development, Drosophila asymmetrically-dividing neural stem cells, called neuroblasts (NBs), sequentially express two antagonistic RNA-binding proteins, Imp and Syncrip (Syp), that respectively promote and repress NB self-renewal. Genetic perturbation of NB asymmetric division cause NB amplification and malignant tumors. By using lineage tracing, clonal analysis and stochastic mathematical modeling of tumor growth, we demonstrated that Imp+ progenitors act as CSCs. They are able to self-renew endlessly and differentiate in Syp+ progenitors, that have a limited self-renewal potential and the high tendency to undergo quiescence. NB tumors follow a rigid hierarchical organization, where the Imp-to-Syp transition is irreversible. Hence, Syp+ progenitors cannot revert to an Imp+ malignant state. Transcriptomic analysis revealed that the Imp-to-Syp transition in tumors induces a downregulation of glycolytic and respiratory genes that exhausts the growth and self-renewing potential of Syp+ progenitors. The striking conservation of these RNA-binding proteins opens the exciting possibility that analogous Imp-Syp hierarchies may exist in human cancers.

Estudos com células tronco (CT) têm despertado grande interesse devido ao seu promissor potencial terapêutico. Neste contexto, as CT mesenquimais (CTm) representam uma alternativa para o tratamento de diversas patologias em diferentes órgãos, inclusive o rim e as glomerulopatias que o acometem. As doenças glomerulares constituem uma freqüente causa de doença renal crônica e se caracterizam por apresentar proteinúria. Neste processo, os podócitos são células que apresentam um papel crucial, sendo que as podocitopatias se associam com o aparecimento de proteinúria e desenvolvimento de esclerose glomerular. A obtenção de um modelo de podocitopatia através da administração de aminonucleosídeo de puromicina (PAN), permite a melhor compreensão dessas células altamente diferenciadas que não possuem potencial de proliferação ou regeneração. O presente projeto teve como objetivo estabelecer o modelo experimental de nefropatia crônica induzida por PAN associado à nefrectomia unilateral (UniNx) para induzir lesões glomerulares mais exuberantes e, neste modelo experimental, avaliar o efeito da infusão de CTm derivadas da medula óssea. Ratos Wistar (n=52) foram divididos em três grupos: Controle (UniNx), PAN (PAN+UniNx) e PAN+CTm (PAN+UniNx+CTm). As CTm foram inoculadas na região subcapsular renal no dia 0 e os animais foram sacrificados após 30 e 60 dias. O efeito da infusão das CTm no tecido renal foi avaliado através de parâmetros clínicos e laboratoriais, além de análise histológica, imunohistoquímica, microscopia eletrônica e PCR em tempo real. Paralelamente, o projeto analisou a diferenciação in vitro de CTm em podócitos através do estímulo com colágeno tipo IV e através de cocultura de glomérulos isolados de ratos com CTm. A diferenciação celular das CTm foi analisada por citometria de fluxo, imunocitoquímica e PCR em tempo real para genes de proteínas podocitárias. No modelo in vivo foi possível observar a presença de CTm até 15 dias após a inoculação na região subcapsular renal. As CTm foram capazes de diminuir significativamente a proteinúria e a albuminúria com 30 e 60 dias, assim como a pressão arterial aos 60 dias. Não houve diferença nos valores de creatinina, uréia sérica, glomeruloesclerose e fibrose intersticial entre o grupo PAN e o grupo PAN+CTm. As CTm foram responsáveis pela diminuição significativa da fusão dos pedicelos à microscopia eletrônica, com melhora da expressão relativa de WT1 aos 60 dias e melhora parcial da expressão gênica de nefrina, podocina e sinaptopodina. A expressão proteica de WT1 também foi significativamente maior no grupo PAN+CTm em comparação ao grupo PAN. Além disso, houve melhora significante da expressão relativa de IL-4 e IL-10, e diminuição de IL-1? e TNF-? no grupo tratado. Ainda, as CTm promoveram aumento significativo da expressão gênica de VEGF aos 60 dias. Nos resultados in vitro não houve diferenciação das CTm em podócitos quando cultivadas com colágeno IV, assim como a cocultura com glomérulos não proporcionou alteração na expressão de marcadores de superfície das CTm. Concluímos que a terapia celular com CTm foi capaz de induzir proteção renal caracterizada por diminuição da proteinúria, da albuminúria e da pressão arterial, associado a menor fusão dos pedicelos, maior expressão gênica de proteínas podocitárias e de expressão celular de WT1. As citocinas inflamatórias IL-1?, TNF-?, IL-4 e IL-10, em conjunto com o VEGF, foram os possíveis mediadores responsáveis por estes resultados / Stem cells (SC) have emerged as a potential therapeutic approach for several diseases. In this context, the mesenchymal SC (mSC) are considered an alternative for the treatment of kidney diseases such as glomerulopathies. Glomerular diseases are an important cause of chronic kidney disease (CKD) and are characterized by proteinuria. In this process, the podocytes are cells that have a critical role, and the podocytopathies are associated with the onset of proteinuria and glomerular sclerosis. The achievement of a podocytopathy model through administration of puromycin (PAN) allows a better understanding of these highly differentiated cells which do not have the potential for proliferation or regeneration. The aim of the present study was to establish an experimental model of chronic nephropathy induced by PAN associated with unilateral nephrectomy (UniNx), to induce early and marked glomerular lesions and, in this experimental model, to evaluate the effect of bone marrow mSC infusion. Wistar rats (n=52) were randomly divided into three groups: Control (UniNx), PAN (PAN+UniNx) and PAN+mSC (PAN+UniNx+mSC). mSC were inoculated into the subcapsular renal region on day 0, and the animals were sacrificed after 30 and 60 days. The mSC infusion effects in renal tissue were evaluated by clinical and laboratory parameters, histology, immunohistochemistry, electron microscopy and real-time PCR. In parallel, we analyzed whether mSC could differentiate in vitro into podocytes through stimulation with collagen type IV or by means of co-culture of isolated rat glomeruli with mSC. The cell differentiation was analyzed by flow cytometry, immunocytochemistry and real-time PCR. In the in vivo model, mSC were detected until 15 days after inoculation in the renal subcapsular region. mSC were able to significantly reduce the proteinuria and albuminuria with 30 and 60 days, as well as blood pressure at 60 days. There was no difference in the values of creatinine, BUN, glomerulosclerosis and interstitial fibrosis between the groups PAN and PAN+mSC. The treated group showed lower effacement of foot process by electron microscopy, with significant improvement in the relative expression of WT1 in 60 days and partial improvement of nephrin, podocyn and synaptopodin. The WT1 protein expression was also significantly higher in the PAN+mSC group compared to the PAN group. In addition, mSC treatment significantly reduced gene expression of IL-1? and TNF-?, as well as increased the expression of IL-4 and IL-10. At 60 days mSC promoted significant increase of VEGF relative expression. In vitro results, mSC cultived with collagen type IV did not show differentiation to podocytes and the co-culture with glomeruli provided no change in expression of mSC surface markers. In conclusion, mSC therapy in the PAN model was able to induce renal protection characterized by the reduction of albuminuria, proteinuria and blood pressure, associated with a lower effacement of foot process, increased gene expression of podocytes proteins and cellular expression of WT1. Inflammatory cytokines IL-1?, TNF-?, IL-4 and IL-10 associated with VEGF were the probable mediators of these results, promoting podocyte protection