Haematopoietic stem cells (HSCs) emerge during embryogenesis and maintain hematopoiesis in the adult organism. Qualitative and quantitative assessment of HSCs can only be performed functionally using the in vivo long-term repopulation assay. Due to the lack of such data, little is known about the development of HSCs in the human embryo, which is a prerequisite for the development of new therapeutic strategies. Employing the xenotransplantation assay, I have performed here the spatio-temporal mapping of HSC activity within the human embryo and have shown that human HSCs emerge first in the aorta-gonad-mesonephros (AGM) region, specifically in the ventral wall of the dorsal aorta, and only later appear in the yolk sac, liver and placenta. Human AGM region HSCs transplanted into immunodeficient mice provide long-term high-level multilineage haematopoietic repopulation. These cells, although present in the AGM region in low numbers, exhibit a very high self-renewal potential. A single HSC derived from the AGM region generates around 600 daughter HSCs in primary recipient mice, which disseminate throughout the entire recipient bone marrow and are retransplantable. These findings highlight the vast regenerative potential of the earliest human HSCs and set a new standard for in vitro generation of HSCs from pluripotent stem cells for the purpose of regenerative medicine. I have also established a preliminary immunophenotype of the earliest human HSC. These data will be useful for my future studies on the mechanisms underlying the high potency of human embryonic HSCs and on the characterisation of embryonic HSC niche.

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

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 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

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

Yau, Ka Long. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 97-113). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Current usage of human embryonic stem cells (hES) and induced pluripotent stem cells (iPS) in clinical therapies and personalized medicine are limited as a result of ethical, technical and medical problems that arise from isolation and generation of these cells. Isolation of hES cells faces ethical problems associated with their derivation from human pre-implantation embryos. The most controversial aspect of hES cell isolation targets the generation of autologous hES cell lines which requires the transfer of a somatic-cell nucleus from the patient to an enucleated oocyte. While already established embryonic stem cell lines from IVF embryos can be used in a similar manner, lack of genetic identity can cause therapy rejection from the host, and prevent their use in personalized medicine. Induced pluripotent stem cells on the other hand, are generated from somatic cells that have been reprogrammed in vitro to behave like stem cells. While these cells can potentially be used for personalized medicine without the risk of rejection by the host system, derivation methods prevent their therapeutic use. The most efficient method used to generate iPS cells involves usage of viral particles which can result in viral DNA being integrated in the host cell’s genome and render these cells non-compliant for clinical therapies. Other methods not involving viral particles exist as well, but the reprogramming efficiency is too low and technical problems with generating large enough numbers of cells prevent these methods from being feasible approaches for clinical therapies. Direct reprogramming of a differentiated cell into a developmentally more plastic cell would offer alternatives to applications in regenerative medicine that currently depend on either embryonic stem cells (ES), adult stem cells or iPS cells. We hypothesize that Xenopus laevis egg cytoplasmic extract contains critical factors needed for reprogramming that may allow for non-viral, chemically defined derivation of human induced pluripotent/multipotent cells which can be maintained by addition of exogenous FGF2. In this thesis we investigated a new method for generation of multipotent cells through determining the ability of select fractions of Xenopus laevis egg extract to induce multipotency in already differentiated cells. We were able to identify select fractions from the extract that in combination with exogenously added FGF2 can reprogram and maintain the reprogrammed cells in an undifferentiated state. The findings of this work also determined that Xenopus laevis egg extract mRNA is required for achieving full reprogramming. The body of work presented in this thesis showed the ability of FGF2 isoforms to bind and activate select FGF receptor tyrosine kinases, act as extracellular mitogenic factors to support growth of hES cells in an undifferentiated state as well bind to nuclear DNA and affect expression of endogenous genes. Moreover, we showed that all FGF2 isoforms can induce expression of stem cell specific proteins in human dermal fibroblasts as well as extend lifespan of human dermal fibroblasts in vitro. In this work we identified HECW1, the gene coding for E3 ubiquitin ligase NEDL1, as a novel nuclear target for all FGF2 isoforms and showed that overexpression of recombinant FGF2 isoforms in human dermal fibroblasts can down regulate expression of HECW1 gene.

"Human umbilical cord blood (CB) hematopoietic stem cells (HSCs) have well established applications for cellular therapy. Current protocols for isolating HSCs from bone marrow or cord select for CD34 + cells, however some CD34 - populations have recently been shown to also contain strong HSC activity. Thus the positive selection of HSCs based on cell surface markers remains controversial. However, it is clear from the literature that differentiated hematopoietic cells (lineage positive, Lin + ), representing the vast majority (>90%) of most blood populations, contain no long-term reconstitution potential. Thus Viacell Inc. (Worcester, MA) expands and enriches its populations of cells containing HSCs by removing only those Lin + cells known not to contain HSCs. This is accomplished on two separation columns (post-sep-1, and post-sep-2) (separated by 7 days of cell growth) that contain a variety of antibodies to known differentiation surface markers. Although this process strongly enriches functional HCSs, these primitive cell populations remain biochemically uncharacterized. Because HSC populations containing long chromosomal telomeres and high telomerase activity (which helps maintain telomeres) have been shown to display the strongest long-term reconstitution potential, the purpose of this thesis was to investigate these two parameters in selected samples of Viacell’s ex vivo amplification procedure. Two specific hypotheses were tested: 1. the removal of Lin + cells will appear to increase the telomerase activity and telomere lengths in the remaining cell population, and 2. these two parameters will decrease upon hematopoietic cell differentiation and proliferation. Telomerase activity was assayed using a telomeric repeat amplication protocol (TRAP), and normalized relative to a cancer cell line positive control. Relative to fresh cord blood, telomerase activity was found to increase significantly in post-sep-1 (from 8.5 ± 1.5% to 76.2 ± 4.9%, p = 0.0001, n = 5) and post-sep-2 (8.5 ± 1.5% to 111.3 ± 4.9%, p = 0.0001, n = 5) fractions following the removal of Lin + cells. This increase was found to be highly reproducible, showing very low intra-cord and inter-cord variability. Telomere lengths were assayed using a telomere length assay (TLA). Relative to fresh cord blood, telomere lengths increased significantly in post-sep-1 (from 10 to 12 kb, n = 2) and post-sep- 2 (from 10 to 14 kb, p = 0.001, n = 2) fractions. These apparent increases likely result from the direct removal of cells low in telomerase activity with short telomeres since the Lin + cells from the post-sep-1 column were found to contain relatively low telomerase activity (32.1 ± 15%, p = 0.001, n = 2) and short telomeres (7.5 kb, p = 0.001), which supports our first hypothesis. Finally, we show that telomerase activity and telomere lengths decreased in Day-14 cells (expanded and differentiated 14 days) relative to post-sep-2 (from 111.8 ± 19.6% to 54 ± 21.2%, p = 0.001, n = 3 for the TRAP, and from 14 kb to 9 kb, p = 0.0001, n = 2 for the TLA). Those two parameters also decreased in pre-sep-3 cells (terminally differentiated by treatment with All Trans Retinoic Acid for 14 days) relative to post-sep-2 (from 111.3 ± 4.9% to 14.8 ± 1.7%, p = 0.0001, n = 6 for the TRAP, and from 14 kb to 7.5 kb, p = 0.001 for the TLA), supporting our second hypothesis. Telomerase activity was found to not directly correlate with CD34 + CD38 - content, supporting recent observations that a significant portion of HSCs reside outside this population."