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Differentiation plasticity of hematopoietic cells
Polgárová, Kamila ; Stopka, Tomáš (advisor) ; Otáhal, Pavel (referee) ; Šálek, Cyril (referee)
Hematopoiesis has been for many years seen as a straightforward process based on sequential restriction of cell fate potential leading to production of mature blood cells. In the last decade, however, several works documented an unexpected plasticity of hematopoietic cells with expanded potential of myeloid development from lymphoid progenitors and vice versa. Under physiologic conditions hematopoiesis is tightly controlled and the definite cell fate is denominated by multiple factors that all lead to changes in regulatory networks that include transcription factors, epigenetic changes and post-transcriptional modulations. Any disruption of this strict regulation, caused by mutations or other events, affects the proliferation and lineage fidelity of hematopoietic precursors. This may lead to clonal growth of variable significance or leukemogenesis and may possibly affect the treatment sensitivity of the hematological malignancies. For better understanding of hematopoietic regulation we described gene expression changes during physiological development of lymphoid and myeloid lineages and in leukemic specimens using our own simplified real-time PCR based platform. We investigated expression of 95 genes connected with lymphoid and myeloid differentiation or with leukemogenesis in sorted hematopoietic...
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Lineage plasticity in normal and malignant lymphocyte precursors
Rezková Řezníčková, Leona ; Froňková, Eva (advisor) ; Otáhal, Pavel (referee)
Klasické schéma vývoje hematopoetických buněk předpokládá časné oddělení lymfoidního a myeloidního prekurzoru. V poslední době jsou navrhovány složitější modely, které předpokládají větší flexibilitu hematopoezy a navrhují existenci progenitorů s lymfoidním i myeloidním potenciálem. Akutní hybridní leukémie jsou malignity, které podle různých kritérií nelze jednoznačně zařadit k lymfoidní nebo k myeloidní linii a jejichž chování spíše dává za pravdu novým modelům hematopoezy. Předkládaná práce se zabývala především výzkumem dětských leukémií s přesmykem z lymfoidní do myeloidní linie během indukční léčby. Jedná se o rozsáhlý projekt, v jehož rámci si diplomová práce si kladla za úkol určit liniové zařazení leukemických blastů pomocí detekce přestaveb genů pro imunoglobuliny a T-buněčné receptory (TCR). Potvrdili jsme, že myeloidní buňky derivované v průběhu léčby pochází u všech pacientů z původního lymfoidního klonu. Dále jsme u těchto případů zkoumali expresi vytipovaných genů ve srovnání s běžnými druhy leukémií. Třetí částí práce byl výzkum prognostického významu přítomnosti přestaveb TCR (a tedy příslušnosti k lymfoidní linii) u leukémií z T-lymfoidní řady.
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The role of stem and progenitor cells in regeneration of hematopoietic tissue
Faltusová, Kateřina ; Nečas, Emanuel (advisor) ; Hofer, Michal (referee) ; Filipp, Dominik (referee)
Tissue regeneration is a complex and highly orchestrated process dependent on cells with the potential to restore structures and functions and on controlling factors from the tissue microenvironment. Hematopoietic tissue has a high ability to regenerate, which is attributed to the presence of stem cells, but the regeneration of severely damaged adult tissue is still only partially understood. Hematopoietic tissue provides a unique opportunity to study tissue regeneration due to its well-established steady- state structure and function, easy accessibility, advanced research methods, and well-defined embryonic, fetal, and adult stages of development. Embryonic/fetal liver hematopoiesis and adult hematopoiesis recovering from damage share the needto expand populations of progenitors and stem cells in parallel with increasing production of mature blood cells. We analyzed adult hematopoiesis in mice subjected to a submyeloablative dose (6 Gy) of gamma radiation, in which only a few cells with reconstituting capacity survived. We targeted the period of regeneration characterized by the renewed massive production of mature blood cells and the ongoing expansion of immature hematopoietic cells. Cells from the top of the hematopoietic hierarchy, hematopoietic stem cells, and multipotent progenitors are almost missing...
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The role of Kit ligands in hematopoiesis of Danio rerio
Oltová, Jana
Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...
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Lineage plasticity in normal and malignant lymphocyte precursors
Rezková Řezníčková, Leona
The classical scheme of hematopoiesis presumes early separation of lymphoid and myeloid precursors. Recently, more complex models are put forward, suggesting greater flexibility of hematopoiesis with progenitors sharing lymphoid and myeloid potential. Acute hybrid leukemia is a malignancy, in which it is not possible to assess unambiguously myeloid or lymphoid lineage of origin. The behaviour of those malignancies favors new models of hematopoiesis. Our work concentrated mainly on the research of childhood leukemias with lineage switch from lymphoid to myeloid lineage during induction treatment. Our task within this extensive project was to determine lineage assignment of leukemic blasts using detection of immunoglobulin and T-cell receptor gene rearrangements. We confirmed that myeloid cells derived during the treatment in all patients descend from the original lymphoid clone. We also investigated the expression of selected genes in those cases compared to common leukemia types. Lastly, we explored prognostic impact of TCR rearrangements (and thus lymphoid lineage commitment) in T-lineage leukemia.
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The utility of Toll-like receptor 2 in defining the progenitors of definitive embryonic hematopoiesis
Šplíchalová, Iva ; Filipp, Dominik (advisor) ; Nečas, Emanuel (referee) ; Kalina, Tomáš (referee)
Hematopoiesis is a vital process in which red blood cells and cells of the immune system are formed. It is initiated during early embryonic development when we find hematopoietic progenitors in separate anatomical sites. Embryonic hematopoiesis comprises three successive and partly overlapping waves of progenitors with a different hematopoietic potential. The primary anatomical place where hematopoiesis takes place shortly before the birth is the bone marrow (BM). Since at this time point of development BM is already populated by hematopoietic stem cell (HSCs) progenitors, it becomes also the site of hematopoiesis in adulthood. However, the bone marrow is not the only place where hematopoietic progenitors emerge and develop. The Yolk sac (YS) and the Aorta-Gonad-Mesonephros (AGM) region are the initial sites of the appearance of the three waves of progenitors in the early embryogenesis. These progenitors and their descendants play an indispensable role during the development of an individual. Because there are no specific markers that would unambiguously characterize progenitors of these individual waves, their physical separation and hence also functional characterization is still incomplete. Recent studies have shown that Toll-like receptors (TLRs) are expressed on adult HSCs. The stimulation of...
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Hematopoiesis in the models of zebrafish and medaka as a recipient for human HSC xenograft
Pravcová, Naďa ; Drbal, Karel (advisor) ; Svoboda, Ondřej (referee)
Danio rerio (zebrafish) and Oryzias latipes (medaka) have recently become popular model organisms to study hematopoiesis. These model organisms present several advantages in comparison to other commonly used models, the most common being Mus musculus (mouse). The advantages are shorter generation time, large offspring production, frequent spawning, external fertilization and development, the optical transparency of embryos amenable to genetic manipulation on the background of vast numbers of transgenic lines (mainly in zebrafish) and inbred strains (in medaka). Moreover, most of the mechanisms behind zebrafish and medaka hematopoiesis are conserved in higher vertebrates. Most importantly, the optical transparency in early development and in adult mutant transparent strains allows for observation of hematopoietic stem cell (HSC) development in vivo. Therefore, it is possible to generate humanized fish using xenotransplanted human HSCs for studies of the engraftment, differentiation, and trafficking of human HSC in vivo. Currently, the most popular organism for human HSC xenotransplantation is mice. This model system is not suitable for in vivo imaging of HSC engraftment. Moreover, a prior immunodepletion step is necessary. The process of immunodepletion includes genetic manipulation or irradiation...
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The role of Kit ligands in hematopoiesis of Danio rerio
Oltová, Jana ; Bartůněk, Petr (advisor) ; Živný, Jan (referee) ; Divoký, Vladimír (referee)
Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...
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Role of Rnf207b in zebrafish hematopoiesis
Vondráková, Zuzana ; Bartůněk, Petr (advisor) ; Živný, Jan (referee)
Hematopoiesis is the process of proliferation, differentiation and self-renewal of hematopoietic stem cells. Regulation of hematopoiesis is a complex process, which takes place on many different levels and is directed by many signals. RNF207 is one of the perspective genes chosen based on a screen in chicken model, where obtained data show its role in hematopoiesis. The aim of this work was to confirm the role of rnf207b as a new regulator of hematopoiesis in Danio rerio and to find out on which level of hematopoiesis is active. Danio rerio is an excellent model to study the function of genes in vivo, thanks to the easy manipulation of genetic expression and wide range of phenotypes during the development. To study the effect of rnf207b in hematopoiesis of Danio rerio we performed the knock-down of this gene by microinjection of morpholino oligonucleotides into one cell stage embryos. In these injected fish, we saw the effect in both thrombocyte and erythroid lineage, suggesting that rnf207b could be a regulator at the hierarchical level of progenitors or even more upstream. The results of developmental and tissue specific expression analysis then show that expression of rnf207b begins as early as 18 hpf, at the time of primitive hematopoiesis. Although rnf207b is expressed in the kidney (an...
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Epigenetic Aspects of normal and malignant hematopoiesis: role of chromatin remodeling ISWIATPase.
Zikmund, Tomáš ; Stopka, Tomáš (advisor) ; Dráber, Peter (referee) ; Otáhal, Pavel (referee)
Chromatin remodeling protein Smarca5 participates on many cellular processes, which are important for tissue development and tumorigenesis. Among these processes utilizing ATPase activity of Smarca5 belong also transcription, replication and DNA repair. We hypothesized that Smarca5 represents essential molecule for chromatin modulation primarily at early developmental stages at the level of fast-dividing progenitors of many origins, in whose the ATPase is highly expressed. To such tissues may belong also hematopoiesis, in which the Smarca5 has highest expression. The subject of my doctoral thesis is therefore analysis of the effect Smarca5 depletion on proliferation and differentiation of hematopoietic progenitors in vivo and a search for mechanisms behind the resulted developmental defects. We utilized conditionally knockout allele of Smarca5 in blood precursors to study in a mouse model how depletion of the ISWI ATPase causes accumulation of earliest progenitors inhibited from further maturation to erythroid and other myeloid lines. The proerythroblasts became dysplastic and the majority of basophilic erythroblasts ceased cycling around the G2/M stage. An expected mechanism for observed changes appeared the activation of stress pathway of protein p53 that is often associated with unrepaired DNA...
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