Characterization of STAT1-/- mice & Investigation of IFN alpha/beta production in murine bone marrow in response to injury

Yulia Kirpicheva
2008
Projet étudiant

Résumé

The hematopoietic system is a regenerative tissue with high cell turnover. Normal homeostasis of the hematopoietic system is insured by hematopoietic stem cells (HSCs). The important features of HSCs are the capacity to self-renew and multipotency. Multipotent HSCs perpetuate through life and are able to form all differentiated cell lineages of the blood. Most HSCs with long-term self-renewal capacity are quiescent and divide infrequently. However quiescent HSCs can be activated in response to injury. The signals responsible for the activation of quiescent HSCs are poorly investigated. The Trumpp laboratory has recently identified a role for IFNα in activation of quiescent HSCs in vivo. Activation of HSCs by IFNα involves increased phosphorylation of STAT1. Therefore the role of STAT1 in the homeostasis of the hematopoietic system was investigated by characterization of the hematopoietic system in STAT1-/- mice. A 2-fold decrease in short-term HSC (ST-HSCs) and multipotent progenitors (MPPs) was observed in the bone marrow of STAT1-/- mice compared to C57Bl/6 (wild type) mice. Analysis of progenitors of HSCs also revealed a small increase in common myeloid progenitors (CMPs) and a small decrease in common lymphoid progenitors (CLPs) in the bone marrow of STAT1-/- mice. These results indicate that STAT1 play a role in the homeostasis of ST-HSCs and MPPs in bone marrow. Functional analysis of STAT1-/- LT-HSCs using in vivo long-term reconstitution assay suggests that STAT1-/- LT-HSCs can reconstitute lethally irradiated mice. Nevertheless further investigations are necessary to examine whether STAT1 plays a role in HSCs self-renewal. The unexpected role of IFNα in activation of quiescent HSCs leads to the hypothesis that endogenously produced IFNα might be important for activation of HSCs in response to injury. To address this we generated the mouse model MxCre;R26REYFP to monitor endogenous IFNα/β production in the bone marrow after injury. Our results suggest that IFNα/β is produced in response to 5-FU induced myeloid suppression and G-CSF treatment in vivo. The role of IFNα/β during the recovery phase after 5-FU induced myeloid suppression was investigated using IFNAR-/- mice. These results suggest that endogenous IFNα/β might be important for the mobilization of HSCs into the spleen induced in response to 5-FU

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https://infoscience.epfl.ch/record/139124?ln=fr

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Yulia Kirpicheva
2008
Projet étudiant

Résumé

Official source

https://infoscience.epfl.ch/record/139124?ln=fr

À propos de ce résultat

Concepts associés (16)

Concepts associés

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Publications associées (52)

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The role of TIF1 gamma during adult murine hematopoiesis

Konstantin Shakhbazov

Transforming Growth Factor beta (TGFβ) signaling plays an important role in a variety of cellular processes during embryonic development, adult tissue homeostasis and cancer. TGFβI ligand is a potent inhibitor of early hematopoietic progenitor [1] cells in vitro, however the exact role and mechanisms of this pathway during adult hematopoietic homeostasis remains poorly understood. The Transcriptional Intermediary Factor 1 gamma (Tif1γ) gene encodes a recently uncovered nuclear protein in the TGFβ transduction pathway. Tif1γ is defective in the zebrafish moonshine mutant, which exhibits primitive and definitive hematopoiesis defects [2]. In addition, Tif1γ has been shown to influence differentiation of human erythroid cells in vitro [3]. Recent work in our laboratory has provided genetic evidence that Tif1γ is required for the generation and survival of hematopoietic stem/progenitor cells during murine development (C.Adolphe, K.Shakhbazov et al., manuscript under submission). Given that Tif1γ function is required for embryonic development and null mutants are embryonic lethal [4] (R.Losson unpublished data), we took advantage of a conditional Tif1γ flox allele (R.Losson unpublished data) and combined it with the inducible MxCre line [5] to generate mice in which Tif1γ function can be specifically ablated during adult hematopoiesis. MxCre:Tif1γKO/flox mutants exhibit a decrease in erythroblasts and dramatic increase in granulocytes within the bone marrow. This phenotype is attributable to a massive increase in granulocyte-monocyte progenitors (GMPs) at the expense of common myeloid progenitors (CMPs) and megakaryocyte- erythrocyte progenitors (MEPs), which are severely decreased in Tif1γ mutant bone marrow. In addition, pre-B cells were dramatically decreased in numbers, attributable to an increase in cell death, indicating a pro-survival role for Tif1γ in B lymphocytes. In addition, we performed deletion of Tif1γ in non-competitive and competitive bone marrow transplantation settings, which indicated that only the CMP/MEP phenotype is a non-cell autonomous effect. Two distinct models have been reported suggesting how Tif1γ functions within the TGFβ pathway: one indicating that Tif1γ binds and acts through Smad2/3 and the other that Tif1γ functions as a Smad4 mono-ubiquitin ligase [3, 6, 7]. To genetically address this apparent discrepancy, we generated double Tif1γ/Smad4 hematopoietic specific mutant mice, whose phenotype was surprisingly similar to that observed in Tif1γ single mutants. These data suggest that Tif1γ function is independent of Smad4 in hematopoietic cells. To address the mechanism by which Tif1γ functions, we performed microarray profiling of normal and mutant progenitors, which revealed prostaglandin D2 synthase as a hub gene linking Tif1γ and TGFβ signaling. Finally, we were able to mimic the hematopoietic-Tif1γ null phenotype by providing 16,16-dimethyl prostaglandin D2 to wild-type hematopoietic cells in vitro. In summary, our data provide genetic evidence for a key role of Tif1γ during early myeloid development as well as pre-B cell survival. In addition, we have identified that the majority of Tif1γ function is Smad4 independent. Finally, we identified a novel link between Tif1γ function and prostaglandin metabolism/signaling as a likely mechanism by which Tif1γ regulates hematopoiesis.

EPFL2009

Chargement

Bone marrow transplantation is a well-established medical procedure for the treatment of various hematologic diseases. However, the relatively low number of hematopoietic stem cells (HSCs) that can be harvested, especially from umbilical cord blood, limits even broader applicability of the procedure. A better understanding of the biology of HSCs, and in particular how these rare cells are regulated by microenvironmental niches in the bone marrow, could ultimately enable robust in vitro expansion of HSCs. In this thesis, several novel strategies were developed to study and manipulate HSC biology in vitro, through the regulation of key niche factors, cellular metabolism, and the complex multicellular crosstalk in a niche-mimicking context. First, we made use of gastruloids, an embryonic stem cell-based model of early mouse development, to mimic key aspects of embryonic hematopoiesis in vitro. Simultaneously with the establishment of a vascular network, we detected by immunophenotyping the emergence of primitive blood progenitor cells during the late developmental stages of gastruloids. These embryonic blood progenitors were spatially localized close to a vascular-like plexus in the anterior portion of the gastruloid. Colony-forming assays demonstrated an interesting differentiation potential of these cells. These data demonstrate the potential of gastruloids as an easily accessible in vitro model to study blood development in an embryo-like context. Second, a bioengineering approach was used to study HSC fate choices upon exposure to niche-specific ligands in a well-defined artificial environment. A combination of time-lapse microscopy and single-cell multigene expression analysis was used to define differentiation and cell-cycle states of mouse and human HSCs. Strikingly, selected artificial niches reduced proliferation and maintained the long-term multilineage potential of HSCs in vitro. These artificial niches hold significant potential for the study of mechanisms involved in HSC fate regulation. Third, the influence of the metabolism on fate choices of adult HSCs was studied, specifically focusing on fatty acid Î²-oxidation. Malonyl-CoA was used to reversibly block fatty acid Î²-oxidation in mouse HSCs. In vitro treatment of HSCs with malonyl-CoA promoted their expansion and increased lymphoid reconstitution upon in vivo transplantation. This study sheds new light on the role of the metabolic environment in HSC regulation. Surprisingly, exposure to only a single, readily available metabolite was found to be sufficient to strongly influence HSC behavior. Fourth, a miniaturized multicellular culture system was developed to mimic the hallmarks of the dynamics of HSCs in their native bone marrow. Primary human mesenchymal stem/progenitor cells and endothelial cells were aggregated in a high-throughput manner in biomimetic hydrogel microwells to form self-organizing bone marrow organoids. Immunostaining demonstrated the formation of branched vascular networks, rendering the organoids permissive for the homing of human hematopoietic stem and progenitor cells. These results suggest that bone marrow organoids may be a suitable platform for the modeling of physiological and clinically-relevant stem cell dynamics in vitro. Altogether, this thesis presents several innovative approaches for modeling key features of native stem cell microenvironments that will contribute to a better understanding of the biology of HSCs and their regulatory niche.

EPFL2019

Chargement

Prospective identification of hematopoietic and neural stem/progenitor cells using transgenic and lentiviral based approaches

Didier Surdez

Adult stem cells represent only a minor proportion of a given tissue and are difficult to amplify in vitro without affecting their biological properties. To circumvent this problem, we used a transgenic/lentiviral based approach to isolate and characterize these cells by driving a fluorescent reporter protein under the control of Spi2a, H2Kb or MELK promoter/enhancer elements, which were previously shown to be active in various stem cells. The first chapter focuses on Spi2a, a serine protease inhibitor (Serpin), which is known to be preferentially expressed in hematopoietic stem cells (HSCs). Taking advantage of the previously characterized minimal Spi2a promoter, we were able to determine its activity profile in the hematopoietic system. For this purpose, we infected HSCs with a lentivirus driving d2eGFP expression under the control of the minimal Spi2a promoter. These cells were then injected into lethally irradiated recipient mice that were analyzed at different time points (≥2 months) after transplantation. Despite variable d2eGFP expression in the hematopoietic compartment, we found a population of bright d2eGFP-expressing bone marrow cells (Spi2a0.1%) that were mostly restricted to HSCs and early myeloid progenitors. Spi2a0.1% cells displayed multilineage colony forming potential in vitro and exhibited long-term multilineage repopulating capacity in vivo, when a restricted numbers of these cells (≥ 300 cells) were transplanted into secondary recipient mice. Furthermore, we generated transgenic mice using a similar construct as used for the lentiviral approach. These mice indicated that the Spi2a promoter may be activated in non-hematopoietic adult stem cells, such as in the brain, the testis or the pancreas. In the second chapter, we describe a transgenic mouse line that expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs expressed exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2Kb-zFP mice. Up to 90% of zFPbright cells were also c-kithigh, Sca-1high, Linneg, Flk-2neg, which is a bona fide phenotype for long-term HSCs. Double-sorted zFPbright HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2Kb-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs. The last chapter is dedicated to MELK. This kinase of the snf1/AMPK family was recently identified to regulate the proliferation of multipotent neural progenitor cells and was also found to be expressed in hematopoietic stem cells and in spermatogonia. Using a transgenic mouse approach by expressing eGFP under a 3.5kb MELK promoter/enhancer element, we demonstrated that eGFP-expressing cells were localized in the subventricular zone (SVZ) of the lateral ventricle and in the subgranular zone of the dentate gyrus, the two major regions of adult neurogenesis. Moreover, the eGFP expression level in SVZ-derived cells positively correlated with their ability to form neurospheres. We also observed strong and restricted expression of eGFP in spermatogonia cells, indicating that MELK may play an important role in early spermatogenesis. Additionally, activation of the MELK promoter in hematopoietic stem/progenitor cells suggested a widespread implication of MELK in various adult stem cells. To conclude, we have generated three different reporter strains and one lentiviral based approach that allow for in vivo identification of stem/progenitor cells in different compartment such as the brain, the bone marrow or the testis. With the raising interest of stem-cell-based therapies in the neurodegenerative or the cancer field, these mice should provide novel and alternative tools to study these phenomena in vivo.

EPFL2008

Chargement

The role of TIF1 gamma during adult murine hematopoiesis

Konstantin Shakhbazov

EPFL2009

EPFL2019

Prospective identification of hematopoietic and neural stem/progenitor cells using transgenic and lentiviral based approaches

Didier Surdez

EPFL2008