Improved HSC reconstitution and protection from inflammatory stress and chemotherapy in mice lacking granzyme B.

The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4-TRIF-p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress

Haematopoietic stem cells in perisinusoidal niches are protected from ageing.

With ageing, intrinsic haematopoietic stem cell (HSC) activity decreases, resulting in impaired tissue homeostasis, reduced engraftment following transplantation and increased susceptibility to diseases. However, whether ageing also affects the HSC niche, and thereby impairs its capacity to support HSC function, is still widely debated. Here, by using in-vivo long-term label-retention assays we demonstrate that aged label-retaining HSCs, which are, in old mice, the most quiescent HSC subpopulation with the highest regenerative capacity and cellular polarity, reside predominantly in perisinusoidal niches. Furthermore, we demonstrate that sinusoidal niches are uniquely preserved in shape, morphology and number on ageing. Finally, we show that myeloablative chemotherapy can selectively disrupt aged sinusoidal niches in the long term, which is linked to the lack of recovery of endothelial Jag2 at sinusoids. Overall, our data characterize the functional alterations of the aged HSC niche and unveil that perisinusoidal niches are uniquely preserved and thereby protect HSCs from ageing

Publisher Correction: Chemotherapy-induced transposable elements activate MDA5 to enhance haematopoietic regeneration.

Funder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265Funder: Max-Planck-Gesellschaft (Max Planck Society); doi: https://doi.org/10.13039/50110000418

Niche derived netrin-1 regulates hematopoietic stem cell dormancy via its receptor neogenin-1

Funder: Studienstiftung des Deutschen Volkes (German National Academic Foundation); doi: https://doi.org/10.13039/501100004350Funder: Heinrich F.C. Behr StiftungFunder: Dietmar Hopp Stiftung; doi: https://doi.org/10.13039/501100005941Abstract: Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal

In vivo consequences of AML1-ETO fusion protein expression for hematopoiesis

The t(8;21) (q22;q22) translocation fusing the ETO (also known as MTG8) gene on human chromosome 8 with the AML1 (also called Runx1 or CBFα) gene on chromosome 21 is one of the most common genetic aberrations found in acute myeloid leukemia (AML). This chromosomal translocation occurs in 12 % of de novo AML cases and in up to 40 % of the AML-M2 subtype of the French-American-British classification. To date, the in vivo function of aberrant AML1-ETO fusion protein expression has been investigated by several groups. However, in these studies, controversial results were reported and some key issues remain unknown. Importantly, the consequences of aberrant AML1-ETO expression for self-renewing hematopoietic stem cells (HSCs), multipotent hematopoietic progenitors (MPPs) and lineage-restricted precursors are not known. rn The aim of this thesis was to develop a novel experimental AML1-ETO in vivo model that (i) overcomes the current lack of insight into the pre-leukemic condition of t(8;21)-associated AML, (ii) clarifies the in vivo consequences of AML1-ETO for HSCs, MPPs, progenitors and more mature blood cells and (iii) generates an improved mouse model suitable for mirroring the human condition. For this purpose, a conditional tet on/off mouse model expressing the AML1-ETO fusion protein from the ROSA26 (R26) locus was generated. rn Aberrant AML1-ETO activation in compound ROSA26/tetOAML1-ETO (R26/AE) mice caused high rates of mortality, an overall disruption of hematopoietic organs and a profound alteration of hematopoiesis. However, since the generalized activity of the R26 locus did not recapitulate the leukemic condition found in human patients, it was important to restrict AML1-ETO expression to blood cell lineages. Therefore, bone marrow cells from non-induced R26/AE mice were adoptively transplanted into sublethal irradiated RAG2-/- recipient mice. First signs of phenotypical differences between AML1-ETO-expressing and control mice were observed after eight to nine months of transgene induction. AML1-ETO-expressing mice showed profound changes in hematopoietic organs accompanied by manifest extramedullary hematopoiesis. In addition, a block in early erythropoiesis, B- and T-cell maturation was observed and granulopoiesis was significantly enhanced. Most interestingly, conditional activation of AML1-ETO in chimeric mice did not increase HSCs, MPPs, common lymphoid precursors (CLPs), common myeloid progenitors (CMPs) and megakaryocyte-erythrocyte progenitors (MEPs) but promoted the selective amplification of granulocyte-macrophage progenitors (GMPs). rn The results of this thesis provide clear experimental evidence how aberrant AML1-ETO modulates the developmental properties of normal hematopoiesis and establishes for the first time that AML1-ETO does not increase HSCs, MPPs and common lineage-restricted progenitor pools but specifically amplifies GMPs. The here presented mouse model not only clarifies the role of aberrant AML1-ETO for shaping hematopoietic development but in addition has strong implications for future therapeutic strategies and will be an excellent pre-clinical tool for developing and testing new approaches to treat and eventually cure AML.rnDie t(8;21) (q22;q22) chromosomale Translokation führt zu einer Fusion zwischen dem ETO-Gen (auch bekannt als MTG8) auf dem humanen Chromosom 8 und dem AML1-Gen (Runx1 oder CBFα) auf Chromosom 21 und ist eine der häufigsten genetischen Abberationen, welche bei akuten myeloischen Leukämien (AML) gefunden wird. Das AML1-ETO Fusionsprotein tritt etwa bei 12% aller AML Patienten auf und ist gemäß des französisch-amerikanisch-englischen Klassifizierungsschemas (FAB) bei etwa 40% von AML Patienten der AML-M2 Untergruppe nachweisbar. Trotz intensiver Forschung auf diesem Gebiet, ist die genaue in vivo Funktion dieses Fusionsproteins weiterhin größtenteils unbekannt. Vor allem der Einfluss von AML1-ETO auf hämatopoetische Stammzellen (HSZ), multipotente hämatopoetische Vorläuferzellen (MHV) und linienrestringierte Progenitoren ist unbekannt.rnDas Ziel der vorliegenden Arbeit war die Etablierung eines neuen AML1-ETO Mausmodells, welches (a) ein besseres Verständnis für die frühe Pathogenesse von AML mit t(8;21) liefert, (b) eine Untersuchung der Funktion von AML1-ETO in HSZ, MHV, unreifen und reifen Blutzellen ermöglicht und (c) die Situation im Patienten möglichst genau widerspiegelt. Hierfür wurde ein tet on/off Mausmodell entwickelt, in dem die Expression des AML1-ETO Fusionproteins unter dem Einfluss des ROSA26 (R26)-Lokus steht.rnAberrante Expression von AML1-ETO in ROSA26/tetOAML1-ETO (R26/AE) Mäusen führte zu einer hohen Mortalität, einer Atrophie hämatopoetischer Organe und einer gestörten Blutzellbildung. Aufgrund der ubiquitären Aktivität des R26-Lokus im Organismus, war es wichtig, die Induktion von AML1-ETO auf Blutzellen zu beschränken, um so die im Patienten gefundene Situation zu rekapitulieren. Um diese Voraussetzung experimentell umzusetzen, wurden Knochenmarkzellen aus nicht induzierten R26/AE Mäusen in subletal bestrahlte RAG2-/- Mäuse adoptiv transferiert. Nach acht bis neun Monaten zeigten sich in den AML1-ETO induzierten Mäusen erste phänotypische Veränderungen. Diese äußerten sich in einer Atrophie hämatopoetischer Organe sowie der Induktion von extramedullärer Hämatopoese. Des Weiteren wurde durch AML1-ETO Expression die initiale Ausreifung von roten Blutzellen, B- und T-Zellen blockiert, die Granulopoese jedoch verstärkt. Interessanterweise führt die konditionelle Aktivierung von AML1-ETO in chimären Mäusen nicht zu einer Vermehrung von HSZ, MHV, gemeinsamen lymphopoietischen, myeloischen und Megakaryo-/Erythrozytären-Vorläufer, aber zu einer spezifischen Expansion der Granulozyten-/Makrophagen-Vorläuferzellen (GMV).rnDie vorliegende Arbeit beschreibt den Einfluss von AML1-ETO Expression auf die normale Hämatopoese und zeigt erstmals, dass AML1-ETO keine Auswirkung auf HSZ, MHV und gemeinsame linienrestringierte Vorläuferpopulationen hat, sondern zu einer spezifischen Expansion von GMV führt. Im Rahmen dieser Arbeit ist es mit Hilfe des hier etablierten Mausmodells gelungen, die funktionelle Auswirkung aberranter AML1-ETO Aktivierung für die Entwicklung von Blutzellen aufzuklären und gleichzeitig neue Therapiestrategien für die zukünftige Behandlung von AML aufzuzeigen.r

Metabolic Regulation of Hematopoietic Stem Cells

Cellular metabolism is a key regulator of hematopoietic stem cell (HSC) maintenance. HSCs rely on anaerobic glycolysis for energy production to minimize the production of reactive oxygen species and shift toward mitochondrial oxidative phosphorylation upon differentiation. However, increasing evidence has shown that HSCs still maintain a certain level of mitochondrial activity in quiescence, and exhibit high mitochondrial membrane potential, which both support proper HSC function. Since glycolysis and the tricarboxylic acid (TCA) cycle are not directly connected in HSCs, other nutrient pathways, such as amino acid and fatty acid metabolism, generate acetyl-CoA and provide it to the TCA cycle. In this review, we discuss recent insights into the regulatory roles of cellular metabolism in HSCs. Understanding the metabolic requirements of healthy HSCs is of critical importance to the development of new therapies for hematological disorders

VarID2 quantifies gene expression noise dynamics and unveils functional heterogeneity of ageing hematopoietic stem cells

Abstract Variability of gene expression due to stochasticity of transcription or variation of extrinsic signals, termed biological noise, is a potential driving force of cellular differentiation. Utilizing single-cell RNA-sequencing, we develop VarID2 for the quantification of biological noise at single-cell resolution. VarID2 reveals enhanced nuclear versus cytoplasmic noise, and distinct regulatory modes stratified by correlation between noise, expression, and chromatin accessibility. Noise levels are minimal in murine hematopoietic stem cells (HSCs) and increase during differentiation and ageing. Differential noise identifies myeloid-biased Dlk1+ long-term HSCs in aged mice with enhanced quiescence and self-renewal capacity. VarID2 reveals noise dynamics invisible to conventional single-cell transcriptome analysis

Transcriptome-wide Profiling and Posttranscriptional Analysis of Hematopoietic Stem/Progenitor Cell Differentiation toward Myeloid Commitment

Hematopoietic stem cells possess lifelong self-renewal activity and generate multipotent progenitors that differentiate into lineage-committed and subsequently mature cells. We present a comparative transcriptome analysis of ex vivo isolated mouse multipotent hematopoietic stem/progenitor cells (LinnegSCA-1+c-KIT+) and myeloid committed precursors (LinnegSCA-1negc-KIT+). Our data display dynamic transcriptional networks and identify a stem/progenitor gene expression pattern that is characterized by cell adhesion and immune response components including kallikrein-related proteases. We identify 498 expressed lncRNAs, which are potential regulators of multipotency or lineage commitment. By integrating these transcriptome with our recently reported proteome data, we found evidence for posttranscriptional regulation of processes including metabolism and response to oxidative stress. Finally, our study identifies a high number of genes with transcript isoform regulation upon lineage commitment. This in-depth molecular analysis outlines the enormous complexity of expressed coding and noncoding RNAs and posttranscriptional regulation during the early differentiation steps of hematopoietic stem cells toward the myeloid lineage