Myosin-II Plays Central Roles In Cell Life and Death Decisions During Adult Hematopoiesis.

Abstract Abstract 1595 Hematopoietic stem cells (HSCs) are maintained in a quiescent state in a bone marrow niche, and become activated to give rise to different types of blood cells. The hierarchical design of hematopoiesis suggests that cell division-cycle machinery of each subpopulation could be differentially regulated. Our laboratory and others have previously demonstrated that non-muscle myosin-II is one of the major cytoskeletal proteins essential for cell fate decisions via adhesion to matrix and cytokinesis, both of which influence hematopoiesis. While myosin-II is essential for embryonic development, very little is known about its roles in regulating different stages of adult hematopoiesis. To test which stages of human hematopoiesis require myosin-II, bone marrow-derived CD34+ cells were cultured in serum-free medium with different combinations of cytokines followed by pharmacological inhibition of myosin over several cell cycles with blebbistatin. Multi-color flow cytometry analysis reveals that myosin inhibition leads to selective elimination of progenitor subpopulations via apoptotic cell death, while long-term HSC (LT-HSC) subpopulations remain viable. Transplantation of CD34+-derived cells treated with blebbistatin into immuno-deficient xenograft mice (NOD/Shi-scid/IL-2Rnull; NSG) shows normal LT-HSC engraftment comparable to untreated cells, producing both myeloid and lymphoid lineages. Colony-forming assays confirm dose-dependent reduction of multipotent, granulocyte-macrophage and erythroid progenitors by myosin inhibition. Under culture conditions that promote differentiation of megakaryocytic (MK) lineages, myosin inhibition produces a 3–10-fold increase of polyploid MKs in suspension cultures, whereas inhibition of other pathways that also affect contractility, including the Rho-kinase pathway, have lesser effects. Myosin-II inhibition also softens the cell cortex and enhances fragmentation upon aspiration into a micropipette. Such shear flow-induced fragmentation of distensible MK processes is known to be key to how MKs in the marrow shed platelets (plts) into permeating capillaries. To test whether myosin inhibition increases plt generation in vivo, human CD34+-derived cells treated with blebbistatin were transplanted intra-tibially in NSG mice. Transplantation of untreated cells produced detectable circulating human CD41+ plts as early as 8hrs with a peak at 48∼72hrs, followed by a complete decline at 2 wks. Blebbistatin-treated cells produce plts at a more sustained level with a slower rate of decline after a peak. Overall, myosin inhibition enhances the generation of circulating human plts per CD41+ cell transplanted: untreated cells yield 229 ± 114 plts/day whereas blebbistatin treatment yields 804 ± 256 plts/day. Plts from both treated and untreated human cells show activated adhesion on collagen assessed ex vivo. Together these data highlight a dual role for myosin-II in adult hematopoiesis in that: 1. Myosin-II is required for survival of hematopoietic myeloid progenitors 2. Myosin-II inhibition accelerates maturation of MKs and plt generation, while maintaining survival of LT-HSCs. Disclosures: No relevant conflicts of interest to declare.

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Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽

10.1182/blood.v120.21.1234.1234 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1234-1234

Author(s):

Robert S Welner ◽

Giovanni Amabile ◽

Deepak Bararia ◽

Philipp B. Staber ◽

Akos G. Czibere ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cells ◽

Quiescent State ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

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EZH2 Mutations Are Drivers of Clonal Hematopoiesis and Leukemic Transformation in a Mouse Model of Primary Myelofibrosis

Blood ◽

10.1182/blood.v124.21.3211.3211 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3211-3211

Author(s):

Ioanna Triviai ◽

Thomas Stuebig ◽

Anita Badbaran ◽

Silke Zeschke ◽

Victoria Panagiota ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Conflicts Of Interest ◽

Primary Myelofibrosis ◽

Bone Marrow Niche ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Neoplastic Stem Cells ◽

Calr Mutations

Abstract Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by aberrant myeloid differentiation, associated with disruption of the bone marrow niche with subsequent fibrosis development and a high risk of leukemic transformation. The phenotypical complexity observed in PMF likely reflects the heterogeneous mutation profile of the neoplastic stem cells driving the disease. In our former work, we identified a CD133+ hematopoietic stem / progenitor cell (HSPC) population from patient peripheral blood that can drive major PMF morbidity parameters in a xenotransplantation mouse model. Mutational analysis of the JAK2 locus at the single cell level within the CD133+ population showed highly variable levels of cells with a JAK2+/+, JAK2V617F/+, or JAK2V617F/V617F genotype, indicating that clonality is unlikely driven by JAK2 mutations. In two of these patient samples, and in a third patient sample with CALR-fs* mutations, we identified a high load of missense mutations in EZH2 (45 to 95%), suggesting they may be critical for the clonal expansion of the neoplastic stem cell compartment. EZH2 mutations are found in circa 7% of PMF patients and are correlated with poor prognosis. EZH2 is a critical enzymatic subunit of the Polycomb Repressor Complex 2, which initiates gene repression of select genes through its intrinsic activity for methylating lysine-27 of histone H3 (H3K27). To date, the exact contribution of EZH2 mutations to PMF evolution or AML transition has not been clarified. CD133+ HSPC carrying EZH2 mutations either with JAK2 or CALR mutations were transplanted into immunodeficient NOD-scid-gamma (NSG) mice. Mice engrafted with patient samples carrying either EZH2-Y633C and JAK2-V617F or EZH2-Y733* and CALR-fs* mutations showed a strikingly similar phenotype, including high human cell engraftment (10-20%), skewed myelopoiesis, dysplastic human megakaryocytes, splenomegaly, anemia, and fibrosis in either the BM or spleen. In the case of xenotransplanted mice receiving CD133+ cells with a low JAK2 burden and EZH2-D265H mutations, we observed the highest engraftment in our mouse model (62-95%) and in one case AML transition with >50% CD133+ human blasts in murine bone marrow. Notably, AML arose from a CD133+ EZH2D265H/+ cell that lacked JAK2V617Fmutation. We thus conclude that EZH2 mutations confer to CD133+ neoplastic stem cells a predisposition to clonal aberrant hematopoiesis; whereas acquisition of JAK2V617F or CALR mutations likely leads to the observed myeloproliferation and disruption of megakaryocytic and erythroid regulation . Moreover, our results demonstrate that epigenetic mutations (like EZH2D265) and not JAK2V617F are critical for AML transition. Our data underscore the importance of post-transcriptional modifiers of histones in altering the epigenetic landscape of neoplastic stem cells, whose clonal growth sustains aberrant myelopoiesis and expansion of pre-leukemic clones. Disclosures No relevant conflicts of interest to declare.

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The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2020-141149 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 33-33

Author(s):

Adedamola Elujoba-Bridenstine ◽

Lijian Shao ◽

Katherine Zink ◽

Laura Sanchez ◽

Kostandin V. Pajcini ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Receptor Expression ◽

Bone Marrow Niche ◽

Transplant Recipients ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Null Mutants

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells which differentiate to maintain and replenish blood lineages throughout life. Due to these characteristics, HSPC transplants represent a cure for patients with a variety of hematological disorders. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. The nervous system has been shown to indirectly influence hematopoiesis by innervating the niche; however, we present a direct route of HSPC regulation via expression of neurotransmitter receptors on HSPC surface. We have identified Gamma Aminobutyric acid (GABA) receptor B subunit 1 (Gabbr1), a hitherto unknown hematopoietic player, as a regulator of HSPC function. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation remains unknown. Based on published RNA-seq data (Nestorowa et al., Blood 2016), we discovered that Gabbr1 is expressed on a subset of HSPCs. We confirmed this expression using RT-qPCR to assay hematopoietic populations in the bone marrow (BM). Surface receptor expression analysis showed that Gabbr1 protein is expressed on a subset of BM HSPCs. To detect GABA, the ligand for Gabbr1 in the BM microenvironment, we utilized imaging mass spectrometry (IMS). We detected regionally specific GABA signal in the endosteal region of the BM. We further identified B cells as a cellular source of GABA in the BM. To understand the role of Gabbr1 in hematopoiesis, we generated CRISPR-Cas9 Gabbr1 null mutants on a C57/BL6 background suitable for hematopoietic studies and studied their hematopoietic phenotype. We discovered a decrease in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but the long-term hematopoietic stem cells (LT-HSCs) remain unaffected. Further analysis of peripheral blood of Gabbr1 null mutants showed decreased white blood cells due to reduced B220+ cells. This differentiation defect was confirmed in an in vitro differentiation assay where Gabbr1 null HSPCs displayed an impaired ability to produce B cells. We show that Gabbr1 null HSCs show diminished reconstitution ability when transplanted in a competitive setting. Reduced Gabbr1 null HSC reconstitution persisted in secondary transplant recipients indicating a cell autonomous role for Gabbr1 in regulating reconstitution of HSCs in transplant recipients. Our results show a crucial role for Gabbr1 in HSPC regulation and may translate to human health as a rare human SNP within the GABBR1 locus that correlates with altered leukocyte counts has been reported (Astle et al., Cell 2016). Our studies indicate an important role for Gabbr1 in HSPC reconstitution and differentiation into B cell lineages. Disclosures No relevant conflicts of interest to declare.

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Activation of Non-Canonical Immune Signalling Pathways Drives Marrow Failure in a Murine Model of MDS

Blood ◽

10.1182/blood.v124.21.3246.3246 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3246-3246

Author(s):

Rawa Ibrahim ◽

Joanna Wegrzyn ◽

Linda Ya-Ting Chang ◽

Patricia Umlandt ◽

Jeff Lam ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Line ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Signalling Pathway ◽

Gene Set Enrichment Analysis ◽

Mouse Bone Marrow ◽

Bone Marrow Niche ◽

Hematopoietic Stem

Abstract The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineages of the bone marrow, resulting in peripheral cytopenias and the propensity to progress to either acute myeloid leukemia (AML) or bone marrow failure (BMF). The most common cytogenetic aberration associated with MDS is deletion of the long arm of chromosome 5. Many of the molecular events involved in the development of del(5q) MDS have been elucidated including haploinsufficiency of the gene encoding the ribosomal protein RPS14, responsible for the anemia observed, and haploinsufficency of the miRNAs miR-145 and miR-146a, which together target the innate immune signaling pathway, specifically, the Toll-like receptor-4 (TLR-4)signalling pathway. It has been demonstrated that overexpression of a target of miR-146a,TRAF6, in mouse bone marrow can recapitulate the phenotype of del(5q) MDS including the cytopenias and progression to BMF or AML. However, enforced expression of TIRAP, a miR-145 target gene, results in rapid BMF independent of TRAF6. The molecular and cellular mechanisms responsible for the differential outcome of overexpression of two genes that act within the same signalling pathway remain to be fully understood. We have identified several differentially expressed cytokines, including interferon gamma (IFNγ) and interleukin-10 (IL-10), following TIRAP overexpression compared with TRAF6 overexpression. Promoter methylation analysis has shown hypermethylation of key adaptors and signal transducers that lie between TIRAP and TRAF6 in the TLR-4 signalling pathway, suggesting activation of different pathways by TIRAP and TRAF6 overexpression. Indeed, blockade of TRAF6 and MyD88 did not inhibit TIRAP induced expression of these cytokines, suggesting that IFNγ and IL-10 production occurs in a TRAF6 and MyD88 independent manner. We identified IFNγ as the critical effector cytokine responsible for TIRAP mediated marrow failure. Gene set enrichment analysis has shown an enrichment of an IFNγ signature in MDS patients with a low risk of transformation to AML compared to healthy controls. Furthermore, interferon signatures were highly enriched in MDS patients compared to patients with AML, suggesting an important role for IFNγ signaling in driving MDS progression toward marrow failure as opposed to leukemic progression. IFNγ has been shown to inhibit components of the bone marrow niche by blocking RANK signalling in stromal cells such as osteoclast progenitors. Using coculture of TIRAP expressing bone marrow cells with the RAW264.7 monocyte cell line, a cell line that is capable of differentiation into osteoclasts, we found an inhibition in the ability of these cells to form osteoclasts compared to control. This provides the first line of evidence suggesting that immune signalling defects arising from genetic perturbations in the hematopoietic stem cell compartment can result in stem cell niche dysfunction leading to marrow failure. Disclosures No relevant conflicts of interest to declare.

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PDK1 Controls the Differentiation of Hematopoietic Stem Cells Via Modulating ROS Levels

Blood ◽

10.1182/blood.v126.23.896.896 ◽

2015 ◽

Vol 126 (23) ◽

pp. 896-896

Author(s):

Tianyuan Hu ◽

Cong Li ◽

Le Wang ◽

Yingchi Zhang ◽

Luyun Peng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Conditional Knockout ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Counts

Abstract Hematopoietic stem cells (HSCs) exist as a rare population with two essential properties of self-renewal and differentiation. HSCs can give rise to all hematopoietic progenitor and mature cells. While critical for a full understanding of the hematopoietic process and HSC-related clinical applications, the mechanisms of self-renewal and differentiation of HSCs remain elusive. The PI3K-Akt signaling pathway plays essential roles in the regulation of hematopoiesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) activates multiple AGC kinases including Akt and is a pivotal regulator in this pathway. PDK1 phosphorylates Akt at its T308 residue and regulates the functional development of B and T cells during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. In this study, we generated PDK1 conditional knockout mice Vav-Cre;PDK1fl/fl (PDK1Δ/Δ) to explore the roles of PDK1 in HSCs. While PDK1Δ/Δ mice have reduced B and T cell counts as previously described, their LT-HSCs and ST-HSCs were significantly increased in comparison with WT mice while MPPs and CMPs were decreased after PDK1 deletion, indicating that the loss of PDK1 perturbed the steady-state hematopoiesis. Furthermore, although deletion of PDK1 increased the frequency of HSCs, PDK1-deficient HSCs fail to reconstitute the hematopoietic system when PDK1-deficient HSCs were used in bone marrow transplantation and competitive transplantation experiments in comparison to the WT HSCs, indicating that PDK1 is vital for hematopoiesis. To explore the mechanisms by which PDK1 regulates HSC function, we examined the cell cycle status and found the percentage of PDK1Δ/Δ HSCs was decreased significantly in G0 stage while increased in G1 and S/G2/M phases. This suggests an increase in HSC exit from a quiescent state. Since MPPs were significantly decreased in bone marrow, we examined the percentage of Annexin V+ DAPI- PDK1Δ/Δ and WT MPPs and found that they are comparable. This indicates that apoptosis did not cause the decrease in MPPs. In addition, a total of 300 LT-HSCs from PDK1Δ/Δ or WT mice and competitor cells were transplanted into lethally irradiated recipient mice to examine whether the decrease in MPPs is due to a defect in HSC differentiation. We found that less than 1% of MPPs arose from PDK1Δ/Δ HSCs 12 weeks after transplantation, indicating that PDK1 is required for the differentiation from LT-HSCs to MPPs. Because the full activation of Akt requires cooperative phosphorylation at its S473 and T308 residues by mTORC2 and PDK1, respectively, we also investigated the function of HSCs in RictorΔ/Δ PDK1Δ/Δ (DKO) mice in conjunction with RictorΔ/Δ or PDK1Δ/Δ mice to explore how mTORC2 and/or PDK1 influence Akt function in HSCs. The flow cytometric analyses of peripheral blood and bone marrow samples revealed very similar parameters of RictorΔ/Δ PDK1Δ/Δ and PDK1Δ/Δ mice. Interestingly, Rictor seemed to exert a minimal impact on HSCs and MPPs. More importantly, in contrast to RictorΔ/Δ, RictorΔ/Δ PDK1Δ/Δ HSCs failed to reconstitute the hematopoietic system after transplantation as PDK1Δ/Δ HSCs, suggesting that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. To explore the mechanism that leads to the defect in HSCs due to loss of PDK1, we assessed ROS levels in PDK1-deficient HSCs and found that PDK1-deficient LSKs and HSCs exhibit greatly reduced ROS levels when compared with the control HSCs. Treating PDK1-deficient BM cells with BSO in vitro increased cellular ROS levels and the colony counts of PDK1-deficient BM cells significantly. Notably, the recovery effect was only observed with BSO concentrations lower than 0.03 mM. This suggests that ROS levels are precisely controlled in HSCs. Higher or lower ROS levels beyond the normal range are both harmful to normal HSC functions. Since increased SDFα expression is associated with cellular ROS levels in various cells including hematopoietic cells, we also treated PDK1Δ/Δ mice with SDFα and found that it couldpartially rescue the defective differentiation ability of PDK1-deficient HSCs. In addition, we found that PDK1 deletion could significantly prolong the life span and inhibit the leukemia development in murine T-ALL model via altering leukemic cell differentiation and proliferation. Taken together, PDK1 controls HSC differentiation via regulating cellular ROS levels and regulates malignant hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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Selected factors influencing angiogenesis and hematopoietic niche

Acta Haematologica Polonica ◽

10.2478/ahp-2018-0018 ◽

2018 ◽

Vol 49 (3) ◽

pp. 112-120

Author(s):

Mateusz Nowicki ◽

Piotr Stelmach ◽

Anna Szmigielska-Kapłon

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Neoplastic Disease ◽

Quiescent State ◽

Bone Marrow Niche ◽

Multistage Process ◽

Hematopoietic Stem ◽

Factors Influencing ◽

Different Types ◽

Hematopoietic Niche

AbstractAngiogenesis is the vital, multistage process in which new blood vessels are created by sprouting from pre-existing vessels. It takes part in carcinogenesis and contributes to progression, metastases, and dissemination of neoplastic disease. In the bone marrow, angiogenesis influences the hematopoietic stem cells (HSC) proliferation, differentiation, and maintenance of normal hematopoiesis under both physiological and stress conditions. The bone marrow niche contains different types of cells, including macrophages, osteoblasts, mesenchymal stem cells, endothelial progenitors, and endothelial cells. All of these interact and form a unique microenvironment necessary for the appropriate function, and preservation of HSC in the quiescent state, and take a major part in the process of mobilization to peripheral blood and homing after transplantation. Cytokines active in the hematopoietic niche as well as miRNAs regulating hemato- poiesis, and angiogenesis have a significant influence on processes occurring in the bone marrow. The aim of this review was to present selected proteins, and molecules associated with angiogenesis as well as bone marrow niche processes: VEGF, ANGPT1, ANGPT2, MMP-9, SDF-1, miRNA-15a, miRNA-16, miRNA-126, miRNA-146a, and miRNA-223.

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MT1-MMP Regulates Hematopoiesis Through HIF-Mediated Chemo-/Cytokine Release From the Bone Marrow Niche,

Blood ◽

10.1182/blood.v118.21.3409.3409 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3409-3409

Author(s):

Chiemi Nishida ◽

Kaori Kusubata ◽

Yoshihiko Tashiro ◽

Ismael Gritli ◽

Aki Sato ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Cell Fate ◽

Conflicts Of Interest ◽

Stem Cell Differentiation ◽

Cell Phenotype ◽

Bone Marrow Niche ◽

Matrix Remodeling ◽

Hematopoietic Stem

Abstract Abstract 3409 Stem cells reside in a physical niche, a particular microenvironment. The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation, stem cell maintenance and regeneration. Various stem cell niches have been shown to be hypoxic, thereby maintaining the stem cell phenotype, e.g. for hematopoietic stem cells (HSCs) or cancer stem cells. The bone marrow (BM) niche is a rich reservoir for tissue-specific pluripotent HSCs. Proteases, such as matrix metalloproteinases (MMPs) can modulate stem cell fate due to their proteolytic or non-proteolytic functions (abilities). We have investigated the role of membrane-type1 matrix metalloproteinase (MT1-MMP), known for its role in pericellular matrix remodeling and cell migration, in hematopoiesis. MT1-MMP is highly expressed in HSCs and stromal cells. In MT1-MMP−/− mice, release of kit ligand (KitL), stromal cell derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo) and interleukin-7 were impaired resulting in erythroid, myeloid and T and B lymphoid differentiation. Addition of exogenous rec. KitL and rec. SDF-1 restored hematopoiesis in vivo and in vitro. Further mechanistic studies revealed that MT1-MMP in a non-proteolytic manner activates the HIF-1 pathway, thereby inducing the transcription of the HIF-responsive genes KitL, SDF-1 and Epo. These results suggested MT1-MMP as a critical regulator of postnatal hematopoiesis, which as a modulator of the HIF pathway alters critical hematopoietic niche factors necessary for terminal differentiation and or migration. Thus, our results indicate that MT1-MMP as a key molecular link between hypoxia and the regulation of vital HSC niche factors. Disclosures: No relevant conflicts of interest to declare.

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Functional Influence of CXCL12 Regulating miRNAs in Mesenchymal Stem Cells (MSCs).

Blood ◽

10.1182/blood.v120.21.2350.2350 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2350-2350

Author(s):

Laleh S. Arabanian ◽

Fernando Fierro ◽

David M. Poitz ◽

Ruth H. Strasser ◽

Martin Bornhaeuser ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Transforming Growth Factor ◽

Conflicts Of Interest ◽

Luciferase Assay ◽

Human Mscs ◽

Bone Marrow Niche ◽

Cxcl12 Expression ◽

Hematopoietic Stem

Abstract Abstract 2350 CXCL12 is a chemokine known to be critical for the regulation of the interaction between hematopoietic stem cells (HSCs) and their niche in the bone marrow, e.g. mesenchymal stem cells (MSCs). MicroRNAs (miRNAs) are post-transcriptional regulators recently shown to mediate a variety of cellular processes in the bone marrow niche. However, identification of specific miRNAs and their regulatory role in the crosstalk between HSCs and MSCs are still poorly understood. From a library of 470 miRNAs, 26 miRNAs were shown to downregulate the levels of CXCL12 in the supernatant of the human MSC line SCP-1. Eight of them (miR-23, 130b, 135, 200b, 200c, 216, 222, 602) were chosen for further investigation according to their significant interaction with the 3'UTR of CXCL12 as determined by luciferase assay. Among them, miR-23a,130 and 222 were expressed in 46 human primary MSCs, whereas the other miRs show negligible expression in resting MSCs. However, we observed, that MSCs that underwent adipogenic and osteogenic differentiation showed strongly decreased CXCL12 protein values early (day 5) and at later stages (day 14). The later drop in CXCL12 expression was clearly associated with an increased expression of miR-23a and miR-200. We furthermore tested a subset of stimuli (proinflammatory cytokines, cytotoxic drugs, chemokines) for their ability to modulate the described miRNAs. Amongst them, exclusively the application of transforming growth factor ß1 (TGF-ß1), resulted in the induction of miR-23a and at the same time reduction of CXCL12. The effect was counteracted by transfection of anti-miR-23 molecules. Taken together, we have shown for the first time that CXCL12-targeting miRNAs (in particular miR23a) have a significant potential to regulate the properties of the stem cell niche. Moreover, miR-23 is implicated in the signalling pathway of TGF-ß1 in human MSCs. Disclosures: No relevant conflicts of interest to declare.

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Murine Fetal Bone Marrow HSPCs Undergo a Dramatic Shift in Frequency at Birth

Blood ◽

10.1182/blood-2019-129073 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2471-2471

Author(s):

Trent Hall ◽

Pramika Sriram ◽

Shannon McKinney-Freeman

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Absolute Number ◽

Global Changes ◽

Bone Marrow Niche ◽

Mouse Development ◽

Hematopoietic Stem ◽

Fetal Bone ◽

And Function

Hematopoietic stem cells (HSCs) give rise to all cells of the hematopoietic system and are classically defined by their ability to stably engraft and reconstitute the blood system of ablated recipients after transplantation. The first transplantable HSCs arise from hemogenic endothelium at embryonic day 10.5 (E10.5) of mouse development and migrate to the fetal liver (FL), where they undergo a robust expansion followed by a second migration to the fetal bone marrow (FBM) at E15.5. The dynamics of hematopoiesis within the FBM has been largely unexplored. To gain a better understanding of FBM hematopoiesis, we catalogued the frequency, absolute numbers, phenotype and function of HSPCs in murine FBM from E15.5 through post-natal day 28 (P28). To avoid assumptions regarding HSPC location during fetal and neonatal development, we pooled bone marrow from the entire fetal skeleton for these studies. HSCs were rare in the FBM, ranging from 70-150 total HSCs at E15.5-E17.5, followed by a burst at E18.5 to 2,200 total HSCs. The frequency and absolute number of HSCs in the bone marrow steadily increased from E18.5 to P6, followed by a continual increase in the absolute number of HSCs from P6 to adulthood. This may reflect the dynamics of HSC cycling, or an influx or expansion of more differentiated progenitors in the fetal and neonatal bone marrow. We also found that the most prevalent hematopoietic stem and progenitor cell (HSPC) population within Lineage-Sca-1+c-Kit+ (LSK) cells in E15.5-E18.5 FBM was Flt3-CD48+CD150+ cells (MPP2). MPP2 cells, which are a megakaryocyte-biased multipotent progenitor population, comprised up to 75% of the LSK compartment at these time points, compared to 3% in adult bone marrow. The percentage of MPP2 cells dropped abruptly and dramatically right before birth (e.g. to 17% at E19) and continued to drop until adulthood. Transplantation of limiting numbers of MPP2 cells revealed that E16.5, E18.5, and P0 MPP2s displayed no repopulating potential, while adult MPP2s showed transient reconstitution of irradiated recipients. Fetal bone marrow MPPs displayed no colony potential in single-cell methylcellulose colony assays until E18.5 and P0, with a bias for erythroid-megakaryocyte colonies. Therefore, fetal bone marrow MPP2s are functionally distinct from their adult counterparts. Whole fetal bone marrow transplants showed that the first transplantable FBM HSCs appeared at E16.5, with up to 75% reconstitution in the peripheral blood (PB) of irradiated recipients. E16.5 FBM HSCs also displayed secondary transplantation activity, while E15.5 FBM HSCs displayed limited ability to reconstitute the PB of primary recipients. In sum, our studies reveal that until birth the predominant HSPC population in the fetal bone marrow is an immunophenotypic MPP2 that is functionally distinct from adult MPP2s, and that HSCs do not begin to accumulate in significant numbers until right before birth. These studies suggest the presence of key mechanisms during birth that influence the HSPC landscape of the fetal and neonatal bone marrow, and work is currently underway to systematically characterize global changes in the bone marrow niche during parturition. Disclosures No relevant conflicts of interest to declare.

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Cell Intrinsic and Extrinsic Mechanisms Mediate the Role of c-Myc Haploinsufficiency in Controlling HSC Fate

Blood ◽

10.1182/blood-2018-99-116975 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2561-2561

Author(s):

Yue Sheng ◽

Chunjie Yu ◽

Rui Ma ◽

Zhijian Qian

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

White Blood Count ◽

Bone Marrow Niche ◽

Severe Anemia ◽

Self Renewal ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract It has been shown that loss of c-Myc leads to accumulation of hematopoietic stem cells (HSCs) and severe cytopenia as a consequence of a blockage of HSC differentiation. Here we report a role of c-Myc haploinsufficiency in regulating HSC quiescence and self-renewal. We showed that c-Myc haploinsufficient mice displayed decreased white blood count and number of lymphocytes with normal myeloid cell differentiation. The number of HSCs and hematopoietic progenitor cells (HPCs) were all decreased significantly in c-Myc haploinsufficient mice as compared with control mice. We found that c-Myc haploinsufficiency inhibited HSC self-renewal capacity, increased proliferation and decreased quiescence of HSCs in vivo. By transplantation assays, we showed that c-Myc haploinsufficiency has extrinsic and intrinsic effects on the maintenance of HSCs in vivo. Our study suggests that loss of c-Myc activity and reduced dosage of c-Myc have distinct effects on HSC functions. c-Myc is a critical downstream mediator of the Wnt/b-catenin pathway. We showed that c-Myc haploinsufficiency is sufficient to prevent severe anemia in Apc heterozygous mice, and to significantly prolong the survival of Apc heterozygous mice. In addition, treatment of Apc haploinsufficient mice by a c-Myc inhibitor significantly reversed anemia in Apc-deficient mice. By transplantation assay, we further demonstrated that reduced expression of c-Myc in the bone marrow niche is responsible for prevention of severe anemia in Apc-deficient mice. However, we found that reduction of c-Myc by loss of a single allele of c-Myc did not rescue defective self-renewal capacity of Apc haploinsufficient HSCs. Taken together, our studies indicate that c-Myc mediates the function of the Wnt/b-catenin signaling pathway in bone marrow niche but not in HSCs. Disclosures No relevant conflicts of interest to declare.

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