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

Blood ◽  
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.

scholarly journals Molecular Modulation of Fetal Liver Hematopoietic Stem Cell Mobilization into Fetal Bone Marrow in Mice

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Huihong Zeng ◽  
Jiaoqi Cheng ◽  
Ying Fan ◽  
Yingying Luan ◽  
Juan Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Vascular Endothelial Cells ◽  
Fetal Liver ◽  
Bone Marrow Niche ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Bone

Development of hematopoietic stem cells is a complex process, which has been extensively investigated. Hematopoietic stem cells (HSCs) in mouse fetal liver are highly expanded to prepare for mobilization of HSCs into the fetal bone marrow. It is not completely known how the fetal liver niche regulates HSC expansion without loss of self-renewal ability. We reviewed current progress about the effects of fetal liver niche, chemokine, cytokine, and signaling pathways on HSC self-renewal, proliferation, and expansion. We discussed the molecular regulations of fetal HSC expansion in mouse and zebrafish. It is also unknown how HSCs from the fetal liver mobilize, circulate, and reside into the fetal bone marrow niche. We reviewed how extrinsic and intrinsic factors regulate mobilization of fetal liver HSCs into the fetal bone marrow, which provides tools to improve HSC engraftment efficiency during HSC transplantation. Understanding the regulation of fetal liver HSC mobilization into the fetal bone marrow will help us to design proper clinical therapeutic protocol for disease treatment like leukemia during pregnancy. We prospect that fetal cells, including hepatocytes and endothelial and hematopoietic cells, might regulate fetal liver HSC expansion. Components from vascular endothelial cells and bones might also modulate the lodging of fetal liver HSCs into the bone marrow. The current review holds great potential to deeply understand the molecular regulations of HSCs in the fetal liver and bone marrow in mammals, which will be helpful to efficiently expand HSCs in vitro.

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

Blood ◽  
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.

scholarly journals An Ex Vivo Bioengineered Human Liver Microenvironment to Support Hematopoietic Stem Cell Maintenance and Expansion

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-10
Author(s):  
Na Yoon Paik ◽  
Grace E. Brown ◽  
Lijian Shao ◽  
Kilian Sottoriva ◽  
James Hyun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Human Umbilical Cord ◽  
Hematopoietic Stem ◽  
Main Site

Over 17,000 people require bone marrow transplants annually, based on the US department of Health and Human Services (https://bloodcell.transplant.hrsa.gov). Despite its high therapeutic value in treatment of cancer and autoimmune disorders, transplant of hematopoietic stem cells (HSC) is limited by the lack of sufficient source material due primarily inadequate expansion of functional HSCs ex vivo. Hence, establishing a system to readily expand human umbilical cord blood or bone marrow HSCs in vitro would greatly support clinical efforts, and provide a readily available source of functional stem cells for transplantation. While the bone marrow is the main site of adult hematopoiesis, the fetal liver is the primary organ of hematopoiesis during embryonic development. The fetal liver is the main site of HSC expansion during hematopoietic development, furthermore the adult liver can also become a temporary extra-medullary site of hematopoiesis when the bone marrow is damaged. We have created a bioengineered micropatterned coculture (MPCC) system that consists of primary human hepatocytes (PHHs) islands surrounded and supported by 3T3-J2 mouse embryonic fibroblasts. Long-term establishment of stable PHH-MPCC allows us to culture and expand HSC in serum-free medium supplemented with pro-hematopoietic cytokines such as stem cell factor (SCF) and thrombopoietin (TPO). HSCs cultured on this PHH-MPCC microenvironment for two weeks expanded over 200-fold and formed tight clusters around the periphery of the PHH islands. These expanded cells also retained the expression of progenitor markers of Lin-, Sca1+, cKit+, as well as the long-term HSC phenotypic markers of CD48- and CD150+. In addition to the phenotypic analysis, the expanded cells were transplanted into lethally irradiated recipient mice to determine HSC functionality. The expanded cells from the PHH-MPCC microenvironment were able to provide multi-lineage reconstitution potential in primary and secondary transplants. With our bioengineered MPCC system, we further plan to scale up functional expansion of human HSC ex vivo and to better understand the mechanistic, cell-based niche factors that lead to maintenance and expansion HSC. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
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.

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

Blood ◽  
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.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1595-1595
Author(s):  
Jae-Won Shin ◽  
Kyle R Spinler ◽  
Joe Swift ◽  
Dennis E Discher
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Myosin Ii ◽  
Cytoskeletal Proteins ◽  
Cell Cortex ◽  
Quiescent State ◽  
Bone Marrow Niche ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions

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.

Ontogeny of Hematopoiesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-4-SCI-4
Author(s):  
Elaine Dzierzak
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Transcription Factors ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Developmental Time ◽  
Hematopoietic Cell ◽  
Conditional Knockout ◽  
Hematopoietic Stem ◽  
And Function

Abstract The current challenge in hematopoietic transplantation and regeneration therapies is acquiring and/or producing a reliable and plentiful source of hematopoietic stem cells (HSCs). Given that HSCs from bone marrow, peripheral, or umbilical cord blood undergo only limited/no expansion ex vivo, there is a high interest in understanding how the adult cohort of multipotent self-renewing HSCs are generated and expanded during embryonic development. The development of HSCs in vertebrate embryos begins in the major vasculature. HSCs are generated in a short window of developmental time starting at embryonic day E10.5 until E12 in the mouse embryo, and from gestational weeks four to six in the human embryo. The first HSCs, which are as potent as bone marrow HSCs in transplantation procedures, are generated in the aorta-gonad-mesonephros (AGM) region. HSCs are found in the major vasculature – aorta, vitelline artery, and umbilical artery – subsequent to the appearance of hematopoietic cell clusters closely associated with the lumenal walls of these vessels. The relationship of HSCs to these clusters and the identification of the precursors to HSCs have been recently established through genetic, phenotypic, and real-time imaging studies. Remarkably, HSCs and hematopoietic progenitors arise directly from a subset of endothelial cells (hemogenic endothelial cells) in a natural transdifferentiation event. They are made through a process called endothelial to hematopoietic cell transition (EHT). EHT and HSC generation is in part regulated through ventral-derived developmental signals and a group of pivotal (core) transcription factors, including Runx1 and Gata2. Conditional knockout strategies show that these transcription factors are required for the generation of vascular hematopoietic clusters and HSCs, suggesting a role in hematopoietic fate induction and/or cell expansion. Interestingly, whereas both Runx1 and Gata2 are required for HSC generation, only Gata2 remains essential in HSCs after their production. We are profiling hemogenic endothelial and HSCs by RNA sequencing so as to understand the complete genetic program that leads to generation of HSCs. These results will be discussed in the context of developmental signaling pathways (BMP4, Hedgehog, etc.) that appear to impact HSC generation and expansion, and the localized dynamic expression and function of Gata2 and Runx1 in vascular endothelial and hematopoietic cluster cells. Disclosures: No relevant conflicts of interest to declare.

Heterogeneity in the Making of Blood

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-27-SCI-27
Author(s):  
David T. Scadden
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Niche ◽  
Cell Selection ◽  
Hematopoietic Progenitor ◽  
Marrow Space ◽  
And Function ◽  
Osteolineage Cells

It is increasingly clear that the bone marrow is comprised of a heterogeneous complex of niches for hematopoietic cells, some for stem cells in the perivascular space and some for progenitors. We have used two approaches to define the role of specific cells in the marrow. First, single cell selection and characterization based on in vivo proximity to HSPC. This method has defined a subset of endosteal lining cells that can be immunophenotypically defined and isolated and reveals IL-18 as a regulator of hematopoietic progenitor quiescence. Second, candidate cell depletion that revealed mature osteolineage cells expressing osteocalcin as regulating the production of thymic emigrants through the expression of Dll4. Deletion of these cells reduces the number and function of T-biased lymphoid progenitors in the marrow space as well as thymic populations and mature T cells in the blood. These data suggest that specific niche subsets can be defined and through them, novel molecular regulators of HSPC function. The bone marrow niche is a heterogeneous composite of distinctive niches. Disclosures No relevant conflicts of interest to declare.

scholarly journals GATA-2 Plays Two Functionally Distinct Roles during the Ontogeny of Hematopoietic Stem Cells

2004 ◽  
Vol 200 (7) ◽  
pp. 871-882 ◽  
Author(s):  
Kam-Wing Ling ◽  
Katrin Ottersbach ◽  
Jan Piet van Hamburg ◽  
Aneta Oziemlak ◽  
Fong-Ying Tsai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Hematopoietic Stem Cells ◽  
Gene Dosage ◽  
Fetal Liver ◽  
Mouse Development ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Adult Bone

GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs), whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac, fetal liver, and adult bone marrow. However, HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also, cytotoxic drug-induced regeneration studies show a clear GATA-2 dose–related proliferation defect in adult bone marrow. Thus, GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow.

A Novel Nr4a1GFP Reporter System Reveals That Nr4a1 Expression Identifies Long-Term Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2339-2339
Author(s):  
Ruben Land ◽  
Trevor Barlowe ◽  
Shwetha Manjunath ◽  
Sophie Eiger ◽  
Matthew Gross ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Family Members ◽  
Reporter System ◽  
Hematopoietic Stem ◽  
And Function

Abstract Abstract 2339 Recent studies have highlighted the importance of the NR4A nuclear receptor family (Nur77 (Nr4a1), Nurr1 (Nr4a3), Nor1 (Nr4a2)) in the regulation of hematopoiesis. In murine models, NR4A gene deficiencies lead to aberrant proliferation of hematopoietic stem cells, and can lead to acute myeloid leukemia (AML). NR4A gene deficiencies also appear to be a feature in human AML cells. In order to better understand the pattern of expression and function of NR4A family members during normal hematopoiesis, we have developed a novel reporter mouse where the Nr4a1 promoter drives GFP expression (Nr4a1GFP). Our analyses reveal a hierarchy in Nr4a1 expression among bone marrow hematopoietic stem cells: long-term (LT) HSC's (CD150+CD48-LSKs) express the highest levels of Nr4a1GFP, more mature HSC's and multilineage progenitor populations (CD150+CD48+ and CD150-CD48+ LSKs) express intermediate levels, and common myeloid progenitors (CMLs, defined as Lin-c-kit+sca-1-) express no Nr4a1GFP. Interestingly, circulating LSK's in the spleen express Nr4a1GFP at higher levels than their bone marrow counterparts. In support of data suggesting that Nr4a family members regulate quiescence, we find that 1) all hematopoietic stem cells that remain in the bone marrow after acute (36h) 5-FU treatment express Nr4a1GFP, 2) Nr4a1GFP expression decreases among circulating splenic LSKs 48 hours after treatment with PolyI:C, and 3) Nr4a1GFP expression increases markedly when stem cells are cultured in vitro under conditions that promote quiescence. We will use this novel system to more directly address the role of Nr4a1 expression in hematopoiesis by evaluating the cell cycle status and defining the reconstitution potential of HSC's on the basis of their Nr4a1GFP expression. Disclosures: No relevant conflicts of interest to declare.

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