Platelet Depletion Causes Rapid But Transient Activation Of Mouse Bone Marrow Long-Term Hematopoietic Stem Cells To Participate In Stress Thrombopoiesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2436-2436
Author(s):  
Lijian Shao ◽  
Di Hou ◽  
Wei Feng ◽  
Jianhui Chang ◽  
Jerry Ware ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Blood Platelet ◽  
Small Population ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Platelet Depletion

Abstract Hematopoietic stem cells (HSCs) are responsible for the production of various lineages of blood cells throughout life. To ensure the longevity of HSCs and prevent their premature exhaustion, a small population of HSCs termed long-term HSCs (LT-HSCs) is maintained in quiescence under homeostatic conditions. Whether LT-HSCs can exit quiescence rapidly in response to hematopoietic stress to replenish blood cells is not known, and thus was investigated in the present study in an acute but transient platelet depletion mouse model induced by intravenous injection of anti-GPIbα antibody. The results from our study show that in response to platelet depletion LT-HSCs can exit quiescence promptly and then proliferate rapidly to participate in stress thrombopoiesis probably via an alternative differentiation pathway. The mechanism by which platelet depletion causes LT-HSC activation is not due to a direct effect of the antibody on LT-HSCs or activation of platelets, but is attributed to a transient reduction of thrombopoietin (TPO) resulting from the acute depletion of platelets. However, the activated LT-HSCs return to quiescence when blood platelet counts are almost back to normal levels without compromising their function. These findings suggest that there is a very efficient and sensitive feedback regulatory circuitry between quiescent LT-HSCs and platelets, which allows LT-HSCs to directly and promptly respond to hematopoietic stress resulting from an acute loss of platelets. In this response, TPO may function as a switch that can rapidly turn on LT-HSCs to participate in stress thrombopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 74-83 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Marker Gene ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

Mesenchymal Stem Cells from the Wharton’s Jelly of Umbilical Cord Segments Support the Maintenance of Long Term Culture-Initiating Cells from Cord Blood.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3650-3650
Author(s):  
Kent W. Christopherson ◽  
Tiki Bakhshi ◽  
Shamanique Bodie ◽  
Shannon Kidd ◽  
Ryan Zabriskie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Blood Cells ◽  
Hematopoietic Stem ◽  
Cord Blood Cells

Abstract Hematopoietic Stem Cells (HSC) are routinely obtained from bone marrow, mobilized peripheral blood, and umbilical Cord Blood. Traditionally, adult bone marrow has been utilized as a source of Mesenchymal Stem Cells (MSC). Bone marrow derived MSC (BM-MSC) have previously been shown to maintain the growth of HSC obtained from cord blood and have been utilized for cord blood expansion purposes. However, the use of a mismatched BM-MSC feeder stromal layer to support the long term culture of cord blood HSC is not ideal for transplant purposes. The isolation of MSC from a novel source, the Wharton’s Jelly of Umbilical Cord segments, was recently reported (Romanov Y, et al. Stem Cells.2003; 21: 105–110) (Lee O, et al. Blood.2004; 103: 1669–1675). We therefore hypothesized that Umbilical Cord derived MSC (UC-MSC) have the ability to support the long term growth of cord blood derived HSC similar to that previously reported for BM-MSC. To test this hypothesis, MSC were isolated from the Wharton’s Jelly of Umbilical Cord segments and defined morphologically and by cell surface markers. UC-MSC were then tested for their ability to support the growth of pooled CD34+ cord blood cells in long term culture - initiating cell (LTC-IC) assays as compared to BM-MSC. We observed that like BM-MSC, CB-MSC express a defined set of cell surface markers. By flow cytometry we determined that that both UC-MSC and BM-MSC are positive for CD29, CD44, CD73, CD90, CD105, CD166, HLA-A and negative for CD45, CD34, CD38, CD117, HLA-DR expression. Utilizing Mitomycin C treated (200 μM, 15 min.) UC-MSC from multiple donors as a feeder layer we observed that UC-MSC have the ability to support the maintenance of long term hematopoiesis during the LTC-IC assay. Specifically, UC-MSC isolated from separate umbilical cord donors support the growth of 69.6±11.9 (1A), 31.7±3.9 (2B), 67.0±13.5 (3A), and 38.5±13.7 (3B) colony forming cells (CFC) per 1×104 CD34+ cord blood cells as compared to 64.0±4.2 CFC per 1×104 CD34+ cord blood cells supported by BM-MSC (Mean±SEM, N=4 separate segments from three different donors). Thus, Umbilical Cord derived Mesenchymal Stem Cells, a recently described novel source of MSC, have the ability to support long term maintenance of Hematopoietic Stem Cells, as defined by the LTC-IC assay. These results may have potential therapeutic application with respect to ex vivo stem cell expansion of Cord Blood Hematopoietic Stem Cells utilizing a Mesenchymal Stem Cell stromal layer. In addition, these data suggest the possibility of co-transplantation of matched Mesenchymal and Hematopoietic Stem Cells from the same umbilical cord and cord blood donor respectively. Lastly, these results describe a novel model system for the future study of the interaction between Cord Blood Hematopoietic Stem Cells and the appropriate supportive microenvironment represented by the Umbilical Cord - Mesenchymal Stem Cells.

Stability of Self-Renewal in Hematopoietic Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-42-SCI-42
Author(s):  
Norman N. Iscove
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Single Cells ◽  
Gene Expression Profiles ◽  
Short Term ◽  
Blood Leukocytes ◽  
Hematopoietic Stem

Abstract Abstract SCI-42 For many years a distinction was drawn between prospectively separable murine HSC populations with long-term, essentially permanent reconstituting potential (LT-HSC), versus HSC populations yielding short-term engraftment lasting only 4 – 6 weeks after transplantation (ST-HSC). Recent work based on transplantation of single cells shows that highly purified populations of LT-HSC prepared by standard sorting parameters consist in fact predominantly of a distinct, newly recognized class of intermediate- term reconstituting cells (IT-HSC) whose grafts endure longer than short-term HSC but also eventually fail (1). IT-HSC are separable from long-term reconstituting cells on the basis of expression of more alpha2 integrin and less SLAM150. Crucial to recognition of the distinction between LT- and IT-HSC are the endpoints used to evaluate reconstitution. If blood erythroid or myeloid reconstitution is measured, IT reconstitution is readily distinguished by the disappearance of these elements by 16 wk post-transplant. If instead reconstitution is measured simply by presence of blood leukocytes of donor origin, which in the mouse are almost entirely lymphocytes, the distinction is not made because lymphoid elements persist even in fading IT clones to 24 wk or beyond. The observations imply a need for reinterpretation of most of the published descriptions of the biology and gene expression profiles previously attributed to LT-HSC but in fact derived from analysis of populations that consisted mainly of IT-HSC. The capacity now to separate LT- from IT-HSC creates new opportunities for probing the mechanisms that specify and sustain long term function in the former but not the latter. 1. Benveniste P, Frelin C, Janmohamed S, Barbara M, Herrington R, Hyam D, Iscove NN. Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell. 2010;6:48–58 Disclosures: No relevant conflicts of interest to declare.

scholarly journals Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3704-3704
Author(s):  
Aldona A Karaczyn ◽  
Edward Jachimowicz ◽  
Jaspreet S Kohli ◽  
Pradeep Sathyanarayana
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Quiescent State ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

Donor-Cell Derived Aplastic Anemia (AA) with a Small Population of CD55−CD59− Blood Cells after Allogeneic Stem Cell Transplantation: Evidence for the Immune System Attack Against Normal Hematopoietic Stem Cells in AA.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3682-3682
Author(s):  
Chiharu Sugimori ◽  
Kanako Mochizuki ◽  
Hirohito Yamazaki ◽  
Shinji Nakao
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Immune System ◽  
Stem Cell Transplantation ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Donor Cell ◽  
Small Population ◽  
Hematopoietic Stem

Abstract Acquired aplastic anemia (AA) is thought to be caused by the immune system attack against hematopoietic stem cells. However, there is no direct evidence that an immune system attack against normal hematopoietic stem cells leads to development of AA. The immune system attack may be directed toward abnormal stem cells given the fact that some patients with myelodysplastic syndrome respond to immunosuppressive therapy. Although the presence of a small population of CD55−CD59− blood cells represents a reliable marker for the immune pathophysiology of AA, little is known regarding when and how such paroxysmal nocturnal hemoglobinuria (PNH)-type cells appear in patients with AA. The development of AA with a small population of PNH-type cells was recently observed in an allogeneic stem cell transplant (SCT) recipient. This patient, a 59-year-old male, who had been treated with allogeneic peripheral blood stem cell transplantation (PBSCT) from an HLA-compatible sibling for treatment of very severe AA in March 2002, developed severe pancytopenia in December 2005. Late graft failure (LGF) without residual recipient cells was diagnosed based on the results of a chimerism analysis. Sensitive flow cytometry failed to reveal any increase in the proportion of CD55−CD59− PNH-type blood cells. The patient underwent a second PBSCT from the original donor without preconditioning in February 2006. Although his pancytopenia was completely resolved by day 20, his blood counts gradually decreased from day 60 without any apparent complications. Flow cytometry revealed small populations of PNH-type granulocytes in his peripheral blood (Figure 1). Both the PNH-type and normal phenotype granulocytes were of donor origin. PIG-A gene analyses showed the PNH-type granulocytes in the patient to be a clonal stem cell with an insertion of thymine at position 593 (codon 198). Similar results were obtained from the sorted PNH-type granulocytes obtained 6 months later. The patient was treated with horse antithymocyte globulin and cyclosporine. The patient required no further transfusions after 88 days of the therapy and remains well as of August, 2007. The small population of PNH-type cells was not detectable in any of 50 SCT recipients showing stable engraftment or in an AA patient suffering graft rejection after a SCT. These findings suggest that some factors expressed by the patient induced an immune system attack against autologous hematopoietic cells, leading to de novo development of donor-cell derived AA. This is the first evidence that an immune system attack against normal hematopoietic stem cells results in AA associated with a clonal expansion of a PIG-A mutant which may originally be present in the donor bone marrow. Figure Figure

The Endothelial Antigen ESAM Marks Hematopoietic Stem Cells throughout Life

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 727-727 ◽  
Author(s):  
Takafumi Yokota ◽  
Kenji Oritani ◽  
Stefan Butz ◽  
Koichi Kokame ◽  
Paul W Kincade ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Side Population ◽  
Expression Profiles ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSC) are an important cell type with the capacity for self-renewal as well as differentiation into multi-lineage blood cells, maintaining the immune system throughout life. Many studies have attempted to identify unique markers associated with these extremely rare cells. In bone marrow of adult mice, the Lin-c-kitHi Sca1+ CD34−/Lo Thy1.1Lo subset is known to include HSC with long-term repopulating capacity. However, several of these parameters differ between strains of mice, change dramatically during developmental age and/or are expressed on many non-HSC during inflammation. Efficient HSC-based therapies and the emerging field of regenerative medicine will benefit from learning more about what defines stem cells. We previously determined that the most primitive cells with lymphopoietic potential first develop in the paraaortic splanchnopleura/aorta-gonad-mesonephros (AGM) region of embryos using Rag1/GFP knock-in mice. We also reported that Rag1/GFP-c-kitHi Sca1+ cells derived from E14.5 fetal liver (FL) reconstituted lympho-hematopoiesis in lethally irradiated adults, while Rag1/GFPLo c-kitHi Sca1+ cells transiently contributed to T and B lymphopoiesis. To extend those findings, microarray analyses were conducted to search for genes that characterize the initial transition of fetal HSC to primitive lymphopoietic cells. The comparisons involved mRNA from Rag1Lo ckitHi Sca1+, early lymphoid progenitors (ELP) and the HSC-enriched Rag1-ckitHi Sca1+ fraction isolated from E14.5 FL. While genes potentially related to early lymphopoiesis were discovered, our screen also identified genes whose expression seemed to correlate with HSC. Among those, endothelial cell-selective adhesion molecule (ESAM) attracted attention because of its conspicuous expression in the HSC fraction and sharp down-regulation on differentiation to ELP. ESAM was originally identified as an endothelial cell-specific protein, but expression on megakaryocytes and platelets was also reported (J. Biol. Chem., 2001, 2002). Flow cytometry analyses with anti-ESAM antibodies showed that the HSC-enriched Rag1-c-kitHi Sca1+ fraction could be subdivided into two on the basis of ESAM levels. The subpopulation with the high density of ESAM was enriched for c-kitHi Sca1Hi cells, while ones with negative or low levels of ESAM were found in the c-kitHi Sca1Lo subset. Among endothelial-related antigens on HSC, CD34 and CD31/PECAM1 were uniformly present on Rag1-c-kitHi Sca1+ cells in E14.5 FL and neither resolved into ESAMHi and ESAM−/Lo fractions. Expression profiles of Endoglin and Tie2 partially correlate with ESAM. The primitive ESAMHi fraction uniformly expressed high levels of Endoglin and Tie2, but many of the more differentiated ESAM−/Lo cells still retained the two markers. ESAM expression correlated well with HSC activity. Cells in the ESAMHi Rag1-ckitHi Sca1+ fraction formed more and larger colonies than those in the ESAM-/Lo Rag1-ckitHi Sca1+ fraction. Particularly, most CFU-Mix, primitive progenitors with both myeloid and erythroid potential, were found in the ESAMHi fraction. In limiting dilution stromal cell co-cultures, we found that 1 in 2.1 ESAMHi Rag1-ckitHi Sca1+ cells and 1 in 3.5 ESAM−/Lo Rag1-ckitHi Sca1+ cells gave rise to blood cells. However, while only 1 in 125 ESAM−/Lo Rag1-ckitHi Sca1+ cells were lymphopoietic under these conditions, 1 in 8 ESAMHi Rag1-ckitHi Sca1+ cells produced CD19+ B lineage cells. In long-term reconstituting assays, ESAMHi Rag1-ckitHi Sca1+ cells contributed highly to the multi-lineage recovery of lympho-hematopoiesis in recipients, but no chimerism was detected in mice transplanted with ESAM−/Lo Rag1-ckitHi Sca1+ cells. These results suggested that HSC in E14.5 FL are exclusively present in the ESAMHi fraction. Tie2+ c-kit+ lympho-hematopoietic cells of E10.5 AGM also expressed high levels of ESAM. Furthermore, ESAM expression in adult bone marrow was detected on primitive progenitors and cells in the side population within the Lin-ckitHi Sca1+ fraction. Interestingly, the expression was up-regulated in aged mice. Based on these observations, we conclude that ESAM marks HSC throughout life in mice. We also observed that many of human cord blood CD34+ CD38− cells express ESAM, suggesting potential application for the purification of human HSC.

Identification of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment and Self-Renewal.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 816-816
Author(s):  
Faiyaz Notta ◽  
Sergei Doulatov ◽  
John E. Dick
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Human Hscs ◽  
Insight Into

Abstract Abstract 816 A fundamental tenet that has guided our insight into the biology of hematopoietic stem cells (HSCs) over the past 50 years is the principle that an HSC can only be assayed by functional repopulation of an irradiated host1. In its strictest definition, only a HSC can provide long-term reconstitution of all the major lineages following single cell transplantation. However, the existing strategies for human HSC isolation lack quantitation and do not submit to this rigorous standard, thus precluding further biological analysis. Here, we report the prospective and quantitative analysis of human cord blood (CB) HSCs transplanted into female NOD/SCID/IL-2Rgcnull mice. We identify integrin a6 (CD49f) as a novel marker of cord blood (CB) HSCs and report that single Lin-CD34+CD38-CD90+CD45RA-RholoCD49fhi cells can reconstitute myeloid, B-, and T-cell lineages for 18 weeks. 5 of 29 mice transplanted with single cells gave rise to human cells indicating that approximately 20% of cells in this fraction are HSCs. This advance finally enables utilization of near-homogeneous populations of human HSCs to gain insight into their biology and to harness them for stem cell-based therapeutics. Disclosures: No relevant conflicts of interest to declare.

Noncanonical Wnt Signaling Maintains Hematopoietic Stem Cells in Different Zones

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 639-639
Author(s):  
Ryohichi Sugimura ◽  
Linheng Li
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Wnt Signaling ◽  
Conflicts Of Interest ◽  
Quiescent State ◽  
Perivascular Niche ◽  
Hematopoietic Stem ◽  
Knockout Mouse Model ◽  
Quiescent Hscs

Abstract Abstract 639 Hematopoietic stem cells (HSCs) are maintained in a balance between quiescent state and proliferating state. While proliferating HSCs are critical for supporting the routine blood production, quiescent HSCs are essential for long term maintenance and can also be activated to replenish lost proliferating or active HSCs. How the different states of HSCs are regulated is a fundamental question. Accumulated evidence supports a model that quiescent HSCs are located in the endosteal zone and active HSCs are in the perivascular zone. The underlying signaling to regulate the quiescence and activation in different niches remains largely unknown. To address this question, we have analyzed the expression profile of Wnt receptors, Frizzleds, in HSCs. We found that noncanonical Wnt signaling via receptor Frizzled8 (Fz8) and co-receptor Flamingo presents in and functionally maintains quiescent HSCs in the endosteal zone (Sugimura, et al., Cell 2012). However, it has not been clear whether and how active HSCs in the perivascular zone are regulated by Wnt signaling. Recently, we detected that another noncanonical Wnt receptor, Frizzled5 (Fz5), is expressed in metabolically active (indicated by Mitotracker) HSCs and also in Nestin-GFP+ mesenchymal stem cells (MSCs) in the perivascular zone of central marrow. Fz5 expresses neither in H2B-GFP label-retaining quiescent HSCs nor in endosteal cells and nor in sinusoidal cells as well. Using an Mx1-Cre:Fz5 knockout mouse model, we found a 60% decrease of HSCs isolated from central marrow, but no change in the number of HSCs isolated from endosteum. Functionally, hematopoietic reconstitution was not affected in the primary transplantation, but was substantially decreased (by 80%) in the secondary transplantation compared to the control. This indicates that Fz5 maintains HSCs in the perivascular zone. To examine the role of Fz5 in Nestin+ MSCs for HSC maintenance, we examined the Nestin-Cre:Fz5 model. We observed a large loss of CD49hiHSCs that were reported to represent intermediate (IT) HSCs. We further found a correlation of the quiescent vs. active HSCs respectively to long term (LT) HSCs vs. IT-HSCs with the latter population sensitive to 5FU treatment. Mechanistically we observed that Fz5 inactivation also led to a loss of Cdc42 polarity in the HSCs residing in the perivascular niche. The results suggest that Fz5-mediated noncanonical Wnt signaling regulates polarity of active HSCs in the perivascular zones. Future study is required to see whether the Fz5-Cdc42 mediated polarity in HSCs is associated with symmetric vs. asymmetric division. We propose that noncanonical Wnt signaling maintains quiescent and active HSCs reside respectively in the endosteal zone and the perivascular zone. In these zones, Fz8 and Fz5 are differentially expressed and mediate noncanonical Wnt signaling for HSC maintenance in the endosteal niche and to regulate active HSC action in the perivascular niche. Disclosures: No relevant conflicts of interest to declare.

Development of Multi-Lineage Hematopoiesis in Two Novel Zebrafish runx1 mutants

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2373-2373
Author(s):  
Erica Bresciani ◽  
Blake Carrington ◽  
Erika Mijin Kwon ◽  
Marypat Jones ◽  
Stephen Wincovitch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Time Lapse ◽  
Loss Of Function ◽  
Transcription Activator ◽  
Zebrafish Model ◽  
Hematopoietic Stem ◽  
Definitive Hematopoiesis

Abstract Long term hematopoietic stem cells are essential for the life-long maintenance of the hematopoietic system of an organism. The transcription factor RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during the embryo development. Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, we previously showed that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval "bloodless" phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish model, which requires verification in additional, independent models. In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated two new runx1 mutants, a deletion of 8 bp (runx1del8/del8) and a deletion of 25 bp (runx1del25/del25) within exon 4 of runx1, respectively, using the Transcription activator-like effector nucleases (TALENs) technology. These mutations cause frameshifts and premature terminations within the runt-homology domain,, resulting in loss of function of runx1 (runx1-/-). Both runx1del8/del8 and runx1del25/del25 mutant embryos had normal primitive hematopoiesis but failed to develop definitive hematopoiesis. Time-lapse recordings with confocal microscopy revealed that, indeed, there was no emergence of HSCs from the ventral wall of dorsal aorta in the runx1-/- embryos. The runx1-/- larvae gradually lost circulating primitive blood cells and became bloodless between 8 and 14 days post fertilization (dpf). However they gradually regained circulating blood cells between 15 and 20 dpf. Eventually, about 40% of runx1del8/del8 and runx1del25/del25 mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data is consistent with the previously described runx1W84X/W84X phenotype and supports the possibility of a runx1-independent mechanism for HSC formation and definitive hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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