G-CSF Induced Expansion and Mobilization of Hematopoietic Stem Cells Is Altered by the Matrix Protein Osteopontin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1400-1400
Author(s):  
Randolf Forkert ◽  
Yon Ko ◽  
Thomas Neuhaus ◽  
Elisabeth Gruenewald ◽  
Silke Schoeneborn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Stem Cell Niche ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Mobilization ◽  
Cell Niche

Abstract Stem cells reside in a physical microenvironment or niche where a balance of signals controls their proliferation, differentiation and death. Components of the specialized microenvironment have generally been defined in terms of cells and signaling pathways affecting stem cell maintenance or expansion. We have defined a role for a matrix glycoprotein that provides a constraining function on hematopoietic stem cells within the bone marrow microenvironment. Osteopontin (OPN) is an abundant glycoprotein in bone that modifies primitive hematopoietic cell number and function in a stem cell non-autonomous manner. Here we analyzed the role of OPN for regulating stem cell mobilization and pool size in times of G-CSF induced marrow stress, a context close to the clinical setting of stem cell mobilization not well understood so far. Bone marrow stromal cells show an enhanced expression of OPN under stimulation with G-CSF, which prompted us to analyze the role of OPN in G-CSF mediated activation of the stem cell niche. First we treated OPN deficient mice and their wild-type littermates with G-CSF for 5 days. We could observe a significant increased stem cell fraction in the peripheral blood and in the bone marrow in the absence of OPN in comparison to the wild-type controls. To evaluate, if this effect is stroma dependent, we first transplanted wild-type bone marrow into wild-type or OPN-deficient recipients. 6 weeks after transplantation we treated these mice with G-CSF for 5 days and analyzed the peripheral blood and the bone marrow for the contents of primitive hematopoietic cells. Here we could detect a significantly increased stem cell fraction in peripheral blood and bone marrow of the OPN−/− recipients in comparison to wild type controls detected by FACS and functional in vitro stem cell assays. We then transplanted the stressed bone marrow in a competitive repopulation assay into wild-type recipients and observed a significant increase of CD45.2 cells from OPN−/− recipient mice up to 12 weeks after transplantation in comparison to wild-type controls, demonstrating an enhanced G-CSF induced expansion of hematopioetic stem cells in the OPN-deficient stem cell niche. Furthermore, we could observe an enhanced expression of Angiopoietin and N-Cadherin in OPN-deficient bone marrow stromal cells after stimulation with G-CSF in comparison to wild-type controls, supporting the stroma dependent expansion of stem cells in the absence of OPN in the G-CSF stimulated stem cell niche. Therefore, OPN is a restricting element of the stem cell niche limiting the size of the stem cell pool and may provide a dynamic mechanism by which excess stem cell expansion is prevented during times of niche stimulation. These findings may provide new insight into expansion and mobilization of hematopoietic stem cells by G-CSF mediated by components of the stem cell niche.

Does primary myelofibrosis involve a defective stem cell niche? From concept to evidence

Blood ◽  
2008 ◽  
Vol 112 (8) ◽  
pp. 3026-3035 ◽  
Author(s):  
Jean-Jacques Lataillade ◽  
Olivier Pierre-Louis ◽  
Hans Carl Hasselbalch ◽  
Georges Uzan ◽  
Claude Jasmin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Stem Cell Niche ◽  
Primary Myelofibrosis ◽  
Hematopoietic Stem ◽  
Stem Cell Proliferation ◽  
Cell Niche

Abstract Primary myelofibrosis (PMF) is the rarest and the most severe Philadelphia-negative chronic myeloproliferative syndrome. By associating a clonal proliferation and a mobilization of hematopoietic stem cells from bone marrow to spleen with profound alterations of the stroma, PMF is a remarkable model in which deregulation of the stem cell niche is of utmost importance for the disease development. This paper reviews key data suggesting that an imbalance between endosteal and vascular niches participates in the development of clonal stem cell proliferation. Mechanisms by which bone marrow niches are altered with ensuing mobilization and homing of neoplastic hematopoietic stem cells in new or reinitialized niches in the spleen and liver are examined. Differences between signals delivered by both endosteal and vascular niches in the bone marrow and spleen of patients as well as the responsiveness of PMF stem cells to their specific signals are discussed. A proposal for integrating a potential role for the JAK2 mutation in their altered sensitivity is made. A better understanding of the cross talk between stem cells and their niche should imply new therapeutic strategies targeting not only intrinsic defects in stem cell signaling but also regulatory hematopoietic niche–derived signals and, consequently, stem cell proliferation.

Hematopoietc Stem Cell Targeted Neonatal Gene Therapy Cures oc/oc Mice from Osteopetrosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 456-456
Author(s):  
Johan Richter ◽  
Maria Johansson ◽  
Teun J. de Vries ◽  
Mats Ehinger ◽  
Vince Everts ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Bovine Bone ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Infantile malignant osteopeterosis (IMO) is a progressive, rare autosomal recessive disorder affecting osteoclast function. 50% of the affected children have a mutation in the Tcirg1 gene coding for one subunit of an osteoclast specific proton pump, OC116. The non-resorbed dense, sclerotic bones cause symptoms including pancytopenia and progressive visual loss and ultimately death. So far, the only curative treatment is hematopoietic stem cell (HSC) transplantation. The oc/oc mouse has a mutation in the gene homologous to Tcirg1 giving rise to similar symptoms as in patients leading to death of the mice at the age of 3–4 weeks. We have previously shown that the oc/oc mouse can be successfully treated with neonatal transplantation of normal HSC leading to prolonged survival and reversal of osteopetrosis (M. Johansson et al., Exp. Hematology34;242, 2006). In the current study we set out to develop HSC directed gene therapy for osteopetrosis in the oc/oc mouse model. As the bone marrow compartment is severely reduced in the oc/oc mouse fetal liver (FL) cells depleted of Ter119+ erythroid cells were used as a source of hematopoietic stem cells. We first established that wild type Ter119 depleted FL cells marked with a GFP vector and transplanted to newborn oc/oc mice i.p. could correct the osteopetrotic phenotype just as was shown for fresh bone marrow cells previously. Subsequently, Ter119 depleted FL cells from oc/oc mice were transduced with a retroviral vector expressing OC116 and GFP. In vitro transduction efficiency was 60–85%. One-day-old oc/oc mice were irradiated (400cGy) and transplanted i.p. with the transduced FL cells (1–3.5x106). 7 out of 14 mice survived past the expected lifespan and had 8–53% GFP+ cells in the peripheral blood at 3, 6 and 12 weeks. Analysis of bone structure with X-ray and histopathology showed an improvement at 8 weeks and an almost normal structure at 18 weeks, indicating induction of osteoclast activity. In vitro culture of osteoclasts from bone marrow from transplanted animals on bovine bone slices showed GFP marked osteoclasts and bone resorption, albeit at lower levels than for wild type cells. In the oc/oc mouse there is a block in B-lymphopoiesis leading to a reduced number of B-lymphocytes in the peripheral blood. In treated mice a reversal of this deficiency was observed. In summary we have demonstrated that the osteoclast defect seen in oc/oc mice can be successfully corrected by neonatal transplantation of gene modified hematopoietic stem cells and that this can lead to long-term survival of treated mice. This represents a significant step towards the development of gene therapy for osteopetrosis.

scholarly journals Hemmule: A Novel Structure with the Properties of the Stem Cell Niche

2020 ◽  
Vol 21 (2) ◽  
pp. 539
Author(s):  
Vitaly Vodyanoy ◽  
Oleg Pustovyy ◽  
Ludmila Globa ◽  
Randy J. Kulesza ◽  
Iryna Sorokulova
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Niche ◽  
Hematopoietic Stem ◽  
Novel Structure ◽  
Hematopoietic Stem Cell Niches ◽  
Cell Niche ◽  
Stem Cell Niches ◽  
Rat Bone

Stem cells are nurtured and regulated by a specialized microenvironment known as stem cell niche. While the functions of the niches are well defined, their structure and location remain unclear. We have identified, in rat bone marrow, the seat of hematopoietic stem cells—extensively vascularized node-like compartments that fit the requirements for stem cell niche and that we called hemmules. Hemmules are round or oval structures of about one millimeter in diameter that are surrounded by a fine capsule, have afferent and efferent vessels, are filled with the extracellular matrix and mesenchymal, hematopoietic, endothelial stem cells, and contain cells of the megakaryocyte family, which are known for homeostatic quiescence and contribution to the bone marrow environment. We propose that hemmules are the long sought hematopoietic stem cell niches and that they are prototypical of stem cell niches in other organs.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 861-861 ◽  
Author(s):  
Viktor Janzen ◽  
Heather E. Fleming ◽  
Michael T. Waring ◽  
Craig D. Milne ◽  
David T. Scadden
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Caspase 3 ◽  
Brdu Incorporation ◽  
Hematopoietic Stem ◽  
Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

In Vivo Selection of Wild-Type Hematopoietic Stem Cells in a Murine Model of Fanconi Anemia

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 2151-2158 ◽  
Author(s):  
Kevin P. Battaile ◽  
Raynard L. Bateman ◽  
Derik Mortimer ◽  
Jean Mulcahy ◽  
R. Keaney Rathbun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Peripheral Blood ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Serial Transplantation

Fanconi anemia (FA) is an autosomal recessive disorder characterized by birth defects, increased incidence of malignancy, and progressive bone marrow failure. Bone marrow transplantation is therapeutic and, therefore, FA is a candidate disease for hematopoietic gene therapy. The frequent finding of somatic mosaicism in blood of FA patients has raised the question of whether wild-type bone marrow may have a selective growth advantage. To test this hypothesis, a cohort radio-ablated wild-type mice were transplanted with a 1:1 mixture of FA group C knockout (FACKO) and wild-type bone marrow. Analysis of peripheral blood at 1 month posttransplantation showed only a moderate advantage for wild-type cells, but upon serial transplantation, clear selection was observed. Next, a cohort of FACKO mice received a transplant of wild-type marrow cells without prior radio-ablation. No wild-type cells were detected in peripheral blood after transplantation, but a single injection of mitomycin C (MMC) resulted in an increase to greater than 25% of wild-type DNA. Serial transplantation showed that the selection occurred at the level of hematopoietic stem cells. No systemic side effects were observed. Our results show that in vivo selection for wild-type hematopoietic stem cells occurs in FA and that it is enhanced by MMC administration.

scholarly journals Niche Recycling through Division-Independent Egress of Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 79-79
Author(s):  
Agnieszka Czechowicz ◽  
Deepta Bhattacharya ◽  
Lisa Ooi ◽  
Derrick J Rossi ◽  
David Bryder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Normal Animal ◽  
Cell Types ◽  
Therapeutic Interventions ◽  
Wild Type ◽  
Advisory Committees ◽  
Hematopoietic Stem

Abstract Abstract 79 Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion. However, previous studies through unfractionated bone marrow transplantation experiments, have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning. The need for ablating host HSCs prior to transplantation to achieve high levels of donor HSC engraftment has been a hotly debated issue over the years, with a number of groups claiming efficient HSC replacement in the absence of prior cytoreductive conditioning of the host, while experimental and clinical studies from our group and others found little evidence for extensive HSC replacement in unconditioned recipients. We specifically examined the intrinsic behavior and replacement properties of HSCs rather than that of unfractionated bone marrow, which contains a number of different cell types that have been reported to influence engraftment and replacement, such as host-reactive T cells and stromal cells. In order to study the physiologic properties of peripheral blood HSCs, we isolated KLS CD27+ IL-7Ra- CD150+ CD34- cells from peripheral blood and were the first to our knowledge to identify these cells as peripheral blood HSCs. We calculated then, through cell surface phenotyping and transplantation of unfractionated blood, that up to 1-5% of the total pool of HSCs enter into the circulation each day. Bromodeoxyuridine (BrdU) feeding of 3, 6, 9 and 12 days demonstrated that HSCs in the bone marrow incorporate BrdU at the same rate as do HSCs in the peripheral blood, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches. Indeed, transplantation of over 10,000 purified HSCs, representing approximately 50% of the total number of HSCs in a normal animal, into unconditioned wild type mice led to the occupancy of 2-5% of the total number of appropriate niches by the donor HSCs, a similar percentage as was estimated for the fraction of HSCs that egress daily. Additionally, repetitive daily transplantations of small numbers of HSCs administered as new niches became available over the course of 7 days led to significantly higher levels of engraftment than did large single bolus transplantations of the same total number of HSCs. These data demonstrate that niche saturation following transplantation is transient and provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches. We, for the first time, have specifically assessed the number of available HSC niches in normal wild type animals and the rate of their emptying under steady-state conditions. Moreover, our study provides a model that is consistent with host HSC replacement following donor HSC transplantation in unconditioned recipients, yet is also consistent with data suggesting the existence of a physically discrete niche which effectively retains and regulates HSCs. Importantly these data suggest therapeutic interventions that capitalize upon physiological HSC egress, and allow for increased levels of HSC engraftment with non-myeloablative conditioning. Disclosures: Weissman: Affiliations that might be perceived to have biased this work are as follows: I.L.W. owns significant Amgen stock, cofounded and consulted for Systemix, is a cofounder and director of Stem Cells, Inc., and cofounded and is a director of Cellerant, Inc. Al: Employment, Research Funding; Affiliations that might be perceived to have biased this work are as follows: I.L.W. owns significant Amgen stock, cofounded and consulted for Systemix, is a cofounder and director of Stem Cells, Inc., and cofounded and is a director of Cellerant, Inc. Al: Consultancy; Affiliations that might be perceived to have biased this work are as follows: I.L.W. owns significant Amgen stock, cofounded and consulted for Systemix, is a cofounder and director of Stem Cells, Inc., and cofounded and is a director of Cellerant, Inc. Al: Equity Ownership; Affiliations that might be perceived to have biased this work are as follows: I.L.W. owns significant Amgen stock, cofounded and consulted for Systemix, is a cofounder and director of Stem Cells, Inc., and cofounded and is a director of Cellerant, Inc. Al: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Hemmule: A Novel Structure with the Properties of the Stem Cell Niche

2019 ◽  
Author(s):  
Vitaly Vodyanoy ◽  
Oleg Pustovyy ◽  
Ludmila Globa ◽  
Randy J Kulesza Jr ◽  
Iryna Sorokulova
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Niche ◽  
Hematopoietic Stem ◽  
Novel Structure ◽  
Hematopoietic Stem Cell Niches ◽  
Cell Niche ◽  
Stem Cell Niches ◽  
Rat Bone

Stem cells are nurtured and regulated by a specialized microenvironment known as stem cell niche. While the functions of the niches are well defined, their structure and location remain unclear. We have identified in rat bone marrow, the seat of hematopoietic stem cells, extensively vascularized node-like compartments that fit the requirements for stem cell niche and which we called hemmules. Hemmules are round or oval structures of about one millimeter in diameter that are surrounded by a fine capsule, have afferent and efferent vessels, are filled with the extracellular matrix and mesenchymal, hematopoietic, endothelial stem cells, and contain cells of the megakaryocyte family, which are known for homeostatic quiescence and contribution to the bone marrow environment. We propose that hemmules are the long sought hematopoietic stem cell niches and that they are prototypical of stem cell niches in other organs.

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