Reversible Transplantable Chronic Phase CML-Like Disease in SCLtTA/BCR-ABL Transgenic Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1002-1002
Author(s):  
Mirle Schemionek ◽  
Jörg Stypmann ◽  
Sven Hermann ◽  
Christian Elling ◽  
Nicole Bäumer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Small Intestine ◽  
Stem Cell ◽  
Fdg Pet ◽  
Bone Marrow Cells ◽  
Chronic Phase ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Chronic myeloid leukemia (CML) is a disorder arising from the transformation of hematopoietic stem cells (HSC). Treatment with kinase inhibitors eradicates BCR-ABL positive progenitors but spares quiescent leukemic HSC (Copland et al. Blood 2006, Hu et al. PNAS 2006). The exact mechanism of this discrepancy is unknown. To better characterize the biology of CML stem cells in vivo, we have previously generated an inducible transgenic mouse model in which stem-cell specific expression of BCR-ABL leads to chronic phase CML-like disease. Here, we followed these mice non-invasively using positron emission tomography (FDG-PET) and abdominal high-resolution ultrasound. Moreover, we performed bone marrow transplants to analyze whether the disease is cell-autonomous and whether the phenotype of the disease is affected by the scheduling of BCR-ABL induction or the donor cell type. Splenomegaly was detectable as early as day 7 in induced double-transgenic SCLtTA/BCR-ABL mice, with an increase of the percentage of Gr-1+/Mac-1+ myeloid cells in spleen and bone marrow. Splenomegaly and myeloid cell proliferation progressed, and there was a close correlation between in vivo ultrasound measurements of the spleen and splenic weights upon autopsy. FDG-PET analysis demonstrated enhanced glucose uptake in the bone marrow suggestive of hyperproliferation. In addition, both FDG-PET and ultrasound revealed abnormalities of the small intestine, characterized by increased FDG uptake and distension of the intestinal wall. Upon autopsy, the small intestine showed an increased infiltration by granulocytic cells. These phenotypic changes were also evident in mice transplanted with cells from the bone marrow of double-transgenic sibling mice and were reversible upon tetracycline re-administration, demonstrating that this abnormality arises from bone marrow cells and is not due to expression of the oncogene outside of the hematopoietic system. We analyzed whether pre-transplant induction of BCR-ABL affected the repopulation potential of HSC or the disease phenotype. When recipient mice receiving unfractionated bone marrow cells from 3-week induced donor mice were compared with non-induced donors, there was no difference in the development of neutrophilia, myeloproliferation, or splenomegaly. However, when FACS-sorted LinnegSca-1+c-kit+ HSC were used as donor cells, the disease latency increased from 8 to 11 weeks post-transplant, and the increase of Gr-1+Mac-1+ cells in the spleen was less pronounced than in mice receiving unfractionated bone marrow. In conclusion, this model reliably and efficiently demonstrates transplantable reversible chronic phase CML-like disease and may thus be valuable for the in vivo analysis of CML stem cell biology and susceptibility to stem-cell directed anti-leukemic therapies.

scholarly journals Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1758-1763 ◽  
Author(s):  
T Nakano ◽  
N Waki ◽  
H Asai ◽  
Y Kitamura
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Electrophoretic Pattern ◽  
Phosphoglycerate Kinase ◽  
X Chromosomes ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.

scholarly journals Divisional Memory Drives Hematopoietic Stem Cell Functional Diversity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 20-20
Author(s):  
James Bartram ◽  
Baobao (Annie) Song ◽  
Juying Xu ◽  
Nathan Salomonis ◽  
H. Leighton Grimes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Functional Decline ◽  
Functional Heterogeneity ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Marrow Cells

Abstract Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We have shown that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state, and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by a doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSC with varying GFP intensity. HSC were separated into an H2B-labeled retention population and an H2B-labeled population. Interestingly, within the H2B-labeled retention population, HSC could be further subdivided into GFP high, medium, and low. MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium populations leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5). We examined the repopulation activity of each population in a serial competitive transplant assay. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts showed myeloid/lymphoid balanced engraftment whereas the G3 graft was myeloid-bias, suggesting that myeloid skewing can be acquired upon HSC division. We further examined lineage fate maps of bone marrow cells derived from G1 or G3 population in vivo, using single cell RNA sequencing, 10X genomics. Surprisingly, G3-derived bone marrow cells displayed a distinct myeloid cell trajectory from G1-derived bone marrow cells, in which G3 gave rise to increased immature neutrophils but fewer myeloid precursors. Remarkably, each lineage population derived from G3 donor cells had different gene expression signatures than those derived from G1 donor cells. Therefore, HSC that have divided in vivo in the same bone marrow microenvironment are intrinsically and molecularly different such that not only do they exhibit lineage potential differences but they also produce progeny that are transcriptionally different. These findings imply that cellular division rewires HSC and that this rewiring is passed down to their fully differentiated progeny. When G1 and G3 single HSC were cultured in-vitro, G1 had a slower entry into cell-cycle which has been associated with increased stemness. Additionally, when single HSC from G1 and G3 were assessed for their multipotency in a lineage differentiation assay, G1 HSC had a higher propensity to produce all four myeloid lineages (megakaryocytes, neutrophils, macrophages, and erythroid), further supporting increased stemness in G1 compared to G3 HSC. Finally, HSC from G1, G2, G3 and G4 populations carried mitochondria that were morphologically different, and express distinct levels of Sca-1, CD34 and EPCR, with Sca-1 high, CD34-, EPCR+ cells more enriched in G1. In summary, this study suggests that HSC transition into distinct metabolic and functional states with division history that may contribute to HSC diversity and functional heterogeneity. It also suggests the existence of a cell-autonomous mechanism that confers HSC divisional memory to actively drive HSC functional heterogeneity and aging. Disclosures No relevant conflicts of interest to declare.

Hematopoietic Stem Cell Engraftment in Bone Marrow Is Dependent upon Gsα.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 857-857
Author(s):  
Gregor B. Adams ◽  
Ian R. Alley ◽  
Karissa T. Chabner ◽  
Ung-il Chung ◽  
Emily S. Marsters ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Conditional Knockout ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

scholarly journals Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow.

1992 ◽  
Vol 175 (1) ◽  
pp. 175-184 ◽  
Author(s):  
N Uchida ◽  
I L Weissman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Stem Cell Activity ◽  
Marrow Cells

Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.

scholarly journals In vivo prostaglandin E2 treatment alters the bone marrow microenvironment and preferentially expands short-term hematopoietic stem cells

Blood ◽  
2009 ◽  
Vol 114 (19) ◽  
pp. 4054-4063 ◽  
Author(s):  
Benjamin J. Frisch ◽  
Rebecca L. Porter ◽  
Benjamin J. Gigliotti ◽  
Adam J. Olm-Shipman ◽  
Jonathan M. Weber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Prostaglandin E2 ◽  
Bone Marrow Cells ◽  
Negative Impact ◽  
Hematopoietic Progenitors ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Microenvironmental signals can determine hematopoietic stem cell (HSC) fate choices both directly and through stimulation of niche cells. In the bone marrow, prostaglandin E2 (PGE2) is known to affect both osteoblasts and osteoclasts, whereas in vitro it expands HSCs and affects differentiation of hematopoietic progenitors. We hypothesized that in vivo PGE2 treatment could expand HSCs through effects on both HSCs and their microenvironment. PGE2-treated mice had significantly decreased number of bone trabeculae, suggesting disruption of their microarchitecture. In addition, in vivo PGE2 increased lineage− Sca-1+ c-kit+ bone marrow cells without inhibiting their differentiation. However, detailed immunophenotyping demonstrated a PGE2-dependent increase in short-term HSCs/multipotent progenitors (ST-HSCs/MPPs) only. Bone marrow cells transplanted from PGE2 versus vehicle-treated donors had superior lymphomyeloid reconstitution, which ceased by 16 weeks, also suggesting that ST-HSCs were preferentially expanded. This was confirmed by serial transplantation studies. Thus in vivo PGE2 treatment, probably through a combination of direct and microenvironmental actions, preferentially expands ST-HSCs in the absence of marrow injury, with no negative impact on hematopoietic progenitors or long-term HSCs. These novel effects of PGE2 could be exploited clinically to increase donor ST-HSCs, which are highly proliferative and could accelerate hematopoietic recovery after stem cell transplantation.

scholarly journals Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 337-343 ◽  
Author(s):  
CA Corey ◽  
AD DeSilva ◽  
CA Holland ◽  
DA Williams
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Genetic Sequences ◽  
Marrow Cells

Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFRr, in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate- induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRr cDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.

High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1758-1758
Author(s):  
Axel Schambach ◽  
Bernhard Schiedlmeier ◽  
Jens Bohne ◽  
Dorothee von Laer ◽  
Geoff Margison ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hiv 1 ◽  
Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

scholarly journals Long-term in vivo expression of a murine adenosine deaminase gene in rhesus monkey hematopoietic cells of multiple lineages after retroviral mediated gene transfer into CD34+ bone marrow cells

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 1975-1980 ◽  
Author(s):  
DM Bodine ◽  
T Moritz ◽  
RE Donahue ◽  
BD Luskey ◽  
SW Kessler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Rhesus Monkey ◽  
Adenosine Deaminase ◽  
Bone Marrow Cells ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Marrow Cells

Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.

scholarly journals Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Michael Laupheimer ◽  
Anna Skorska ◽  
Jana Große ◽  
Gudrun Tiedemann ◽  
Gustav Steinhoff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Stem Cell Marker ◽  
Stem Cell Markers ◽  
Hematopoietic Stem ◽  
Spontaneous Migration ◽  
Marrow Cells

Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes.

Genistein Protects Hematopoietic Stem Cells Against G-CSF Induced DNA Damage

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3486-3486
Author(s):  
Liliana Souza ◽  
Erica Silva ◽  
Elissa Calloway ◽  
Michael Rossi ◽  
Omer Kucuk ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Oral Supplementation ◽  
Marrow Cells

Abstract Abstract 3486 Granulocyte colony-stimulating factor (G-CSF) is widely utilized in multiple clinical settings to lessen the effects of neutropenia. Although clearly beneficial, there are concerns about the long term effects of G-CSF. A particular concern is that G-CSF therapy may increase the risk of MDS and or AML. The most striking example is that of Severe Congenital Neutropenia (SCN). While G-CSF clearly improves survival, there are several lines of evidence to suggest that G-CSF treatment contributes to development of leukemia in these patients. First, the risk of leukemia appears to correlate with the cumulative dose of G-CSF. Second, of all the congenital marrow failure syndromes predisposed to AML, SCN alone does not appear to be a hematopoietic stem cell disorder. Since AML appears to rise from sequential mutations in hematopoietic stem cells, this would suggest that therapy, not the intrinsic cell defect is causal. It has been demonstrated that G-CSF does initiate signaling pathways in hematopoietic stem cell (HSC). We hypothesize that G-CSF induced excessive HSC proliferation can lead to DNA damage and genome instability. To test our premise, mice were treated with G-CSF for 4 months and bone marrow cells were analyzed. Our results demonstrated a 3 fold increase in linage negative, Sca positive and cKit positive (LSK) population and a 2 fold increase in the amount of DNA double strand breaks via the presence of nuclear pH2AX in the LSK population. To determine if the G-CSF induced proliferation lead to chromosome alterations, we performed array-comparative genomic hybridization analyses (CGH). DNA from lineage negative bone marrow cells from animals treated with G-CSF for 4 months were compared to untreated mice. Our results demonstrate variations in gains and losses of several chromosome regions. Fluorescence in situ hybridization (FISH) of Lin-Sca+ bone marrow cells confirmed loss on regions of chromosome 2 (6%) and 17 (30%). Since prolonged G-CSF exposure promotes genomic instability in HSCs we hypothesize that an alternative strategy would be to co-administer a drug that selectively blocks the effect of G-CSF on HSCs. Previous studies suggested genistein as an attractive compound. Genistein is a natural soy isoflavone with excellent bioavalibity that has anti-oxidant and anti-proliferative properties. In this study, we utilized a dose of genistein that can easily be obtained through oral supplementation. Mice were concomitant treated with G-CSF and genistein 3 times a week. Genistein partially blocked the G-CSF induced expansion of LSK cells and reduced pH2AX levels in this population by 40%. This was also accompanied by a reduction in LSK cells with an abnormal FISH signal (50% reduction). Importantly, genistein did not block the G-CSF driven expansion of mature neutrophils as total number of neutrophils in mice treated with G-CSF and genistein are the same as those treated with G-CSF alone. Our results suggest that genistein's effects are mediated primarily through inhibition of HSC proliferation. We demonstrate that G-CSF treatment induces GSK3β phosphorylation and Cyclin D1 and D3 expression. Genistein blocked GSK3β phosphorylation and Cyclin D1 and D3 induction. Inhibition of GSKβ3 has been demonstrated to delay HSC entry into cell cycle by promoting degradation of β-catenin, while HSCs from the triple cyclin knock out mouse (Cyclins D1, D2, and D3) display delayed cell cycle entry. Collectively, our results imply that prolonged G-CSF treatment induces DNA damage in HSCs by initiating cell cycle progression. HSCs are long lived, quiescent cells that preferentially utilize non-homologus end joining for DNA repair when progressing from G0 to G1. NHEJ is a relatively error prone DNA repair mechanism. Its preferential use by HSCs has been postulated as reason chromosomal deletions and translocations are often seen and many times are causal in the development of acute leukemia. Importantly, we demonstrate, that genistein, at levels obtainable through oral supplementation, is able to reduce DNA damage by attenuating G-CSF induced HSC proliferation without compromising G-CSFs ability to accelerate terminal neutrophilic differentiation. These results suggest that genistein may be an effective therapeutic agent in patients with SCN who require prolonged G-CSF support. Disclosures: No relevant conflicts of interest to declare.

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