Studies of hematopoietic stem cells spared by 5-fluorouracil.

Mouse marrow cells were exposed to 5-fluorouracil (FU) either in vivo or in vitro and the effects on the hematopoietic stem cell compartment were studied. The drug was highly toxic to bone marrow cells including the spleen colony-forming unit (CFU-S) population. The small population of stem cells surviving FU, however, caused a different pattern of spleen colony growth when injected into lethally irradiated mice. Whereas numbers of spleen colonies caused by normal marrow cells remained constant during an 8-14 d period after transplantation, spleen colonies derived from FU-treated marrow cells increased by as much as 100-fold during this time. This effect on stem cells was dose dependent both in vitro and in vivo. When FU was given in vivo, the day 14/day 8 ratio of colonies was greatest 1 d after injection and, over the next 7 d, returned to a near-normal value, that is, unity. A number of studies have shown that the stem cell compartment is heterogeneous with respect to self-replicative capacity and developmental potential. An age structure for the stem cell compartment has been proposed wherein cells with a short mitotic history are more likely to self-replicate than they are to differentiate; hence they are more primitive. 'Older' stem cells with a longer mitotic history are, according to the hypothesis, more likely to differentiate. 5-fluorouracil may be toxic to the older stem cells and selectively spare the more primitive subpopulation. Although the surviving cells may not themselves be able to form spleen colonies, they may give rise to an older cohort of cells more likely to differentiate and form spleen colonies. It is the requisite developmental maturation within the stem cell compartment that may be responsible for the delay in appearance of spleen colonies derived from FU-treated marrow. Our results support this explanation and identify the locus of at least part of this activity as the bone marrow. We found that the FU-treated marrow did not cause an increase in spleen colony numbers between 8 and 14 d in hosts with a long-standing marrow aplasia, due to the incorporation of 89Sr into bone. I propose that the delayed spleen colony appearance in normal hosts is the result of developmental maturation of the primitive stem cell compartment that survives FU and is responsible for spleen colonies arising around day 14. This maturation, at least initially, occurs in the marrow and leads to the replenishment of the more differentiated CFU-S subsets ablated by FU, which are normally responsible for spleen colonies appearing earlier after transplantation.

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Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽

10.1182/blood.v122.21.2476.2476 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2476-2476

Author(s):

Kasia Mierzejewska ◽

Ewa Suszynska ◽

Sylwia Borkowska ◽

Malwina Suszynska ◽

Maja Maj ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Sex Hormones ◽

Peripheral Blood ◽

Hematopoietic Stem ◽

Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

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Stem Cell Factor Sall4 Regulates Normal and Malignant Hematopoiesis In an Isoform-Specific Manner

Blood ◽

10.1182/blood.v122.21.3728.3728 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3728-3728

Author(s):

Samuel Milanovich ◽

Jeremy Allred ◽

Jonathan Peterson ◽

Cary Stelloh ◽

Sridhar Rao

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem ◽

Empty Vector ◽

Bmi1 Expression ◽

Colony Forming Units ◽

Colony Forming Capacity

Abstract Stem cells play key roles in early normal development (e.g. embryonic stem cells (ESCs)), maintenance of adult organs (e.g. hematopoietic stem cells (HSCs)) and in some cancers (e.g. leukemia stem cells). To what degree these different types of stem cells rely upon shared versus distinct transcriptional programs remains controversial. Sall4 is a zinc finger transcription factor that exists in two distinct splice isoforms, Sall4a (long) and Sall4b (short). Sall4 has been implicated in embryonic, hematopoietic and malignant stem cell transcriptional regulation. Additionally, Sall4 has been proposed as a potential means of ex-vivo hematopoietic stem cell expansion prior to transplantation. Sall4 isoform-specific differences have been described in ESCs, with Sall4b shown to be critical for maintaining ESC “stemness”. Here we investigate the role of Sall4 isoforms in pediatric acute myeloid leukemia (AML) and murine hematopoiesis to unravel shared versus unique transcriptional programs across different stem cell types. Quantitative real time PCR shows that Sall4b is the predominant Sall4 isoform in murine HSCs and lin-, Sca1+, cKit+ (LSK) cells. Sall4b expression decreases in early lineage-committed progenitors, while Sall4a expression is minimal to absent across murine HSCs and progenitors. Next, we evaluated seven pediatric AML samples and found highly variable Sall4 expression across AML cases. All samples had measurable Sall4a and Sall4b; in 3/7 cases Sall4a and Sall4b expression was similar to that of ESCs, in the other 4 cases Sall4 expression was minimal (<3% of ESCs). To study overexpression of Sall4, we used a murine stem cell retrovirus system to express Sall4a or Sall4b. Bone marrow was harvested from C57/BL6 mice and lineage-committed cells were removed by magnetic column separation. Lineage-negative bone marrow was infected with either empty vector, Sall4a or Sall4b. Transduced bone marrow was then cultured in methylcellulose media to assess colony forming capacity and proliferation in vitro or transplanted in syngeneic mice to assess engraftment and hematopoietic reconstitution in vivo. Sall4a or Sall4b overexpression caused diminished colony forming capacity and cellular proliferation in vitro compared to bone marrow transduced with empty vector (Figure 1). In bone marrow transplant assays, all mice (4/4) transplanted with Sall4b-transduced bone marrow following lethal irradiation succumbed to bone marrow failure within 10 days of transplant. Transplantation of Sall4b-transduced bone marrow into sublethally irradiated mice failed to contribute to hematopoiesis as measured by peripheral blood leukocyte GFP expression (encoded by the viral vector). Together, this data shows that Sall4b-transduced hematopoietic cells fail to engraft and reconstitute hematopoiesis in vivo. We postulated that this phenotype might be mediated through the interaction of Sall4 with Bmi1. Bmi1 is a member of the polycomb complex necessary for normal hematopoiesis, and is known to be bound by Sall4. In preliminary experiments, we have found that overexpression of Sall4 leads to decreased Bmi1 expression at 48 hours post-infection compared to bone marrow infected with empty vector.Figure 1Lin- bone marrow expressing Sall4a, Sall4b or empty vector was cultured in methylcellulose; plates were flushed and replated out to three generations. Colony forming units were assessed (A) and viable cells were counted (B) after 7-10 days in culture.Figure 1. Lin- bone marrow expressing Sall4a, Sall4b or empty vector was cultured in methylcellulose; plates were flushed and replated out to three generations. Colony forming units were assessed (A) and viable cells were counted (B) after 7-10 days in culture. In conclusion, our data shows that Sall4b is expressed in murine hematopoietic stem cells and progenitors, suggesting that Sall4b but not Sall4a influences a hematopoietic cell fate. Additionally, Sall4 expression is variable in AML specimens, implicating a potential pathogenic role in some leukemias, while others are Sall4-independent. Lastly, Sall4 overexpression is associated with decreased expression of the critical hematopoietic gene Bmi1. Together this data suggests that hematopoiesis is dependent upon appropriately regulated Sall4 expression with alterations leading to impaired proliferation and self-renewal. These effects on hematopoiesis appear to be mediated at least in part through a dose-dependent effect on Bmi1 expression. Future studies will evaluate other genes targeted by Sall4 in hematopoiesis and leukemia to define Sall4-dependent gene signatures in normal versus malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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The kinetics of murine hematopoietic stem cells in vivo in response to prolonged increased mature blood cell production induced by granulocyte colony-stimulating factor

Blood ◽

10.1182/blood.v86.8.2986.2986 ◽

1995 ◽

Vol 86 (8) ◽

pp. 2986-2992 ◽

Cited By ~ 26

Author(s):

G de Haan ◽

B Dontje ◽

C Engel ◽

M Loeffler ◽

W Nijhof

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Blood Cells ◽

Granulocyte Colony Stimulating Factor ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Cell Numbers ◽

Stem Cell Compartment ◽

Stimulating Factor

Abstract Because of the complexity of appropriate stem cell assays, little information on the in vivo regulation of murine stem cell biology or stemmatopoiesis is available. It is unknown whether and how in vivo the primitive hematopoietic stem cell compartment is affected during a continued increased production of mature blood cells. In this study, we present data showing that prolonged (3 weeks) administration of granulocyte colony-stimulating factor (G-CSF), which is a major regulator of mature granulocyte production, has a substantial impact on both the size and the location of various stem cell subset pools in mice. We have used the novel cobblestone area forming cell (CAFC) assay to assess the effects of G-CSF on the stem cell compartment (CAFC days 7, 14, 21, and 28). In marrow, in which normally 99% of the total number of stem cells can be found, G-CSF induced a severe depletion of particularly the most primitive stem cells to 5% to 10% of normal values. The response after 7 days of G-CSF treatment was an increased amplification between CAFC day 14 and 7. However, this response occurred at the expense of the number of CAFC day 14. It is likely that the resulting gap of CAFC day 14 cell numbers was subsequently replenished from the more primitive CAFC day 21 and 28 compartments, because these cell numbers remained low during the entire treatment period. In the spleen, the number of stem cells increased, likely caused by a migration from the marrow via the blood, leading to an accumulation in the spleen. The increased number of stem cells in the spleen overcompensated for the loss in the marrow. When total body (marrow and spleen) stem cell numbers were calculated, it appeared that a continued increased production of mature granulocytes resulted in the establishment of a higher, new steady state of the stem cell compartment; most committed stem cells (CAFC day 7) were increased threefold, CAFC day 14 were increased 2.3-fold, CAFC-day 21 were increased 1.8-fold, and the most primitive stem cells evaluated, CAFC day 28, were not different from normal, although now 95% of these cells were located in the spleen. Four weeks after discontinuation of the G-CSF treatment, the stem cell reserve in the spleen had returned to a normal level, whereas stem cell numbers in marrow had recovered to values above normal. This study shows that the primitive stem cell compartment is seriously perturbed during an increased stimulation of the production of mature blood cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures

Blood ◽

10.1182/blood.v93.1.80 ◽

1999 ◽

Vol 93 (1) ◽

pp. 80-86 ◽

Cited By ~ 17

Author(s):

Shai Erlich ◽

Silvia R.P. Miranda ◽

Jan W.M. Visser ◽

Arie Dagan ◽

Shimon Gatt ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Progenitor Cells ◽

Fetal Liver ◽

Acid Sphingomyelinase ◽

Hematopoietic Stem

Abstract The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.

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Hematopoietic stem cell depletion by restorative growth factor regimens during repeated high-dose cyclophosphamide therapy [see comments]

Blood ◽

10.1182/blood.v80.1.77.bloodjournal80177 ◽

1992 ◽

Vol 80 (1) ◽

pp. 77-83 ◽

Cited By ~ 1

Author(s):

RL Hornung ◽

DL Longo

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Cell Depletion ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Gm Csf ◽

Stem Cell Compartment ◽

Marrow Cells

We studied the effects of six cycles of repeated cyclophosphamide (CTX) therapy followed by restorative therapy with either granulocyte- macrophage colony-stimulating factor (GM-CSF) or G-CSF on the hematopoietic stem cell compartment. Stem cell function was assessed by serially transferring bone marrow cells from CTX-CSF-treated mice into lethally irradiated recipient mice. Bone marrow cells from mice that initially received either G-CSF or GM-CSF after CTX therapy more rapidly lost the ability to repopulate the recipient lymphoid organs, showed a dramatic loss of hematopoietic progenitors, a more rapid loss of CFU-S capacity, and a 40% to 50% reduction in marrow repopulating ability (MRA). Interleukin-1 (IL-1) appeared to have little effect on the CTX-treated mice when used alone. However, when administered before the CTX-CSF regimen, IL-1 prevented the stem cell depletion as determined by CFU-C, CFU-S, and MRA through the serial transplantation procedures. These results support the hypothesis that repeated treatments with myelosuppressive drugs followed by stimulation with the CSFs may induce damage to the host stem cell compartment, and further suggest that pretreatment with IL-1 before CTX therapy may prevent this stem cell damage.

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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 ◽

10.1182/blood.v104.11.1758.1758 ◽

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.

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Leukemic Stem Cell Assessment in Remission Bone Marrow of Acute Myeloid Leukemia Patients Is a New Prognostic Parameter.

Blood ◽

10.1182/blood.v114.22.399.399 ◽

2009 ◽

Vol 114 (22) ◽

pp. 399-399 ◽

Cited By ~ 3

Author(s):

Monique Terwijn ◽

Angèle Kelder ◽

Arjo P Rutten ◽

Alexander N Snel ◽

Willemijn Scholten ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Prognostic Information ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Stem Cell Compartment ◽

Acute Myeloid

Abstract Abstract 399 In acute myeloid leukemia (AML), relapses originate from the outgrowth of therapy surviving leukemic blasts know as minimal residual disease (MRD). Accumulating evidence shows that leukemia initiating cells or leukemic stem cells (LSCs) are responsible for persistence and outgrowth of AML. Monitoring LSCs during and after therapy might thus offer accurate prognostic information. However, as LSCs and hematopoietic stem cells (HSCs) both reside within the immunophenotypically defined CD34+CD38- compartment, accurate discrimination between LSCs and HSCs is required. We previously showed that within the CD34+CD38- stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers (leukemia associated phenotype, LAP), including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen, Leukemia 2007, Blood 2007). In addition, we reported that flowcytometer light scatter properties add to even better detection of LSCs, allowing LSCs detection in AML cases lacking LAP (ASH abstract 1353, 2008). Using this gating strategy, we determined LSC frequency in 64 remission bone marrow samples of CD34+ AML patients. A stem cell compartment was defined as a minimum of 5 clustered CD34+CD38- events with a minimal analyzed number of 500,000 white blood cells. After first cycle of chemotherapy, high LSC frequency (>1 × 10-3) clearly predicted adverse relapse free survival (RFS, figure 1a). LSC frequency above cut-off led to a median RFS of 5 months (n=9), while patients with LSC frequency below cut-off (n=22) showed a significantly longer median RFS of >56 months (p=0.00003). In spite of the relatively low number of patients, again a high LSC frequency (>2 × 10-4) after the second cycle and after consolidation therapy predicted worse RFS: after second cycle, median RFS was 6 months (n=9) vs. >43 months for patients with LSC frequency below cut-off (p=0.004). After consolidation, these figures were 6 months (n=7) vs. >32 months (n=6, p=0.03). Although total blast MRD (leukemic blasts as % of WBC) is known to predict survival (N.Feller et al. Leukemia 2004), monitoring LSCs as compared to total blast MRD has two major advantages: the specificity is higher (van Rhenen et al. Leukemia 2007) and well-known LSC makers like CLL-1, CD96 and CD123 can in principle be used for LSC monitoring, but not for total blast MRD detection since these markers are also expressed on normal progenitor cells. On the other hand, LSCs constitute only a small fraction of all leukemic blasts and therefore monitoring total blast MRD may have the advantage of a higher sensitivity. We thus tested the hypothesis that even more accurate prognostic information could be obtained by combining LSC frequency with total blast MRD. Total blast MRD after first cycle was predictive for survival with borderline significance (p=0.08): a cut-off of 0.3% resulted in two patient groups with median RFS of 9 months vs. >56 months. Figure 1b shows the result of the combined data of LSC and MRD frequency after first cycle therapy. We used the terms LSC+ and MRD+ for cell frequencies above cut-off and LSC- and MRD- for those below cut-off. We could clearly identify that apart from LSC+/MRD+ patients, LSC+/MRD- patients too have very poor prognosis, while MRD+/LSC- patients show an adverse prognosis as compared to LSC-/MRD- patients. These results from the first study on the in vivo fate of LSCs during and after therapy, strongly support the hypothesis that in CD34+ AML the leukemia initiating capacity originates from the CD34+CD38- population and is important for tumor survival and outgrowth. These results show that LSC frequency might be superior in predicting prognosis of AML patients in CR as compared to MRD total blast frequency, while the combination of both may offer the most optimal parameter to guide future intervention therapies. This work was supported by Netherlands Cancer Foundation KWF. Disclosures: No relevant conflicts of interest to declare.

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

Bone Marrow Research ◽

10.1155/2014/182645 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 4

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.

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

Blood ◽

10.1182/blood.v110.11.1002.1002 ◽

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.

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Hematopoietic stem cells: effect of preirradiation, bleeding, and erythropoietin on thrombopoietic differentiation

Blood ◽

10.1182/blood.v49.2.253.253 ◽

1977 ◽

Vol 49 (2) ◽

pp. 253-261 ◽

Cited By ~ 4

Author(s):

R Goodman ◽

H Grate ◽

E Hannon ◽

S Hellman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Proliferative Activity ◽

Bone Marrow Cells ◽

Cell Compartment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Stem Cell Compartment ◽

Cell Assays

Abstract A method of measuring differentiation of stem cells towards platelets is described using syngeneic bone marrow injected into lethally irradiated mice. Fourteen days after such injection, the platelet counts are found to be proportional to the number of bone marrow cells injected and can be used as a measure of platelet progenitors. Perturbation of the milieu in which the transplanted marrow is placed by host preirradiation, bleeding, or erythropoietin administration leads to enhanced thrombopoiesis. It has been shown previously that similar perturbation favors erythropoiesis at the expense of granulopoiesis. The data from these and other experiments appear to be consistent, with a model of the stem cell compartment as a continuum with proliferative activity increasing as commitment is restricted. These functions vary inversely with the capacity for self-renewal. The various stem cell assays measure different ranges of stem cells, but overlap within this continuum.

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