Pivotal Role for Gsk3 in Hematopoietic Stem Cell Homeostasis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1292-1292
Author(s):  
Jian Huang ◽  
Peter S. Klein
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Signaling Pathways ◽  
Hematopoietic Stem Cell ◽  
Pivotal Role ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Gsk3 Inhibitor

Abstract Abstract 1292 Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our recent study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a μ-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by μ-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. These findings identify unexpected functions for GSK3 in HSC homeostasis, suggest a therapeutic approach to expand HSCs in vivo using currently available medications that target GSK3 and mTOR, and provide a compelling explanation for the clinically prevalent hematopoietic effects of lithium, a widely prescribed GSK3 inhibitor. In the following study, we found that the combination of Gsk3 inhibitor and mTOR inhibitor can expand phenotypic HSCs in vivo and maintain functional HSC in ex vivo culture. This study will provide the basis for a new clinical approach to improve the efficiency of bone marrow transplantation. Disclosures: Klein: Follica: Consultancy.

Pleiotrophin Signaling Is Necessary and Sufficient for Hematopoietic Stem Cell Self-Renewal In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 404-404 ◽  
Author(s):  
Heather A Himburg ◽  
Pamela Daher ◽  
J. Lauren Russell ◽  
Phuong Doan ◽  
Mamle Quarmyne ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Signaling Pathways ◽  
Fold Increase ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Human Hscs ◽  
Necessary And Sufficient

Abstract Abstract 404 Several signaling pathways have been elucidated which regulate hematopoietic stem cell self-renewal, including the Notch, Wnt, HOX and BMP signaling pathways. However, several of these pathways (e.g. Notch, Wnt) may not be necessary for maintenance of HSCs in vivo. We recently demonstrated that treatment of murine and human HSCs with the heparin binding growth factor, pleiotrophin (PTN), was sufficient to induce self-renewal of murine and human HSCs in culture (Himburg, Nat Med, 2010). In order to determine if PTN signaling is necessary for HSC self renewal and normal hematopoiesis in vivo, we examined the bone marrow HSC content and hematopoietic profile of mice bearing a constitutive deletion of PTN (PTN−/− mice) as well as mice bearing constitutive deletion of the PTN receptor, receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) (courtesy of Dr. Gonzalo Herradon, Spain and Dr. Sheila Harroch, L'Institut Pasteur, Paris, FR). PTN−/− mice demonstrated no significant differences in total bone marrow (BM) cells or BM colony forming cells (CFCs) but had significantly decreased bone marrow CD34(-)c-kit(+)sca-1(+)lin(-) (34-KSL) cells compared to littermate controls which retained PTN (PTN+/+) mice (0.007% vs. 0.02%, p=0.03). Consistent with this phenotype, PTN−/− mice also contained 2–fold decreased CFU-S12 compared to control PTN+/+ mice (p= 0.003). PTN−/− mice also demonstrated an 11-fold reduction in long-term repopulating HSC content compared to PTN+/+ mice as measured via competitive repopulating assay (12 week CRU frequency: 1 in 6 cells vs. 1 in 66 cells). Taken together, these data demonstrate that PTN signaling is necessary for maintenance of the BM HSC pool in vivo. Since PTN is known to antagonize the phosphatase activity of RPTPβ/ζ, we hypothesized that deletion of RPTPβ/ζ would increase BM HSC self-renewal and result in expansion of the BM HSC pool in vivo. Consistent with this hypothesis, RPTPβ/ζ−/− mice displayed a 1.3-fold increase in total BM cells (p= 0.04), 1.8-fold increase in BM 34-KSL cells (p=0.03), 1.6-fold increase in BM CFCs (p= 0.002) and 1.6–fold increase in BM CFU-S (p< 0.0001). RPTPβ/ζ−/− mice also demonstrated 1.4–fold higher long-term repopulating capacity (12 weeks) following competitive repopulating assay compared to RPTPβ/ζ+/+ mice (Donor CD45.1+ cell engraftment: 4.2% vs. 1.5%). Interestingly, RPTPβ/ζ −/− mice had significantly increased PB white blood cell counts, hemoglobin and platelet counts compared to RPTPβ/ζ+/+ mice coupled with splenomegaly. The RPTPβ/ζ−/− mice also had significantly increased BM vascular density (via quantitative mouse endothelial cell antigen staining) compared to RPTPβ/ζ+/+ mice, suggesting that PTN/RPTPβ/ζ signaling may augment the HSC pool size directly and also indirectly via activation of the BM vascular niche. These results demonstrate that PTN signaling is necessary and sufficient for induction of HSC self-renewal in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

G-CSF Treatment Induces Hematopoietic Stem Cell Quiescence but Loss of Repopulating Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1206-1206
Author(s):  
Joshua N. Borgerding ◽  
Priya Gopalan ◽  
Matthew Christopher ◽  
Daniel C. Link ◽  
Laura G. Schuettpelz
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Inflammatory Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
A Cell

Abstract Abstract 1206 There is accumulating evidence that systemic signals, such as inflammatory cytokines, can affect hematopoietic stem cell (HSC) function. Granulocyte colony stimulating factor (G-CSF), the principal cytokine regulating granulopoiesis, is often induced in response to infection or inflammation. Additionally, G-CSF is the most commonly used agent for HSC mobilization prior to stem cell transplantation. Recently there has been a renewed interest in the use of “G-CSF primed bone marrow” for stem cell transplantation, so understanding the affect of G-CSF on bone marrow HSCs is clinically relevant. Because the G-CSF receptor is expressed on HSCs, and G-CSF creates biologically relevant modifications to the bone marrow microenvironment, we hypothesized that increased signaling through G-CSF may alter the repopulating and/or self-renewal properties of HSCs. Due to G-CSF's role as an HSC mobilizing agent, we predicted that the number of HSCs in the bone marrow would be reduced after 7 days of G-CSF treatment. Surprisingly, we observe that stem cell numbers markedly increase, regardless of which HSC-enriched population is analyzed. C-kit+lineage−sca+CD34− (KLS-34−), KLS CD41lowCD150+CD48− (KLS-SLAM), and KLS-SLAM CD34− increase by 6.97±2.25 fold, 1.79±0.29 fold, and 2.08±0.39 fold, respectively. To assess HSC repopulating activity, we conducted competitive bone marrow transplants. Donor mice were treated with or without G-CSF for 7 days, and bone marrow was transplanted in a 1:1 ratio with marrow from untreated competitors into lethally irradiated congenic recipients. Compared to untreated HSCs, we found that G-CSF treated cells have significantly impaired long-term repopulating and self-renewal activity in transplanted mice. In fact, on a per cell basis, the long-term repopulating activity of KLS-CD34− cells from G-CSF treated mice was reduced approximately 13 fold. The loss of repopulating activity per HSC was confirmed by transplanting purified HSCs. Homing experiments indicate that this loss of function is not caused by an inability to home from the peripheral blood to the bone marrow niche. As HSC quiescence has been positively associated with repopulating activity, we analyzed the cell cycle status over time of KLS-SLAM cells treated with G-CSF. This analysis revealed that after a brief period of enhanced cycling (69.8±5.0% G0 at baseline; down to 55.9±4.1% G0after 24 hours of G-CSF), treated cells become more quiescent (86.8±2.8% G0) than untreated HSCs. A similar increase in HSC quiescence was seen in KLS-34− cells. Thus our data show that G-CSF treatment is associated with HSC cycling alterations and function impairment. Because G-CSF is associated with modifications to the bone marrow microenvironment, and the microenvironment is known to regulate HSCs at steady state, we asked whether the G-CSF induced repopulating defect was due to a cell intrinsic or extrinsic (secondary to alterations in the microenvironment) mechanism. To do this, we repeated the competitive transplantation experiments using chimeric mice with a mixture of wild-type and G-CSF receptor knockout (Csf3r−/−) bone marrow cells. We find that only the repopulating activity of HSCs expressing the G-CSF receptor is affected by G-CSF, suggesting a cell-intrinsic mechanism. To identify targets of G-CSF signaling that may mediate loss of stem cell function, we performed RNA expression profiling of sorted KSL-SLAM cells from mice treated for 36 hours or seven days with or without G-CSF. The profiling data show that G-CSF treatment is associated with activation of inflammatory signaling in HSCs. Studies are in progress to test the hypothesis that activation of specific inflammatory signaling pathways mediates the inhibitory effect of G-CSF on HSC function. In summary, G-CSF signaling in HSCs, although associated with increased HSC quiescence, leads to a marked loss of long-term repopulating activity. These data suggest that long-term engraftment after transplantation of G-CSF-primed bone marrow may be reduced and requires careful follow-up. Disclosures: No relevant conflicts of interest to declare.

Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2340-2342 ◽  
Author(s):  
Stéphane J. C. Mancini ◽  
Ned Mantei ◽  
Alexis Dumortier ◽  
Ueli Suter ◽  
H. Robson MacDonald ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Notch Signaling ◽  
Hematopoietic Stem Cell ◽  
Stromal Cells ◽  
Essential Role ◽  
Self Renewal ◽  
Hematopoietic Stem

AbstractJagged1-mediated Notch signaling has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. Unexpectedly, we report here that inducible Cre-loxP–mediated inactivation of the Jagged1 gene in bone marrow progenitors and/or bone marrow (BM) stromal cells does not impair HSC self-renewal or differentiation in all blood lineages. Mice with simultaneous inactivation of Jagged1 and Notch1 in the BM compartment survived normally following a 5FU-based in vivo challenge. In addition, Notch1-deficient HSCs were able to reconstitute mice with inactivated Jagged1 in the BM stroma even under competitive conditions. In contrast to earlier reports, these data exclude an essential role for Jagged1-mediated Notch signaling during hematopoiesis.

CD26 Inhibitor Treated and CD26−/− Recipient Mice Exhibit Improved Transplant Efficiency as Quantitated by Increased Short-Term Homing and Long-Term Engraftment

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 361-361 ◽  
Author(s):  
Laura A. Paganessi ◽  
Stephanie A. Gregory ◽  
Henry C. Fung ◽  
Kent W. Christopherson
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Inhibitor Treatment

Abstract A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/ HPC) trafficking is believed to be critical for the development of methodologies to improve transplant efficiency and subsequently immune reconstitution during hematopoietic stem cell transplantation in the clinical setting. Through the use of CD26 inhibitors and CD26 deficient mice (CD26−/−), we have previously generated data in mice suggesting that suppression of CD26/DPPIV (dipeptidylpeptidase IV) enzymatic activity on the transplant donor cell population can be utilized as a method of increasing transplant efficiency (Christopherson, KW 2nd, et al, Science 2004. 305:1000–3). However, the clinical importance of the transplant recipient should not to be overlooked given the potential importance of the bone marrow microenvironment in regulating the transplant process. We therefore investigated here whether inhibition or loss of CD26 activity in recipient mice would have an effect on transplant efficiency utilizing an in vivo congenic mouse model of transplantation. The short-term homing and long-term engraftment of BoyJ donor cells (expressing CD45.1+) into lethally irradiated control C57BL/6, CD26 inhibitor (Diprotin A) treated C57BL/6, or CD26−/− mice (expressing CD45.2+) was monitored by flow cytometric analysis of the bone marrow and peripheral blood at 24 hours and 6 months post-transplant respectively. Twenty-four hours post-transplant of 20×106 BoyJ mononuclear cells, we observed 8.85±0.58%, 10.69±1.01%, and 12.45±1.33% donor derived Sca-1+lin− cells in the bone marrow of recipient mice for control, Diprotin A treated, and CD26−/− recipient mice respectively. As compared to control mice, this represents a 20.8% increase (p=0.01) with CD26 inhibitor treatment and a 40.7% increase (p£0.05) resulting from the use of a CD26−/− recipient in short-term homing (N=5 mice per group). Six months post-transplant of 1×105 BoyJ mononuclear cells, we observed 39.90± 4.38%, 70.22± 3.72%, and 92.51± 1.04% donor contribution to hematopoiesis in the peripheral blood of control, Diprotin A treated, and CD26−/− recipient mice respectively. This represents a 76.0% increase (p£0.01) with CD26 inhibitor treatment and a 131.9% increase (p£0.01) as a result of the CD26−/− recipient in long-term engraftment as compared to control recipient mice (N=14 mice per group). These results provide pre-clinical evidence of the importance of CD26 expression within the transplant recipient with regard to regulating hematopoietic stem cell homing and engraftment. Our results also support the potential use of CD26 inhibitors to treat transplant patients during hematopoietic stem cell transplantation as a method of improving transplant efficiency. Lastly, our use of inhibitor treated C57BL/6 and CD26−/− recipient mice, which are also on a C57BL/6 background, in conjunction with a congenic model of transplantation provides a accurate and convenient model system for the in vivo testing of the efficacy of existing and new CD26 inhibitors in transplant recipients.

A Functional In Vivo RNAi screen Involving Jumonji C Domain Containing Candidates Unravels Kdm5b As a Negative Modulator of Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 385-385
Author(s):  
Sonia Cellot ◽  
Kristin J Hope ◽  
Martin Sauvageau ◽  
Jalila Chagraoui ◽  
Eric Deneault ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Expansion ◽  
Rt Pcr ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem Cell Expansion ◽  
Time Point

Abstract Abstract 385 Epigenetic modifications influence chromatin accessibility, impacting on cell fate decisions, such as stem cell self-renewal and differentiation, in both normal and leukemic stem cells (LSC). To investigate the putative role of histone demethylases (HDM) in modulating primary hematopoietic stem cell (HSC) fate, an in vivo functional screen was performed, using an RNAi based strategy, involving 25 members of the Jumonji (JmjC) domain protein family. As a first step, expression profile studies of these gene candidates were undertaken. Transcripts of all these enzymes were detected in isolated HSC populations (frequency 1:2) from fetal liver (n=1) and bone marrow (n=2), except for Hairless. As compared to unsorted bone marrow (BM), stem cells harboured higher expression of Jarid1b (relative-fold enrichment (RQ) of 3.9±1.7), Jmjd2d (RQ3.8±1.9), and Jhdm1b (3.1±1.7). Next, 5shRNA were designed against each of the 25 JmjC containing proteins, and cloned into a retroviral LMP vector encoding GFP to permit tracking of transduced cells in vivo. HSC-enriched CD150+CD48−Lin−cells (∼60 LT-HSC) were infected over 5 days by co-culture with retroviral producer cells in an arrayed 96-well format, with one shRNA per well. Directly after infection, the in vivo reconstituting potential of ¼ of each well was evaluated through duplicate competitive repopulation assays involving the co-transplantation of 1.5 × 105 congenic BM competitor cells into irradiated recipients. The remaining cell fraction served to asses gene transfer by GFP epifluorescence measurements, and RNA isolated from sorted GFP+ cells was used to evaluate gene knockdown levels by Q-RT-PCR analysis. Blood reconstitution was evaluated at an early (4wks) and late time point (16–20wks), tracking the contribution of the donor CD45.1+ transduced (GFP+) cells to recipient hematopoiesis over time. As baseline references, sh-RNA to Luciferase (no effect) and the histone acetyl transferase Myst3 (stem cell loss) were used, as well as Hoxb4 over-expression (stem cell expansion). The primary screen, followed by validation experiments, unravelled one positive (Jhdm1f/Phf8) and two negative (Jarid1b, Hif1an) regulators of HSC activity. The strongest impact was seen with Jarid1b knockdown, and the resulting gain in HSC activity. As a confirmation step, cells were kept in culture for one week, to better contrast an increase in HSC activity, compared to control HSC. After 7 days in vitro, 1/8 equivalents of single well cultures were transplanted into 3 mice, and blood reconstitution levels serially assessed. Cells transduced with sh-RNA against Jarid1b contributed more significantly to host hematopoiesis than sh-RNA Luciferase transduced cells (58±16% vs 26±3% GFP), or Hoxb4 over-expressing cells (37±2% GFP), at comparable gene transfer rates, at the 16 week time point and beyond (3 independent experiments). Long-term HSC frequencies were evaluated from these cultures, and found to be 6–10 fold increased in shJari1d1b-cell cultures. In long-term recipients, differentiation potential of these cells was preserved, as evidenced by CD4+CD8+ thymic cells, B220+ splenic cells and CD11b+ bone marrow cells in the GFP positive contingent. Clonality studies on DNA isolated from these sorted populations confirmed oligoclonality of the stem cell expansion, and HSC pluripotency. There were no cases of leukemic transformation in all of the transplant recipients (n>30). As assessed by Q-RT-PCR, levels of HoxA5, HoxA9, HoxA10 and CxCl5 were increased in day7 sh3Jarib1b-cells (vs ctl), while the levels of the tumor suppressors Cav1, Sash1 and Egr1 were decreased. A more detailed assessment of the HoxA cluster revealed predominant expression of 5' cluster genes in expanding shJarib1b-cells, from HoxA5 to HoxA11, with a concomitant increase in the level of H3K4 tri-methylation, as assessed by ChIP-CHIP. In conclusion, HDM of the JmjC family can modulate HSC activity, both positively and negatively. These data suggest that the H3K4 demethylase Jarid1b (KDM5b) restrains stem cell self-renewal, acting as a co-repressor, possibly via epigenetic regulation of the HoxA gene cluster, among other target genes. This observation could be further exploited as an HSC expansion strategy. Disclosures: No relevant conflicts of interest to declare.

p16INK4a Is a Key Downstream Mediator of the Deleterious Effects of FoxO Deficiency on Maintenance of the Hematopoietic Stem Cell Compartment.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1405-1405
Author(s):  
Zuzana Tothova ◽  
Stephen M Sykes ◽  
Dena S Leeman ◽  
James W Horner ◽  
Norman Sharpless ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Accelerated Aging ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Downstream Mediator

Abstract Regulation of oxidative stress in the hematopoietic stem cell (HSC) compartment is critical for the maintenance of HSC self-renewal. A number of reports have previously implicated p16 in aging of HSCs, pancreatic β-islet cells and subventricular zone progenitors in the brain [1–3]. In the context of the hematopoietic system, p16INK4a expression in HSCs increases with age, and correlates with decreased HSC repopulating ability, decreased self-renewal, and increased apoptosis with stress [1]. We and others have recently reported that FoxO play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment [4, 5]. Young mice deficient in FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system, with striking similarity to aging wild-type mice, show a defect in bone marrow repopulating ability, decrease in self-renewal, myeloid skewing in differentiation and increased levels of apoptosis. Furthermore, young FoxO-deficient HSC show increased levels of p16 when compared to their wildtype counterparts. These collective findings suggested the possibility that FoxO loss could result in accelerated aging of HSC due to increased expression of p16 as a consequence of increased ROS. To test the hypothesis that p16 is one of the key mediators of FoxO loss responsible for accelerated aging of HSC, we deleted FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system of mice deficient in p16INK4a. Young mice deficient in FoxO and p16 shared the same characteristics of their HSC(Lin−Sca1+c-kit+) compartment as mice deficient in FoxO only, including decreased number of HSC, increased percentage of HSC entering S/G2/M and apoptosis, and increased levels of ROS as compared to their wildtype counterparts. However, in a setting of long-term repopulation studies, bone marrow isolated from mice deficient in p16 and FoxO demonstrated a rescue of long-term repopulation for up to 20 weeks, as compared to FoxO deficient bone marrow that showed a severe defect in long-term repopulation. p16 deficiency in the setting of FoxO deficiency did not result in reduction of ROS levels in the HSC compartment. Taken together, these findings indicate that p16 is a critical downstream mediator of FoxO in the maintenance of the HSC compartment, and that it can dissociate the detrimental effects of ROS on HSC self-renewal in a setting of FoxO deficiency.

Alcam Mediated Cellular Interaction Regulates Hematopoietic Stem Cell Repopulation and Self-Renewal

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1291-1291
Author(s):  
Robin Jeannet ◽  
Qi Cai ◽  
Hongjun Liu ◽  
Hieu Vu ◽  
Ya-Huei Kuo
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Surface ◽  
Hematopoietic Stem Cell ◽  
Cellular Interaction ◽  
Mrna Levels ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 1291 Alcam, which encodes the activated leukocyte cell adhesion molecule (CD166), is a cell surface immunoglobulin superfamily member mediating homophilic adhesion as well as heterotypic interactions with CD6. It has recently been shown that Alcam+ endosteal subset in the bone marrow contain hematopoietic niche cells able to support hematopoietic stem cell (HSC) activity. We examined Alcam mRNA levels and cell surface expression by quantitative RT-PCR and flow cytometry in various hematopoietic stem and progenitor subsets. We found that Alcam is highly expressed in long-term repopulating HSC (LT-HSC), multipotent progenitors (MPP), and granulocyte/macrophage progenitors (GMP). We use an Alcam null mouse allele to assess the function of Alcam in HSC differentiation and self-renewal. Clonogenic colony-forming progenitor serial-replating assay show that the replating potential of Alcam-deficient LT-HSCs is impaired. An in vitro single-cell differentiation assay of phenotypic LT-HSCs reveals that Alcam-deficiency leads to an enhanced granulocytic differentiation. In competitive repopulation transplantation, Alcam-deficient cells show a transient engraftment enhancement, however, the engraftment is significantly lower in secondary transplantation, suggesting that the self-renewal capacity of Alcam-deficient HSC is compromised. We performed a limiting-dilution transplantation assay and determined that the frequency of long-term repopulating cells in Alcam-deficient bone marrow is significantly reduced compared to wild type control. We further assessed the engraftment efficiency of phenotypically purified LT-HSCs. We show that the engraftment efficiency of Alcam-deleted LT-HSCs is significantly reduced compared to wild type LT-HSCs. Since Alcam-deleted HSCs are able to home efficiently to the bone marrow cavity, the engraftment defect is not due to inefficient homing upon transplantation. Collectively, These studies implicate Alcam mediated cell-cell interaction in the regulation of hematopoietic transplantation and recovery. Disclosures: No relevant conflicts of interest to declare.

Enhanced Notch Signaling Skews Hematopoietic Stem Cell Differentiation in Fanconi Anemia Murine Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1191-1191
Author(s):  
Wei Du ◽  
Jared Sipple ◽  
Jonathan Schick ◽  
Qishen Pang
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stem Cell ◽  
Notch Signaling ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Gfp Expression ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 1191 Objective: Hematopoietic stem cells (HSCs) can either self-renew or differentiate into various types of cells of the blood lineage. Little is known about the signaling pathways that regulate this choice of self-renewal versus differentiation. We studied the effect of altered Notch signaling on HSC differentiation in mouse models of Fanconi anemia (FA), a genetic disorder associated with bone marrow failure and progression to leukemia and other cancers. Methods: The study used a Notch reporter mouse, in which Notch-driven GFP expression acts as a sensor for HSC differentiation. Long-term hematopoietic stem cell (LT-HSC) and multipotential progenitor (MPP) cell compartments, as well as GFP expression in different cell populations were detected by Flow Cytometry analysis using primary bone marrow cells from Notch-eGFP-WT, Notch-eGFP-Fanca−/− or Notch-eGFP-Fancc−/− mice. Cell Cycle analysis was performed to distinguish the difference of quiescent state in GFP-gated LSK cells from these Notch-eGFP reporter mice. Colony forming units (CFU) assay and bone marrow transplantation (BMT) were utilized to determine HSC self-renew capacity. Gene arrays for pathways involved in DNA repair, cell cycle control, anti-oxidant defense, inflammatory response and apoptotic signaling were employed to define the gene expression signatures of the MPP population. Results and conclusions: In mice expressing a transgenic Notch reporter, deletion of the Fanca or Fancc gene enhanced Notch signaling in MPPs, which was correlated with decreased phenotypic long-term HSCs and increased formation of MPP1 progenitors. Furthermore, we found a functional correlation between Notch signaling and self-renewal capacity in FA hematopoietic stem and progenitor cells (HSPCs). Significantly, we show that FA deficiency in MPPs deregulates a complex network of genes in the Notch and canonical NF-kB pathways. Specifically, enhanced Notch signaling in FA MPPs was associated with the unregulation of genes involved in inflammatory and stress responses (including Rela, Tnfrsf1b, Gadd45b, Sod2, Stat1, Irf1 and Xiap), cell-cycle regulation (including Ccnd1, Cdc16, Cdkn1a, Gsk3b, Notch2 and Nr4a2), and transcription regulation (including Rela, Stat1, Hes1, Hey1, Hoxb4, Notch1 and Notch2). Consequently, TNF-a stimulation enhanced Notch signaling of FA LSK cells, leading to decreased HSC quiescence and compromised HSC self-renewal. Finally, genetic ablation of NF-kB reduced Notch signaling in FA MPPs to nearly wide-type level, and blocking either NF-kB or Notch signaling partially restored FA HSC quiescence and self-renewal capacity. Translational Applicability: The study identifies a functional interaction between the FA pathway and Notch signaling in HSC differentiation and establishes a role of FA proteins in the control of balance between renewal and lineage commitment, hence contributing to hematopoiesis. These findings indicate that the Notch signaling pathway may represent a novel and therapeutically accessible pathway in FA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals No Evidence for Hematopoietic Stem Cell Self-Renewal in-Vivo Following Inflammatory Challenge

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 456-456
Author(s):  
Ruzhica Bogeska ◽  
Paul Kaschutnig ◽  
Stella V Paffenholz ◽  
Julia Knoch ◽  
Jan-Philipp Mallm ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mouse Model ◽  
Hematopoietic Stem Cell ◽  
Normal Aging ◽  
Post Treatment ◽  
Quiescent State ◽  
Self Renewal ◽  
Hematopoietic Stem

Elevated levels of inflammation have been previously linked to both inherited and acquired bone marrow failure (BMF) syndromes, as well as to normal aging, suggesting a role in the etiology of these conditions. One potential explanation for this phenomenon is that repeated inflammation can promote the suppression of hematopoietic stem cell (HSC) function.We have previously demonstrated that interferon-α can accelerate HSC attrition by driving HSCs out of quiescence, leading to the development of BMF in a mouse model of Fanconi anemia (Walter et al. Nature, 2015). To more broadly address the impact of repetitive inflammatory challenge on HSC regeneration, we challenged C57BL6 wild type (WT) mice with polyinosinic:polycytidylic acid (pI:C), a TLR3 agonist that mimics viral infection. Injection with 1-3 rounds of pI:C (8 injections per round) in WT mice had no sustained impact on hematopoiesis, since peripheral blood (PB) and bone marrow (BM) counts were within normal ranges at 5 weeks (5wk) post-treatment. However, in vitro analysis of the clonal proliferation potential of 411 individual sorted long-term (LT)-HSCs revealed a 2-fold reduction (p&lt;0.0001) in the total number of progeny produced per HSC. Additionally, cell fate tracking experiments showed accelerated entry into first division and differentiation following treatment. In line with this data, competitive repopulation assays demonstrated a progressive depletion of functional HSC numbers, with an approximate 2-fold decrease in multi-lineage competitive repopulating activity with each additional round of inflammatory challenge (p&lt;0.01). In order to assess in vivo recovery of HSCs following inflammatory challenge, competitive and limiting dilution transplantation assays were used to quantify HSC frequencies using BM harvested from mice at 5, 10 or 20wk after 3 rounds of pI:C treatment. In both assays we observed a sustained ~18 fold decrease in functional HSCs, with no evidence of recovery within the 20wk window. To exclude microenvironment effects on HSC function, we performed reverse transplantation experiments in which pI.C challenged WT mice were injected with saturating doses of LT-HSCs from non-treated WT donors, in the absence of additional irradiation conditioning. We observed a durable suppression of endogenous HSCs that was sufficient to facilitate robust engraftment of donor LT-HSCs up to 20wk post-treatment. We next used the inducible transgenic Scl-tTA;H2B-GFP mouse model (Wilson et al., Cell, 2008) in order to prospectively segregate quiescent label retaining LT-HSCs (LRCs) from LT-HSCs that proliferate in vivo in response to pI:C (nonLRCs). Following a single round of pI:C challenge, label retention was reduced as a result of LT-HSC proliferation (Table 1). Importantly, the clonal proliferative potential of individual LRCs was preserved upon pI:C challenge while that of nonLRCs was more than halved. This suggests that LT-HSCs fail to undergo self-renewal divisions in vivo under these conditions but rather are functionally compromised in line with increasing proliferative history. We hypothesized that this apparent progressive irreversible depletion of functional HSCs may eventually lead to compromised hematopoiesis. We therefore assessed the hematologic parameters of aged mice that had been exposed to repetitive pI:C treatment in early to mid-life. While these mice had normal PB counts at 5wk post-treatment, upon reaching 2 years of age, treated mice demonstrated mild PB cytopenias, BM hypocellularity and a relative expansion of BM adipocytes (Table 2). Taken together, our data contradict the canonical view that HSCs demonstrate extensive self-regenerative capacity following injury. Rather, in the context of inflammatory challenge, HSCs are progressively and irreversibly depleted as they are driven out of their quiescent state. These findings have broad implications regarding the role of inflammation in the suppression of hematopoiesis that are likely relevant to BMF and also normal aging. Disclosures Lipka: InfectoPharm GmbH: Employment. Frenette:Pfizer: Consultancy; Cygnal Therapeutics: Equity Ownership; Ironwood Pharmaceuticals: Research Funding; Albert Einstein College of Medicine, Inc: Patents & Royalties.

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