The Mll-AF9 Fusion Gene Results in Overexpression of Homeobox Genes and Deregulation of Granulocyte-Monocyte Progenitors.

Abstract Our understanding of the biology of MLL fusion gene leukemias is limited by the lack of knowledge of the effects of the different MLL fusion genes on expression of specific homoebox genes and the specific cell compartment(s) that are subsequently deregulated. In this study we investigated whether cellular deregulation was present in committed myeloid precursors and/or the multi-potent hematopoietic stem cells derived from Mll-AF9 knock-in mice. We used the murine knock-in model since it offers the advantage of a single copy of the Mll fusion gene under the control of the endogenous promoter that is present in every hematopoietic stem/progenitor cell. The Mll-AF9 knock-in mice display expansion of the myeloid compartment as early as 6 weeks of age (young adult) and develop myeloid leukemia at approximately 6 months. We purified hematopoietic stem cells (HSCs) and granulocyte-monocyte progenitors (GMPs) from wild type and Mll-AF9 young adult bone marrow. We depleted lineage positive cells using a magnetic separation system and purified the respective populations using fluorescence activated cell sorting with specific panels of antibodies (HSC=Li−/Thy1.1lo/IL-7R−/C-kit+/Sca-1+; GMP=Lin−/IL-7R−/Sca-1+/C-kit+/CD34+/CD16/32hi). We cultured these cells in methylcellulose supplemented with GM-CSF, IL-3, SCF and IL-6, conditions that promote the growth of myeloid colonies. We assessed growth deregulation by increased colony numbers at the end of 7 days of culture and by the predominance of dense, compact colony morphology, the latter comprised of immature myeloid cells. Culture of HSCs from Mll-AF9 and wild type mice yielded an identical number of colonies (1102 and 1315 colonies per 104 cells respectively, average). In contrast, GMPs from Mll-AF9 mice yielded almost four times the number of colonies compared to wild type GMPs (3331 and 920 colonies per 104 cells respectively, average). Additionally, Mll-AF9 GMPs formed a higher number of dense, compact colonies compared to Mll-AF9 HSCs (1314 and 352 colonies per 104 cells respectively, average). Neither HSCs nor GMPs from wild type mice formed dense, compact colonies. These results indicate a greater deregulation of GMPs compared to HSCs in Mll-AF9 mice. MLL fusion gene leukemias are characterized by over-expression of specific homeobox genes, and we have previously shown that Mll-AF9 bone marrow cells display increased expression of 5′ Hox-a genes and of the Hox co-factor Meis1 compared to wild type counterparts. We hypothesized that these genes are over-expressed in Mll-AF9 GMPs compared to wild type GMPs. Real time quantitative RT-PCR showed that expression levels of Hoxa7, Hoxa9 and Meis1 were increased in Mll-AF9 GMPs compared to wild type (2.7 ± 0.8, 11.7 ± 7.8 and 19 ± 11.3 fold respectively, mean ± SEM). Overall, these data support the hypothesis that the Mll-AF9 gene is “instructive” at the molecular level at least in part via specific homeobox gene over-expression, resulting in deregulation and expansion of specific progenitor/stem cells such as the GMP population. This expanded GMP population then becomes a target for secondary mutations and later development of leukemia. Future studies focused on understanding the biology of this compartment in Mll-AF9 mice will help in our understanding of the pathogenesis of leukemia and aid in the development of newer, more effective therapies.

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Exhaustion of Donor Hematopoietic Stem Cells in Lethally-Irradiated Hosts Can Be Sbstantially Mitigated by Targeting p18INK4C.

Blood ◽

10.1182/blood.v104.11.1200.1200 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1200-1200

Author(s):

Hui Yu ◽

Youzhong Yuan ◽

Xianmin Song ◽

Feng Xu ◽

Hongmei Shen ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Kinase Inhibitor ◽

Hematopoietic Cells ◽

Hematopoietic Stem ◽

Total Period ◽

Over Expression

Abstract Hematopoietic stem cells (HSCs) are significantly restricted in their ability to regenerate themselves in the irradiated hosts and this exhausting effect appears to be accelerated in the absence of the cyclin-dependent kinase inhibitor (CKI), p21. Our recent study demonstrated that unlike p21 absence, deletion of the distinct CKI, p18 results in a strikingly positive effect on long-term engraftment owing to increased self-renewing divisions in vivo (Yuan et al, 2004). To test the extent to which enhanced self-renewal in the absence of p18 can persist over a prolonged period of time, we first performed the classical serial bone marrow transfer (sBMT). The activities of hematopoietic cells from p18−/− cell transplanted mice were significantly higher than those from p18+/+ cell transplanted mice during the serial transplantation. To our expectation, there was no detectable donor p18+/+ HSC progeny in the majority (4/6) of recipients after three rounds of sBMT. However, we observed significant engraftment levels (66.7% on average) of p18-null progeny in all recipients (7/7) within a total period of 22 months. In addition, in follow-up with our previous study involving the use of competitive bone marrow transplantation (cBMT), we found that p18−/− HSCs during the 3rd cycle of cBMT in an extended long-term period of 30 months were still comparable to the freshly isolated p18+/+ cells from 8 week-old young mice. Based on these two independent assays and the widely-held assumption of 1-10/105 HSC frequency in normal unmanipulated marrow, we estimated that p18−/− HSCs had more than 50–500 times more regenerative potential than p18+/+ HSCs, at the cellular age that is equal to a mouse life span. Interestingly, p18 absence was able to significantly loosen the accelerated exhaustion of hematopoietic repopulation caused by p21 deficiency as examined in the p18/p21 double mutant cells with the cBMT model. This data directly indicates the opposite effect of these two molecules on HSC durability. To define whether p18 absence may override the regulatory mechanisms that maintain the HSC pool size within the normal range, we performed the transplantation with 80 highly purified HSCs (CD34-KLS) and then determined how many competitive reconstitution units (CRUs) were regenerated in the primary recipients by conducting secondary transplantation with limiting dilution analysis. While 14 times more CRUs were regenerated in the primary recipients transplanted with p18−/−HSCs than those transplanted with p18+/+ HSCs, the level was not beyond that found in normal non-transplanted mice. Therefore, the expansion of HSCs in the absence of p18 is still subject to some inhibitory regulation, perhaps exerted by the HSC niches in vivo. Such a result was similar to the effect of over-expression of the transcription factor, HoxB4 in hematopoietic cells. However, to our surprise, the p18 mRNA level was not significantly altered by over-expression of HoxB4 in Lin-Sca-1+ cells as assessed by real time PCR (n=4), thereby suggesting a HoxB4-independent transcriptional regulation on p18 in HSCs. Taken together, our current results shed light on strategies aimed at sustaining the durability of therapeutically transplanted HSCs for a lifetime treatment. It also offers a rationale for the feasibility study intended to temporarily target p18 during the early engraftment for therapeutic purposes.

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Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽

10.1182/blood.v124.21.350.350 ◽

2014 ◽

Vol 124 (21) ◽

pp. 350-350

Author(s):

Kyung-Hee Chang ◽

Amitava Sengupta ◽

Ramesh C Nayak ◽

Angeles Duran ◽

Sang Jun Lee ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Negative Regulator ◽

Wild Type ◽

Hematopoietic Stem ◽

P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

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Bclaf1 Promotes Maintenance and Self-Renewal of Fetal Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2018-99-114144 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1269-1269 ◽

Cited By ~ 1

Author(s):

Lynn S. White ◽

Deepti Soodgupta ◽

Rachel L. Johnston ◽

Jeffrey A. Magee ◽

Jeffrey J. Bednarski

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Liver Cells ◽

Lower Percentage ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract Hematopoietic stem cells (HSC) persist throughout life by undergoing extensive self-renewal divisions while maintaining an undifferentiated state. The mechanisms that support HSC self-renewal change throughout the course of development as temporal changes in transcriptional regulators coordinate distinct genetic programs in fetal, post-natal and adult HSCs. These self-renewal programs are often ectopically activated in leukemia cells to drive neoplastic proliferation and high expression of HSC-associated genes predicts a poor prognosis in acute myelogenous leukemia (AML). In this regard, it was recently shown that expression of the transcriptional regulator BCLAF1 (Bcl2 associated transcription factor 1) is increased in AML blasts relative to normal precursor populations and suppression of BCLAF1 causes reduced proliferation and induction of differentiation to a dendritic cell fate. These findings raise the question of whether BCLAF1 may regulate normal as well as neoplastic self-renewal programs. We find that Bclaf1 is highly expressed in HSCs versus committed bone marrow populations consistent with a potential role for this gene in HSC functions. To test whether BCLAF1 regulates HSC development and hematopoiesis, we used germline loss of function mice. Bclaf1-/- mice succumb to pulmonary failure shortly after birth due to poor lung development, so we assessed prenatal hematopoiesis. Bclaf1-deficient mice had significantly reduced HSC and hematopoietic progenitor cell (HPC) frequencies and numbers despite normal fetal liver cellularity. To determine if Bclaf1 is required for HSC function during fetal development, we performed competitive reconstitution assays. Fetal liver cells from Bclaf1+/+or Bclaf1-/-mice were transplanted along with wild-type competitor bone marrow cells into lethally irradiated recipient mice. Compared to recipients of Bclaf1+/+fetal liver cells, recipients of Bclaf1-/-cells had a significantly lower percentage of donor-derived leukocytes at all time points after transplantation as well as reduced percentage of donor HSCs at 16 weeks post-transplant. Notably, all leukocyte populations (B cells, T cells, granulocytes and macrophages) from Bclaf1-/-donors were reduced consistent with an abnormality in HSC repopulating activity rather than a defect in a specific differentiation pathway. Consistent with these findings, Bclaf-deficient cells did not engraft in competitive transplants with limiting numbers of sorted fetal liver HSCs whereas sorted wild-type Bclaf1+/+cells effectively reconstituted hematopoiesis in recipient mice. In addition, Vav-cre:Bclaf1flox/floxmice, which have selective deletion of Bclaf1 in hematopoietic cells, have reduced frequencies and numbers of fetal liver HSCs identical to the findings observed in germline Bclaf1-/-mice. These results show that loss of Bclaf1 leads to defective development and repopulating activity of fetal HSCs. Interestingly, when adult mice are successfully engrafted with Bclaf1-deficient HSCs, the donor HSCs suffer no additional functional impairment. Furthermore, in secondary transplant experiments Bclaf1-deficient HSCs maintain long-term repopulating activity. Thus, Bclaf1 may have distinct functions in fetal versus adult HSC self-renewal. Collectively, our findings reveal Bclaf1 is a novel regulator of fetal HSC function and suggest that it may have distinct functions in different developmental contexts. Disclosures No relevant conflicts of interest to declare.

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Hematopoietic stem cells with controllable tEpoR transgenes have a competitive advantage in bone marrow transplantation

Blood ◽

10.1182/blood.v95.12.3710 ◽

2000 ◽

Vol 95 (12) ◽

pp. 3710-3715 ◽

Cited By ~ 11

Author(s):

Suzanne Kirby ◽

William Walton ◽

Oliver Smithies

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Erythropoietin Receptor ◽

Wild Type ◽

Hematopoietic Stem ◽

Myeloid Progenitor ◽

Colony Forming Units

Abstract In a previous study, it was found that a truncated erythropoietin receptor transgene (tEpoR tg) enables multilineage hematopoietic progenitor amplification after treatment with erythropoietin (epo) in vitro and in vivo. This study used competitive bone marrow (BM) repopulation to show that tEpoR tg facilitates transplantation by hematopoietic stem cells (HSC). Individual multilineage colonies, committed myeloid progenitor colonies, and lymphoid colonies (pre-B colony-forming units) were grown from the marrow of animals 6 months after they received a 50/50 mixture of transgene and wild-type BM cells. In epo-treated recipients, the transgene-bearing cells significantly outcompeted the wild-type cells (84%-100% versus 16%-0%, respectively). In recipients treated with phosphate-buffered saline, the repopulation was minimally different from the donor mixture (49%-64% transgene versus 51%-36% wild-type). The epo-induced repopulation advantage is maintained in secondary transplants. In addition, neither accelerated HSC depletion nor uncontrollable proliferation occurred during epo-stimulated serial transplants of transgene-containing BM. Thus, the tEpoR tg functions in a benign fashion in HSC and allows for a significant and controllable repopulation advantage in vivo without excessive HSC depletion relative to wild-type BM.

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Roles of Histone Acetyltransferase MORF and MOZ in Hematopoiesis.

Blood ◽

10.1182/blood.v114.22.2525.2525 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2525-2525

Author(s):

Takuo Katsumoto ◽

Issay Kitabayashi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Liver Cells ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract Abstract 2525 Poster Board II-502 MOZ (MOnocytic leukemia Zinc finger protein) and MORF (MOz Related Factor), Myst-type histone acetyltransferases, are involved in chromosome translocations associated with FAB-M4/5 subtypes of acute myeloid leukemia. We have reported that MOZ is essential for hematopoietic cell development and self-renewal of hematopoietic stem cells. To explore the possibility MORF also plays important roles in hematopoiesis, we generated Morf-deficient mice with homologous recombination methods. Morf−/− mice were smaller than their wildtype littermates and died within 4 weeks after birth on C57BL/6 background. In MORF−/− fetal liver, Flt3-negative KSL (c-Kit+ Sca-1+ Lineage-) cells containing hematopoietic stem cells were decreased. When fetal liver cells were transplanted into irradiated recipient mice, MORF−/− cells less efficiently reconstituted hematopoiesis than wild-type cells. Additionally, bone marrow cells reconstituted with MORF−/− cells rarely contributed to hematopoiesis in secondary transplants. To reveal relationship between MORF and MOZ in hematopoiesis, we generated double heterozygous (Moz+/− Morf+/−) mouse. Double heterozygous mice were smaller than wild-type littermates and died at least 4 weeks after birth. Numbers of KSL cells, especially Flt3- KSL cells and common myeloid progenitors were decreased in the double heterozygous embryos. The double heterozygous fetal liver cells also displayed less activity to reconstitute hematopoiesis than MOZ+/− or MORF+/− cells. Since MORF−/− mice and MOZ/MORF double heterozygous mice were alive at adult on a mixed C57BL/6/DBA2 genetic background, we investigated adult hematopoiesis in these mice. MORF−/− or MOZ/MORF double heterozygous mice were smaller than their wild-type littermates and had small numbers of thymocytes and splenocytes. However, there were no significant differences in number of bone marrow cells and hematopoietic lineage population in MORF−/− or MOZ/MORF double heterozygous mice. These results suggest that MORF as well as MOZ plays important roles in self-renewal of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Ott1(Rbm15)-Deficient Hematopoietic Stem Cells Are Unable to Maintain Quiescence During Replicative Stress and Display Features of Premature Aging,

Blood ◽

10.1182/blood.v118.21.3433.3433 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3433-3433

Author(s):

Nan Xiao ◽

Kaushal Jani ◽

Jonathan L Jesneck ◽

Glen D Raffel

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Gene Set Enrichment Analysis ◽

Wild Type ◽

Hematopoietic Stem ◽

Replicative Stress

Abstract Abstract 3433 With age, hematopoietic stem cells (HSCs) have numerical expansion, skewing towards myeloid development, loss of lymphoid potential, an underlying pro-inflammatory state and loss of self-renewal potential thus severely limiting responses to hematopoietic stress, ultimately leading to bone marrow failure. The mechanisms and pathways responsible for these changes in aged HSCs are incompletely understood. Using a conditional allele of Ott1, a gene originally isolated as the 5' fusion partner in t(1;22) acute megakaryocytic leukemia, we previously found a global regulatory role for the gene in hematopoiesis. Deletion of Ott1 in adult mice utilizing Mx1-cre recapitulated certain aspects of aging hematopoiesis including increased Lin−Sca1+c-Kit+ (LSK) population, myeloid expansion and decreased lymphopoiesis. The LSK compartment was further characterized using SLAM and CD34/Flk2 markers and demonstrated normal levels of LT-HSCs and increased ST-HSCs. Despite sufficient LT-HSC numbers, Ott1-deleted bone marrow was unable to competitively or non-competitively repopulate irradiated recipients. To exclude a homing or engraftment effect, Ott1flox/null Mx1-cre bone marrow was transplanted with competitor then excised post-engraftment. The rapid loss of the Ott1-deficient graft demonstrated Ott1 is required for maintenance under competitive stress. In contrast, primary mice undergoing Ott1 excision lived a normal lifespan and were able to maintain sufficient hematopoiesis although with a partial reduction in bone marrow clonagenicity showing loss of Ott1 is not limiting under steady state conditions. To test the HSC requirement for Ott1 under replicative stress, Ott1 knockout mice were challenged with 5-fluorouracil (5-FU). Ott1-deleted mice treated with 5-FU displayed delayed peripheral blood neutrophil recovery and showed accelerated bone marrow failure. Cell cycle analysis of steady state Ott1 knockout HSCs showed a similar profile to wild type controls, however, after 5-FU treatment, the G0 fraction was dramatically reduced. The G0 fraction is associated with the quiescent, self-renewing HSC population, therefore, Ott1 is required for maintaining HSC quiescence during replicative stress but not steady state hematopoiesis. To more specifically assess whether the functional hematopoietic changes seen after loss of Ott1 were accompanied by alterations in known aging-associated pathways, Gene Set Enrichment Analysis comparing Ott1-deleted HSCs in steady state to aged HSCs was performed and showed a highly enriched gene expression signature (NES 2.02 p<0.0001). Physiologic sequelae of HSC aging were observed after Ott1 excision including activation of NFκβ, elevation of reactive oxygen species (ROS), increase in DNA damage (γH2A.X levels) and activation of p38Mapk. Although ROS was elevated under steady state conditions, neither apoptosis, senescence or proliferation was significantly different from wild type control HSCs. Furthermore, anti-oxidant treatment with N-acetyl-cysteine was unable to rescue the HSC maintenance defect of the Ott1 knockout, signifying additional requirements in HSCs for Ott1 beyond regulation of ROS. An observed increase of mitochondrial mass in Ott1-deleted HSCs suggests an upstream function for Ott1 in metabolic control, potentially contributing to ROS generation or degradation. In summary, we have demonstrated an essential role for Ott1 in maintaining HSC quiescence during replicative stress and shown loss of Ott1 leads to the acquisition of key gene expression patterns and pathophysiologic changes associated with aging. These data suggest Ott1 functions in part to oppose specific consequences of aging in the hematopoietic compartment. Ott1 and Ott1-dependent pathways therefore represent a potential therapeutic target to prevent the morbidity and mortality arising from age-related defects in hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v94.6.2151.418a22_2151_2158 ◽

1999 ◽

Vol 94 (6) ◽

pp. 2151-2158 ◽

Cited By ~ 1

Author(s):

Kevin P. Battaile ◽

Raynard L. Bateman ◽

Derik Mortimer ◽

Jean Mulcahy ◽

R. Keaney Rathbun ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fanconi Anemia ◽

Peripheral Blood ◽

Wild Type ◽

Hematopoietic Stem ◽

In Vivo Selection ◽

Serial Transplantation

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

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A limited role for p16Ink4a and p19Arf in the loss of hematopoietic stem cells during proliferative stress

Blood ◽

10.1182/blood-2004-06-2242 ◽

2005 ◽

Vol 106 (3) ◽

pp. 827-832 ◽

Cited By ~ 51

Author(s):

Lilia Stepanova ◽

Brian P. Sorrentino

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Dominant Role ◽

Wild Type ◽

Hematopoietic Stem ◽

Serial Transplantation ◽

Marrow Cells

Abstract It has long been known that prolonged culture or serial transplantation leads to the loss of hematopoietic stem cells (HSCs); however, the mechanisms for this loss are not well understood. We hypothesized that expression of p16Ink4a or p19Arf or both may play a role in the loss of HSCs during conditions of enhanced proliferation, either in vitro or in vivo. Arf was not expressed in freshly isolated HSCs from adult mice but was induced in phenotypically primitive cells after 10 to 12 days in culture. When cultured bone marrow cells from either Arf–/– or Ink4a-Arf–/– mice were compared to wild-type cells in a competitive repopulation assay, no significant differences in HSC activity were seen. We then evaluated the role of p19Arf and p16Ink4a in the loss of HSCs during serial transplantation. Bone marrow cells from Ink4a-Arf–/–, but not Arf–/–, mice had a modestly extended life span and, on average, supported reconstitution of one additional recipient compared to wild-type cells. Mice given transplants of Ink4a-Arf–/–cells eventually did die of hematopoietic failure in the next round of transplantation. We conclude that mechanisms independent of the Ink4a-Arf gene locus play a dominant role in HSC loss during conditions of proliferative stress.

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Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cells Via Cell Autonomous and Cell Non-Autonomous Mechanisms

Blood ◽

10.1182/blood.v126.23.1174.1174 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1174-1174

Author(s):

Darlene Monlish ◽

Angela Herman ◽

Molly Romine ◽

Sima Bhatt ◽

Laura G. Schuettpelz

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Immune Cells ◽

Conflicts Of Interest ◽

Wild Type ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Tlr2 Agonist ◽

Tlr2 Signaling

Abstract Toll like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that shape the innate immune system by identifying foreign pathogen-associated molecular patterns (PAMPS) and host-derived damage associated patterns (DAMPS). TLRs are widely expressed on both immune cells and non-immune cells, including hematopoietic stem and progenitor cells (HSPCs). Of clinical significance, both lymphoproliferative and myelodysplastic syndromes have been linked to aberrant TLR signaling (Schuettpelz, et al., Front Immunol 2013; Varney, et al., Exp Hematol 2015). Despite extensive studies focused on the influence of TLRs through committed effector cell populations, more recent evidence suggests that these PRRs may elicit immune regulation from the more primitive level of hematopoietic stem cells (HSCs). As TLR2 is expressed on HSCs, in the present study, we sought to elucidate the effect of TLR2 signaling on HSCs, and determine the cell-autonomous versus non-autonomous effects of this signaling. To this end, we utilized the synthetic TLR2 agonist, PAM3CSK4, to assess the effects of augmented TLR2 signaling on HSC mobilization, function, cycling, and differentiation. In previous studies, we found that TLR2 is not required for HSC function (Schuettpelz et al., Leukemia 2014); however, in the present study, treatment of wild-type mice with PAM3CSK4 led to HSC expansion in both the bone marrow and spleen, and a reduction in bone marrow megakaryocyte-erythroid progenitors (MEPs). Further, we observed increased HSC cycling and loss of function in competitive bone marrow transplantation assays in response to TLR2 agonist exposure. Treatment of chimeric animals (Tlr2-/- + Tlr2+/+ bone marrow transplanted into Tlr2+/+ or Tlr2-/- recipients) showed that these effects are largely cell non-autonomous, with a minor contribution from cell-autonomous TLR2 signaling. Analysis of serum, bone marrow, and spleen samples by cytokine expression arrays revealed an increase in G-CSF (serum) and TNFα (bone marrow) following TLR2 agonist treatment in wild-type mice. To further characterize the influence of these cytokines, respective receptor knockout models were employed. Inhibition of G-CSF enhanced HSC bone marrow expansion in response to PAM3CSK4, but partially rescued the expansion of spleen HSPCs. Likewise, loss of TNFa partially mitigated the expansion of spleen HSPCs in response to PAM3CSK4, and abrogated the PAM3CSK4-induced spleen HSC cycling. Further, we observed that loss of TNFa rescued the PAM3CSK4-mediated loss of bone marrow MEPs. Taken together, these data suggest that TLR2 signaling affects HSCs via both cell cell-autonomous and non-autonomous cues, with G-CSF and TNFa contributing to TLR2 agonist-mediated effects on HSC cycling, mobilization, and function. Ongoing studies aim to determine the particular cell types that are crucial for mediating the effects of TLR2 signaling on HSCs and elucidate the role of this pathway on HSCs in myelodysplastic syndrome (MDS) pathogenesis and other hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v114.22.79.79 ◽

2009 ◽

Vol 114 (22) ◽

pp. 79-79

Author(s):

Agnieszka Czechowicz ◽

Deepta Bhattacharya ◽

Lisa Ooi ◽

Derrick J Rossi ◽

David Bryder ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Normal Animal ◽

Cell Types ◽

Therapeutic Interventions ◽

Wild Type ◽

Advisory Committees ◽

Hematopoietic Stem

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

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