Increase In Number Of Hematopoietic Stem Cells Research Articles

Age-related changes in the hematopoietic stem cell pool revealed via quantifying the balance of symmetric and asymmetric divisions.

Hematopoietic stem cells (HSCs) are somatic stem cells that continuously generate lifelong supply of blood cells through a balance of symmetric and asymmetric divisions. It is well established that the HSC pool increases with age. However, not much is known about the underlying cause for these observed changes. Here, using a novel method combining single-cell ex vivo HSC expansion with mathematical modeling, we quantify HSC division types (stem cell-stem cell (S-S) division, stem cell-progenitor cell (S-P) division, and progenitor cell-progenitor cell (P-P) division) as a function of the aging process. Our time-series experiments reveal how changes in these three modes of division can explain the increase in HSC numbers with age. Contrary to the popular notion that HSCs divide predominantly through S-P divisions, we show that S-S divisions are predominant throughout the lifespan of the animal, thereby expanding the HSC pool. We, therefore, provide a novel mathematical model-based experimental validation for reflecting HSC dynamics in vivo.

Mitochondria-Mediated (MAVS) Innate Immune Signaling Drives Inflammation-Induced Bone Marrow Failure

Bone marrow failure syndromes (BMFS) are life threatening diseases, characterized by impaired fitness of hematopoietic stem cells (HSC) and ineffective hematopoiesis causing the absence of one or more hematopoietic lineages in the peripheral blood. Substantial clinical data indicate that hyperactivity of inflammatory cytokines, including TNFα, IL-6, and transforming growth factor-β (TGFβ), directly contribute to BMF. Inflammatory challenges result in irreversible HSC functional decline, including decreased repopulation potential and myeloid-bias differentiation. We recently showed that the combination of inflammatory challenges, including polyinosinc:polycytidilic acid (pIC - a double stranded RNA [dsRNA]) plus high TGFβ causes chronic pancytopenia, anemia, expanded hematopoietic stem and progenitor cell pools, and enhanced bone marrow dysplasia, mimicking a myelodysplastic syndrome (MDS) that developed 3-4 months after pIC challenge (Javier et.al. Haematologica 2022). However, the mechanisms behind this association and/or causality are still ill-defined. Here, we show that mitochondrial antiviral signaling (MAVS) protein can mediate the negative effect of pIC and TGFβ on HSC functions and BMF/ MDS development. Differential gene expression analyses in HSCs suggested that pIC plus TGFβ altered expression of nuclear-encoded mitochondrial genes. Mitochondria serve as platform for the activation of MAVS and downstream innate immune signaling in response to dsRNA. We thus examined the role of mitochondria and MAVS in pIC plus TGFβ-induced BMF/MDS. We show that overexpression TGFβ and pIC together cause alterations in mitochondrial proteins and membrane potential as well as enhanced caspase1 activity, but not the necroptotic signals p-RIP1 and p-RIP3, in HSCs. pIC exposure caused an increase in MAVS expression, and a redistribution into large aggregates that localize onto mitochondria in HSCs, indicative of MAVS activation (two days after pIC exposure in vivo). The MAVS aggregates resolved into smaller and dispersed clusters by 3 months after pIC exposure in control mice. In contrast, in HSCs from TGFβ overexpressing mice, MAVS aggregates persisted 3 months after pIC exposure. To determine the impact of augmented MAVS in dsRNA+TGFβ-driven BMF, we used MAVS deficient mice crossed with TGFβ overexpressing mice. We found that absence of MAVS protein restored normal white blood cell count, including neutrophils and lymphocytes, restored normal red blood cell but not platelet counts in TGFβ overexpressing mice after pIC challenge, compared to controls. Remarkably, MAVS deficiency partially rescued the dysplastic features of bone marrow myeloid cells in TGFβ overexpressing mice after pIC challenge. MAVS-deficiency also reduced the increased caspase1 activity and lowers mitochondria membrane potential in HSCs of these mice. In serial competitive transplant studies, MAVS-deficiency conferred HSC repopulation advantage. These findings suggest that pIC+TGFβ overexpression mediated BMF/MDS phenotype depends on chronic activation of MAVS. Because MAVS aggregation depends on mitochondrial fusion, we used genetic loss of the mitochondrial fission regulator Drp1. Drp1-deficiency caused severe mitochondrial aggregation in HSCs; this was associated with sustained MAVS aggregation. In response to pIC, Drp1-deficient mice exhibited increased caspase-1 activity, increased mitochondrial membrane potential and increase in HSC numbers 6 months after pIC challenges. Drp1 loss resulted in anemia and mild pancytopenia. Interestingly, Drp1-deficient bone marrow myeloid progenitors exhibited severe dysplastic features after pIC challenged. These findings suggest that chronic mitochondrial and MAVS aggregation are sufficient to drive BMF/MDS phenotype. Finally, we investigated the levels of epigenetic markers (acetyl H3K27 and methyl H3 Lys4) in HSCs after pIC stress. H4K4me3 levels were decreased in HSCs long-term after pIC challenges [6 months], this was further decreased in Drp1-deficient HSCs. In conclusion, these findings indicate that non-genetic factors contribute to the onset of BMF/MDS. Mitochondria and MAVS protein together mediate an innate immune mechanism that drives BMF/MDS. This study opens the possibility to target mitochondria-related proteins as a therapeutic strategy to restore ineffective hematopoiesis due to chronic inflammation.

Hematopoietic Stem Cell Numbers Are Regulated at Both Systemic and Local Levels

Since Schofield proposed the niche model in the 1970s, it has been widely believed that the number of hematopoietic stem cells (HSCs) is determined by the niche size. We and others have identified several components of the HSC niche, including Nestin-GFP + mesenchymal stem cells (MSCs). However, the number of defined niche cells vastly exceeds that of HSCs, raising the question about the actual regulatory mechanisms governing HSC numbers. To address this question, we initially established a mathematical model of niche availability and occupancy. The model predicted that an increase in available niches initially leads to a corresponding rise in the number of occupying HSCs, which aligns with the concept of local, niche availability-driven restriction on HSC numbers. Interestingly, our model also suggested that this increase in HSC numbers saturates when the number of available niches reaches a critical level. One potential mechanism for this saturation is that HSC numbers may also be regulated at the systemic level, with total HSC numbers in the body being restricted. To experimentally test this model and hypothesis, we developed a bone transplantation technique that allows us to increase the number of available HSC niches. When a single femur (referred to as a graft) was transplanted per mouse (called a host), we observed the progressive regeneration of bone marrow (BM) stroma and hematopoietic recovery in the grafts. While the number of HSCs (Lin -Sca-1 +c-Kit +CD150 +CD48 -CD34 -) in the grafts was still 41.7 ± 7.60% of that in the host femurs at 5 months after transplantation (n = 8, P = 0.0002), they were comparable at 3 months after transplantation when granulocyte colony-stimulating factor (G-CSF) was administered to hosts at 1 month after transplantation (n = 8, P = 0.660). By utilizing Nestin-GFP transgenic mice and a CD45 congenic system, we found that MSCs in grafts are derived from the graft, while hematopoietic cells are derived from the host. Given that a single femur harbors only 8.3 ± 0.440% of HSCs in the total body (n = 11), 6 femurs were transplanted per mouse, followed by G-CSF administration, to assess the impact of increased HSC niches on total HSC numbers. Strikingly, HSC numbers per host femur and graft femur were lower than those per femur from sham-operated mice (n = 7-48, P = 0.012). Moreover, we found that the sum of HSC numbers in the bone transplantation host and graft bones was equivalent to that in the sham-operated group (n = 7-8, P = 0.364). Total HSC numbers were also maintained when 6 femurs from wild-type (WT) mice were transplanted into Cdh2 (N-Cadherin)-CreER; Cxcl12fl/fl mice, which have an impaired ability to retain HSCs in the BM (n = 8, P = 0.711). These results indicate that the addition of normal niches does not alter total HSC numbers in the body, regardless of whether the endogenous niche is functional, supporting the conclusion that HSC numbers are indeed restricted at the systemic level (Figure 1A). To determine whether HSC numbers are also limited at the local level, as suggested by our mathematical model, we transplanted a single WT femur to Cxcl12fl/fl or Cdh2-CreER; Cxcl12fl/fl mice. We hypothesized that implanting only one femur would have a minimal impact on the total BM, and therefore HSC numbers in the grafts transplanted to Cxcl12fl/fl mice would reach the physiological level. We reasoned that if there is no local restriction on HSC numbers, there would be more HSCs in the WT grafts transplanted to Cdh2-CreER; Cxcl12fl/fl mice compared with those implanted to Cxcl12fl/fl mice. As expected, HSC numbers per WT graft femur in Cxcl12fl/fl mice were comparable to those per femur of sham-operated Cxcl12fl/fl mice (corresponding to the physiological level) (n = 8, P = 0.433). However, CXCL12 deficiency in the host BM did not affect HSC numbers per WT graft femur (n = 8, P = 0.986). Importantly, the sum of HSC numbers in the host body and the graft femur of Cdh2-CreER; Cxcl12fl/fl recipient mice was lower than that of sham-operated Cxcl12fl/fl mice (n = 8, P = 0.003), indicating that total HSC numbers in the former mice are below the systemic limit. Collectively, these data suggest that the failure to increase HSC numbers per WT graft in Cdh2-CreER; Cxcl12fl/fl hosts is, at least in part, due to a local restriction on HSC numbers (Figure 1B). Our study demonstrates that HSC numbers are restricted at both systemic and local levels, thereby rewriting the long-standing model for the regulation of HSC numbers.

1029 - IRON HOMEOSTASIS-REGULATORY PATHWAYS IN HEMATOPOIETIC STEM CELLS

Iron is an important bioelement essential for catalyzing various electron transfer reactions in cells. It occurs as ferric and ferrous forms mostly incorporated in iron-sulfur clusters, or bound to iron-binding proteins. Only a small fraction of transitory free iron is present at all times limiting the generation of hydroxyl radicals; the amount of this labile iron pool (LIP) depends on the cells’ energetic needs. Our resent study of iron homeostasis-regulatory pathways in hematopoietic stem cells (HSC) has uncovered a previously unrecognized key role of this molecular circuit in hematopoietic stem cell self-renewal and maintenance. Experimental transient reduction of the LIP, mimicking the reduction of free intracellular iron upon cell division, led to a significant two to three-fold increase in HSC numbers using in vivo and ex vivio models. Mechanistically, our study uncovered that a transient reduction of the LIP triggers a compensatory molecular response in HSCs which, besides upregulation of the transferrin receptor and induction of ferritinophagy to restore the LIP, also comprises lipolysis activation, increased fatty acid mobilization and oxidation. This cascade, in turn, reinforces molecular HSC states. Together, our study has uncovered a new iron-dependent rheostat mechanism aiding in sustaining HSC identify upon cell division.

MGTA-145 in Combination with Plerixafor Rapidly Mobilizes High Numbers of Hematopoietic Stem Cells and Graft-Versus-Host Disease Inhibiting Myeloid-Derived Suppressor Cells in Non-Human Primates

Background. The majority of bone marrow transplants (BMTs) are performed with granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood (mPB) as the source of hematopoietic stem cells (HSCs) for patients. Up to 80% of mPB allogeneic recipients, however, will experience graft-versus-host disease (GvHD). Despite higher levels of CD3+ T cells in mPB grafts compared to BM, the level of acute GvHD observed following transplant of HLA-matched mPB is comparable to HLA-matched BM. One explanation is that G-CSF mobilized grafts contain myeloid-derived suppressor cells (MDSCs) possessing potent immunosuppressive properties capable of inhibiting T cell proliferation in vitro. The percentage of MDSCs is variable in grafts mobilized with G-CSF and clinical data suggest that patients transplanted with mPB grafts that contain higher numbers of MDSCs may have better outcomes including lower rates of acute GvHD (Vendramin et al., BBMT 2014). Identification of a mobilizing regimen that consistently produces high numbers of HSCs and MDSCs may be preferred.We recently reported that MGTA-145 (GroβT), a CXCR2 agonist, when combined with the CXCR4 inhibitor, plerixafor, robustly mobilizes HSCs (Blood 2017 130:1920). In this study, non-human primates (NHPs) were mobilized with a single dose of MGTA-145, plerixafor, or MGTA-145/plerixafor versus a multi-dose regimen of G-CSF, and mPB was harvested to allow detailed immune profiling at 0 through 24 hours. We observed a significant and rapid increase in number of HSCs and CD34dim monocytes with potent in vitro and in vivo immunosuppressive properties.Results. MGTA-145/plerixafor consistently produced a 16-fold increase in number of CD34+CD90+CD45RA- HSCs within four hours of dosing (p=0.0003, n=11). Profiling of graft subsets from these primates also showed a 10-fold increase over baseline in the number of CD34dim monocytes at 4 hours post treatment (p<0.0001, n=11, Figure 1A) that corresponded to 2-3-fold higher frequency and number compared to G-CSF or plerixafor alone (p<0.01, n=2-5) and correlated with degree of HSC mobilization (p<0.0001). To determine if this monocytic cell population had immunosuppressive properties, CD34dim cells were sorted from peripheral blood of NHPs treated with MGTA-145/plerixafor and co-cultured with anti-CD2, anti-CD3 and anti-CD28-stimulated autologous T cells. MGTA-145/plerixafor CD34dim monocytes suppressed T cell proliferation, as measured by CFSE staining after four days.To assess whether these immunosuppressive monocytes may prevent GvHD, we developed a xenograft GvHD model in NSG mice. MGTA-145/plerixafor mPB (6 x 106 PBMCs) containing a high percentage of CD34dim monocytes were injected into sublethally irradiated NSG mice. This was compared to unmobilized primate PBMCs (6 x 106 PBMCs) containing relatively low numbers of CD34dim cells. At day 20, all mice (8/8) transplanted with unmobilized PBMCs had died of acute GvHD compared to none of the mice transplanted with MGTA-145/plerixafor mPB. Mice transplanted with unmobilized PBMCs also demonstrated 3-fold higher numbers of T-cells and increased T-cell activation compared to mice transplanted with MGTA-145/plerixafor mobilized PBMCs (p<0.01, n=6-8). At day 60 post-transplant, 7/8 mice remained alive (Figure 1B, p<0.0001). To assess whether this immunosuppressive effect is due to CD34dim monocytes, we sorted these cells and transplanted PBMCs depleted of CD34dim monocytes into NSG mice. In addition, experiments comparing the number and function of primate HSCs mobilized by MGTA-145/plerixafor or G-CSF alone using the NSG engraftment model and using autologous NHP transplant coupled with ex vivo HSC gene therapy are ongoing.Conclusions. Co-administration of MGTA-145/plerixafor in NHPs results in both rapid and efficacious mobilization of highly enriched HSCs and a CD34dim monocyte population with potent immunosuppressive activity compared to cells mobilized with plerixafor alone or with the current standard of care, G-CSF. The increased number of these immunosuppressive monocytes compared to G-CSF has the potential to reduce GvHD in the allogenic transplant setting. Thus, MGTA-145/plerixafor may offer an advantageous graft in the allogeneic setting where the risk of GvHD remains a significant clinical problem. IND-enabling studies of MGTA-145 are in progress to assess this regimen for mPB collection and transplant. [Display omitted] DisclosuresGoncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Falahee:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Li:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Morrow:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Adenosine from Niche-Associated Tregs Maintains Hematopoietic Stem Cell Quiescence

A crucial player in immune regulation, FoxP3+ regulatory T cells (Tregs) are drawing attention for their heterogeneity and noncanonical functions. For example, specific subsets of Tregs in the adipose tissue control metabolic indices; muscle Tregs potentiate muscle repair, and lung Tregs prevent tissue damage. These studies, together with a previous finding that Tregs are enriched in the primary site for hematopoiesis, the bone marrow (BM), prompted us to examine whether there is a special Treg population which controls hematopoietic stem cells (HSCs).We showed that HSCs within the BM were frequently adjacent to distinctly activated FoxP3+ Tregs which highly expressed an HSC marker, CD150. Moreover, specific reduction of BM Tregs achieved by conditional deletion of CXCR4in Tregs, increased reactive oxygen species (ROSs) in HSCs. The reduction of BM Tregs further induced loss of HSC quiescence and increased HSC numbers in a manner inhibited by anti-oxidant treatment. Additionally, this increase in HSC numbers in mice lacking BM Tregs was reversed by transfer of CD150high BM Tregs but not of CD150low BM Tregs. These results indicate that CD150high niche-associated Tregs maintain HSC quiescence and pool size by preventing oxidative stress.We next sought to identify an effector molecule of niche Tregs which regulates HSCs. Among molecules highly expressed by niche Tregs, we focused on CD39 and CD73, cell surface ecto-enzymes which are required for generation of extracellular adenosine, because 1) CD39highCD73high cells within the BM were prevalent among CD150high Tregs and 2) HSCs highly expressed adenosine 2a receptors (A2AR). We showed that both conditional deletion of CD39 in Tregs and in vivo A2AR antagonist treatment induced loss of HSC quiescence and increased HSC pool size in a ROS-dependent manner, which is consistent with the findings in mice lacking BM Tregs. In addition, transfer of CD150high BM Tregs but not of CD150low BM Tregs reversed the increase in HSC numbers in FoxP3cre CD39flox mice. The data indicate that niche Treg-derived adenosine regulates HSCs.We further investigated the protective role of niche Tregs and adenosine in radiation injury against HSCs. Conditional deletion of CD39 in Tregs increased radiation-induced HSC apoptosis. Conversely, transfer of as few as 15,000 CD150high BM Tregs per B6 mouse (iv; day-1) rescued lethally-irradiated (9.5Gy) mice by preventing hematopoiesis failure. These observations indicate that niche Tregs protect HSCs from radiation stress.Finally, we investigated the role of niche Tregs in allogeneic (allo-) HSC transplantation. Our previous study showed that allo-hematopoietic stem and progenitor cells but not allo-Lin+ cells persisted in the BM of non-conditioned immune-competent recipients without immune suppression in a manner reversed by systemic Treg depletion1. This observation suggests that HSCs have a limited susceptibility to immune attack, as germline and embryonic stem cells are located within immune privileged sites. Because the study employed systemic Treg depletion and non-conditioned recipients, it remains unknown whether niche Tregs play a critical role in immune privilege of HSCs and in allo-HSC engraftment following conditioning. We showed here that the reduction of BM Tregs and conditional deletion of CD39 in Tregs abrogated allo-HSC persistence in non-conditioned immune-competent mice as well as allo-HSC engraftment following nonmyeloablative conditioning. Furthermore, transfer of CD150high BM Tregs but not of other Tregs (15,000 cells/recipient; day -2) significantly improved allo-HSC engraftment. This effect of niche Treg transfer is noteworthy given that 1-5 million Tregs per mouse were required in case of transfer of spleen or lymph node Tregs. These observations suggest that niche Tregs maintain immune privilege of HSCs and promote allo-HSC engraftment.In summary, our studies identify a unique niche-associated Treg subset and adenosine as regulators of HSC quiescence, numbers, stress response, engraftment, and immune privilege, further highlighting potential clinical utility of niche Treg transfer in radiation-induced hematopoiesis failure and in allo-HSC engraftment (under revision in Cell Stem Cell).1 Fujisaki, J. et al. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature474, 216-219, doi:10.1038/nature10160 (2011). DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Memory CD8+ T Cells Play a Supporting Role in Hematopoiesis

The bone marrow (BM) has an important function as primary lymphoid organ through the process of hematopoiesis. This process is sustained by hematopoietic stem cells (HSCs) and their life-long production of new blood cells, which is made possible by their unique ability to self-renew. Next to this, BM also functions as a secondary lymphoid organ, as it can mediate primary T cell responses against invading pathogens. We and others have shown that activated T cells can influence the hematopoietic process through the production of pro-inflammatory cytokines. This indicates that adaptive immune responses and hematopoiesis are intertwined in the BM, though most of the cellular and molecular interactions that occur during this crosstalk are yet unknown.Based on previous observations that T cell deficient mice have altered hematopoiesis and that depletion of T cells from allogeneic BM grafts compromises HSC engraftment, we questioned to what extent BM T cells can directly affect the function of HSCs. To test this, we sorted and co-cultured murine BM T cells with HSCs (Lin-Sca-1+c-Kit+CD48-CD150+). We found that particularly BM CD8+ central memory (CD44+CD62L+) T cells (Tcm) enhance the capacity of HSCs to self-renew. Furthermore, we found that TCR-transgenic mice, which do not have memory T cells, have lower numbers of HSCs, which could subsequently be increased by transferring BM CD8+ Tcm. Remarkably, an increase in HSC numbers was also observed when HSCs were cultured with only supernatant derived from BM CD8+ Tcm. Moreover, HSCs cultured with supernatant from BM CD8+ Tcm and later transplanted in myeloablated hosts displayed a strongly enhanced ability to restore hematopoiesis. Importantly, the strong impact of BM T cells on HSCs was not only apparent in the steady state situation, but also following a viral infection with either acute or chronic lymphocytic choriomeningitis virus (LCMV). We could establish that both acute and chronic LCMV-specific CD8+ T cells or supernatant from these cells could increase the HSC self-renewal capacity.In conclusion, our findings demonstrate that BM memory CD8+ T cells can positively influence the function of HSC through soluble mediators. We postulate that this process is particularly relevant after immune activation, in order to protect and/or restore the HSC pool and the subsequent hematopoietic recovery. We are currently using a proteomics approach to identify these soluble mediator produced by CD8+ T cells. DisclosuresNo relevant conflicts of interest to declare.

Mir-199b, a Novel Tumor Suppressor miRNA In Acute Myeloid Leukemia With Prognostic Implications In FAB-M5 Subtype

Acute myeloid leukemia (AML) manifests a marked heterogeneity in both response to therapy and patient survival, observations that likely reflect its varied pathogenesis. Dysregulation of miRNAs that can act as tumor suppressors or oncogenes can result in tumorigenesis. One miRNA of interest is human miR-199b-5p, which is an intragenic miRNA encoded in the Dynamin 1 gene. Previously we demonstrated that miR-199b was significantly downregulated in AML and targets Podocalyxin and Discoidin Domain Receptor 1. Herein we investigated the functional role of miR-199b in AML and its prognostic implications. We first examined the relative miR-199b expression in steady state hematopoiesis and showed CD33+ myeloid progenitors had the highest miR-199b expression (p<0.03). In order to test whether suppression of miR-199b results in myeloproliferation, human bone marrow derived CD34+ cells were transduced with anti-miR-199b or control lentivirus particles. To examine the effect of miR-199b silencing on colony forming abilities, a CFU assay was performed. At day 16, silencing of miR-199b in CD34+ cells resulted in significant increases in CFU-GM colonies. We then investigated the effect of decreased miR-199b on hematopoietic stem cells (HSC) proliferation via aldehyde dehydrogenase assay. Anti-miR-199b CD34+ cells exhibited a significant increase in HSC numbers compared to control CD34+ cells demonstrating a proliferative advantage for HSC with decreased miR-199b. Via The Cancer Genome Atlas (TCGA), we analyzed the molecular and clinical characteristics of 166 AML cases to investigate a prognostic role for miR-199b. The Kaplan-Meier curves for high and low expression values of miR-199b and the observed distribution of miRNA expression revealed the highly expressed group had significantly better survival outcomes (p<0.016, log rank test). In addition, miR-199b expression varied by cytogenetic risk category in the least squared mean plot, with significantly higher miR-199b expression for the favorable category compared to the intermediate and poor categories combined (p<0.0001). Therefore, low expression of miR-199b predicts worse survival outcome and higher cytogenetic risk in AML. Additionally, there was significant difference between miR-199b expression across the AML subtypes with particularly low expression found in the M5 subtype. Furthermore, M5 subtype showed a poor prognosis with a one-year survival rate of only 25%, compared with 51% survival in the overall sample (p<0.024). With AML patients grouped into M5 versus other subtypes to look at the dichotomized miR-199b variable, we found that all of the M5 patients have low miR-199b expression (p<0.0001). Due to miR-199b significantly correlating with M5 subtype, the M5 cell line THP-1 was utilized to determine the epigenetic regulation of miR-199b in AML. Recently, epigenetic alterations such as histone modifications and DNA methylation have been shown to deregulate miRNA expression. Therefore, treatment of THP-1 cells for 24 hours with HDAC inhibitors AR-42 (2uM), Panobinostat (0.7uM), or Decitabine (demethylating agent, 5uM) showed miR-199b expression was significantly elevated upon AR-42 and Panobinostat treatment (p<0.0002 and p<0.0001 respectively). Furthermore, AR-42 and Panobinostat treated cells showed drastic apoptosis in both treatments. Thus, epigenetic regulation of miR-199b appears to be via histone modification. To further understand the hematopathological consequences of decreased miR-199b in the onset and development of myeloid leukemia, we employed a transduce / transplant mouse model. The Lin-Sca-1+Kit+ (LSK) population was isolated from Ly5.2 C57Bl6 mice, transduced with anti-miR-199b or control lentivirus particles, and transplanted into Ly5.1 C57Bl6 recipient mice. Short term (3.5 weeks) peripheral blood analysis revealed that loss of miR-199b results in significant increase of % neutrophils (p<0.001), % monocytes (p<0.04), and % eosinophils (p<0.02). Further analysis to determine the role of miR-199b in leukemogenesis is ongoing via this mouse model. Taken together, for the first time our results demonstrate that in vivo loss of miR-199b can lead to myeloproliferation and in vitro HDAC inhibitors can restore miR-199b-5p expression and promote apoptosis. Lastly, low miR-199b-5p in AML patients leads to worst overall survival and has prognostic significance for FAB-M5 subtype. Disclosures:No relevant conflicts of interest to declare.

The cell fate determinant Llgl1 influences HSC fitness and prognosis in AML

A unique characteristic of hematopoietic stem cells (HSCs) is the ability to self-renew. Several genes and signaling pathways control the fine balance between self-renewal and differentiation in HSCs and potentially also in leukemia stem cells. Recently, studies have shed light on developmental molecules and evolutionarily conserved signals as regulators of stem cells in hematopoiesis and leukemia. In this study, we provide evidence that the cell fate determinant Llgl1 (lethal giant larvae homolog 1) plays an important role in regulation of HSCs. Loss of Llgl1 leads to an increase in HSC numbers that show increased repopulation capacity and competitive advantage after transplantation. This advantage increases upon serial transplantation or when stress is applied to HSCs. Llgl1(-/-) HSCs show increased cycling but neither exhaust nor induce leukemia in recipient mice. Llgl1 inactivation is associated with transcriptional repression of transcription factors such as KLF4 (Krüppel-like factor 4) and EGR1 (early-growth-response 1) that are known inhibitors of HSC self-renewal. Decreased Llgl1 expression in human acute myeloid leukemia (AML) cells is associated with inferior patient survival. Thus, inactivation of Llgl1 enhances HSC self-renewal and fitness and is associated with unfavorable outcome in human AML.

Lnk deficiency partially mitigates hematopoietic stem cell aging

Upon aging, the number of hematopoietic stem cells (HSCs) in the bone marrow increases while their repopulation potential declines. Moreover, aged HSCs exhibit lineage bias in reconstitution experiments with an inclination toward myeloid at the expense of lymphoid potential. The adaptor protein Lnk is an important negative regulator of HSC homeostasis, as Lnk deficiency is associated with a 10-fold increase in HSC numbers in young mice. However, the age-related increase in functional HSC numbers found in wild-type HSCs was not observed in Lnk-deficient animals. Importantly, HSCs from aged Lnk null mice possess greatly enhanced self-renewal capacity and diminished exhaustion, as evidenced by serial transplant experiments. In addition, Lnk deficiency ameliorates the aging-associated lineage bias. Transcriptome analysis revealed that WT and Lnk-deficient HSCs share many aging-related changes in gene expression patterns. Nonetheless, Lnk null HSCs displayed altered expression of components in select signaling pathways with potential involvement in HSC self-renewal and aging. Taken together, these results suggest that loss of Lnk partially mitigates age-related HSC alterations.

Deletion of the Scl +19 enhancer increases the blood stem cell compartment without affecting the formation of mature blood lineages

The stem cell leukemia (Scl)/Tal1 gene is essential for normal blood and endothelial development, and is expressed in hematopoietic stem cells (HSCs), progenitors, erythroid, megakaryocytic, and mast cells. The Scl +19 enhancer is active in HSCs and progenitor cells, megakaryocytes, and mast cells, but not mature erythroid cells. Here we demonstrate that in vivo deletion of the Scl +19 enhancer (SclΔ19/Δ19) results in viable mice with normal Scl expression in mature hematopoietic lineages. By contrast, Scl expression is reduced in the stem/progenitor compartment and flow cytometry analysis revealed that the HSC and megakaryocyte-erythroid progenitor populations are enlarged in SclΔ19/Δ19 mice. The increase in HSC numbers contributed to enhanced expansion in bone marrow transplantation assays, but did not affect multilineage repopulation or stress responses. These results affirm that the Scl +19 enhancer plays a key role in the development of hematopoietic stem/progenitor cells, but is not necessary for mature hematopoietic lineages. Moreover, active histone marks across the Scl locus were significantly reduced in SclΔ19/Δ19 fetal liver cells without major changes in steady-state messenger RNA levels, suggesting post-transcriptional compensation for loss of a regulatory element, a result that might be widely relevant given the frequent observation of mild phenotypes after deletion of regulatory elements.

Absence of E-Selectin at the Vascular Niche Delays Hematopoietic Stem Cell Turn-Over.

Hematopoietic stem cells (HSC) reside in specialized niches in the bone marrow (BM), that regulate their survival, proliferation and differentiation. Two types of HSC niches have been reported:1.endosteal niches in close contact with osteoblasts, and2.endothelial niches near vascular sinuses.Whether these niches have distinct functions in controlling HSC fate remains unknown. One difference between these two niches is the constitutive expression of E-selectin and P-selectin by BM endothelial cells. E- and P-selectin are two cell adhesion molecules that modulate hematopoietic progenitor cell (HPC) survival, proliferation and differentiation in vitro. We now show that deletion of E-selectin, but not P-selectin, delays HSC turn-over in the BM in vivo. Mice lacking either E-selectin (E−/ −), P-selectin (P−/ −) or both (PE−/ −) were given bromo-deoxyuridine (BrdU) in their drinking water for up to 14 days. Lineage-negative c-KIT+ Sca-1+ CD34− (LKS34) cells were sorted from the BM and stained for BrdU incorporation into genomic DNA. Although it took only 3.6 days for 50% of LKS34 cells from wild-type (WT) and P−/ − mice to incorporate BrdU, 9 days were required for 50% BrdU incorporation in LKS34 cells from E−/ − and PE−/ − double KO mice. Thus, HSC cycling time is 2.5 times slower in the absence of E-selectin. To confirm these findings, LKS cells were stained with rhodamine123, a vital dye that is retained by metabolically active cells but effluxed from quiescent HSC. A higher proportion of LKS cells from E−/ − mice were rhodamine dull (34±2%) than WT LKS (23±1%; p=0.037) confirming that a greater proportion of HSC from E−/ − mice are quiescent. To further support these findings, we determined the effect of E-selectin deletion on HSC recovery following cytotoxic stress with a single dose of 5-fluorouracil (5FU 150mg/kg). As KIT is strongly down-regulated in the BM of 5FU-treated mice, we examined frequency and BrdU incorporation in Lin− Sca1+ CD41− CD48− CD150+ long-term reconstituting HSC. We found HSC recovery to be enhanced in E−/ − mice with a 5-fold increase in HSC numbers per femur compared to WT mice at day 7 post-5FU. Despite the more rapid recovery of E−/ − HSC, BrdU incorporation remained significantly lower in E−/ − HSC on days 3 and 7 post-5FU suggesting the decreased HSC turn-over in the absence of E-selectin protects them from the cytotoxic effect of 5FU. To determine whether this effect was mediated by the two described E-selectin receptors PSGL-1 and/or CD44, BrdU incorporation experiments were repeated with mice lacking both the PSGL-1 and CD44 genes. LKS cell turnover in these mice was identical to that of WT suggesting that the effect is mediated by a distinct unknown receptor(s) on HSC. The fact that a novel E-selectin receptor on HSC/HPC is involved was confirmed both using flow cytometry with selectin-IgM chimeras as well as cell adhesion assays using plastic-adsorbed selectin-IgG chimeras. In both assays, 90-95% of LKS cells from CD44−/ − PSGL-1−/ − double KO mice bound E-selectin whereas adhesion to P-selectin was completely lost. Taken together our findings suggest that1.E-selectin, whose constitutive expression is restricted to BM endothelial cells, plays an important role in the regulation HSC turnover in vivo,2.endothelial niches, where E-selectin is expressed, support more rapid HSC turn-over within the BM, and3.this effect is mediated by unknown E-selectin receptors distinct from PSGL-1 or CD44.

Revealing the Interplay of Extrinsic Versus Intrinsic Regulators of Self-Renewal in Hoxb4-Transduced HSCs.

By modulating levels of Pbx-1, a homeodomain protein and DNA binding cofactor of Hoxb4, it is possible to generate pluripotent, ultracompetitive in vivo repopulating Hoxb4hi and Pbx1lo hematopoietic stem cells (HSCs) (Immunity, 2003, Krosl et al). Despite the tremendous regenerative potential demonstrated by these cells, the total in vivo HSC pool in recipients remained within physiological limits (~20 000 HSC/mouse), implying that environmental factors (niche availability?) restricted their expansion. These studies thus implied that bypassing the in vivo constraints of niche availability by culturing the transduced HSCs in vitro might reveal the intrinsic expansion potential of these cells. To study this hypothesis, Hoxb4hiPbx1lo transduced HSCs were generated by co-infecting primary mouse bone marrow (BM) cells with retroviruses encoding antisense Pbx1 cDNA plus YFP, and Hoxb4 plus GFP. At the end of the co-culture with retroviral producers, double gene transfer (Hoxb4hiPbx1lo ) was ~20% as determined by flow cytometric analysis of GFP and YFP co-expression. These cells were then cultured in the presence of serum and cytokines for additional 12–16 days, and the numbers of total cells, clonogenic progenitors and HSCs were determined at regular intervals. To quantify the magnitude of the in vitro HSC expansion, CRU assays for determination of cells with long-term lympho-myeloid repopulation potential were performed after removal of BM cells from co-culture with retroviral producers, and then at various time points. The increase in HSC numbers over time in culture was calculated as the ratio between absolute numbers of Hoxb4hiPbx1lo HSCs at a given time point, and their numbers at initiation of culture. In our initial experiment, numbers of Hoxb4hiPbx1lo CRU increased from 1000 at day 0 to 1.2 x 107 at day 12, for a net 10 000-fold expansion. After transplantation into irradiated mice, these cells underwent an additional 360-fold in vivo expansion to regenerate HSC pools of recipients up to, but not above, normal levels. Southern blot analyses of proviral integrations in DNA isolated from sorted Mac-1+, B-220+ and CD4+CD8+ cells derived from primary and secondary recipients demonstrated that cultured Hoxb4hiPbx1lo HSCs retained their ability to differentiate into all hematopoietic lineages examined. To estimate the numbers of distinct Hoxb4hiPbx1lo HSCs in cultures, BM cells from primary recipients of 1 x 106 cells from a 16-day expansion culture were plated in methylcellulose, and the clonal origin of individual myeloid colonies was determined by Southern blot analysis. These experiments showed that several distinct HSC clones reconstituted each recipient, and that some clones reconstituted at least 2 mice, illustrating the polyclonal nature and self-renewal activity of the cultured HSCs. Together, our experiments show that after unprecedented levels of in vitro expansion, Hoxb4hiPbx1lo HSCs remained capable of reconstituting myeloid and lymphoid systems of primary and secondary recipients, and yet responsive to the in vivo regulatory mechanisms that limit total stem cell pool size, reflecting the interplay between autonomous and non-cell autonomous control of HSC self-renewal. Decreasing Pbx1 levels in Hoxb4 overexpressing HSCs could thus be used as a tool for studying mechanisms of self-renewal and replicative senescence, and may lead to clinically relevant protocols for HSC expansion.