Activity Of Hematopoietic Stem And Progenitor Cells Research Articles

STAT3 protects hematopoietic stem cells by preventing activation of a deleterious autocrine type-I interferon response.

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.

MDia formins form hetero-oligomers and cooperatively maintain murine hematopoiesis.

mDia formin proteins regulate the dynamics and organization of the cytoskeleton through their linear actin nucleation and polymerization activities. We previously showed that mDia1 deficiency leads to aberrant innate immune activation and induces myelodysplasia in a mouse model, and mDia2 regulates enucleation and cytokinesis of erythroblasts and the engraftment of hematopoietic stem and progenitor cells (HSPCs). However, whether and how mDia formins interplay and regulate hematopoiesis under physiological and stress conditions remains unknown. Here, we found that both mDia1 and mDia2 are required for HSPC regeneration under stress, such as serial plating, aging, and reconstitution after myeloid ablation. We showed that mDia1 and mDia2 form hetero-oligomers through the interactions between mDia1 GBD-DID and mDia2 DAD domains. Double knockout of mDia1 and mDia2 in hematopoietic cells synergistically impaired the filamentous actin network and serum response factor-involved transcriptional signaling, which led to declined HSPCs, severe anemia, and significant mortality in neonates and newborn mice. Our data demonstrate the potential roles of mDia hetero-oligomerization and their non-rodent functions in the regulation of HSPCs activity and orchestration of hematopoiesis.

AA2P-mediated DNA demethylation synergizes with stem cell agonists to promote expansion of hematopoietic stem cells

Small molecules have enabled expansion of hematopoietic stem and progenitor cells (HSPCs), but limited knowledge is available on whether these agonists can act synergistically. In this work, we identify a stem cell agonist in AA2P and optimize a series of stem cell agonist cocktails (SCACs) to help promote robust expansion of human HSPCs. We find that SCACs provide strong growth-promoting activities while promoting retention and function of immature HSPC. We show that AA2P-mediated HSPC expansion is driven through DNA demethylation leading to enhanced expression of AXL and GAS6. Further, we demonstrate that GAS6 enhances the serial engraftment activity of HSPCs and show that the GAS6/AXL pathway is critical for robust HSPC expansion.

The Role of Hematopoietic Stem Cells in Lung Cancer Response to Radiation Therapy

Non-small cell lung carcinoma (NSCLC) comprises approximately 85% of all lung cancer cases in the United States. We demonstrated that after radiation therapy (RT), bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) are recruited to the NSCLC tumor microenvironment (TME). These HSPCs seemed to positively affect tumor regrowth as their numbers directly correlated with regrowth rates. The TME is comprised of tumor tissue, stromal cells, immune cells, bone marrow-derived cells, and extra cellular matrix (ECM) proteins and has emerged as one of the predominant defensive weapons with which cancer can resist various forms of therapeutic intervention. The ability of tumor-associated HSPCs to maintain hematopoietic potency in tumors suggested a prominent role of these cells in tumor physiology, however; what was missing was a mechanistic understanding of: 1) how HSPCs participate in tumor growth post-RT and 2) how HSPCs are maintained within tumors. To answer these questions, we established NSCLC tumors in mice using Lewis Lung Carcinoma (LLC) cells and assessed tumor-associated HSPCs activity and molecular changes in the TME following RT. We observed that RT induces the TME to produce colony stimulating factor (CSF)-1, a factor that promotes the differentiation of HSPCs into tumor supportive M2-macrophages. Adoptive transplantation of dsRed labeled HSPCs into tumor bearing mice followed by RT directly showed the differentiation of HSPCs into M2-macrophages and a concomitant increase in the regrowth of tumors post-RT. Use of a therapeutic inhibitor of the CSF-1 receptor (GW2580) prevented HSPC differentiation in to M2-macrophages and significantly decreasing regrowth rates. We next assessed how tumor-associated HSPCs maintain their hematopoietic potency within the TME. Analysis of tumors demonstrated high expression levels of the laminin 5-1-1 isoform, which is known to bind CD49f (integrin alpha 6), a marker of functional HSPCs. Tumors exposed to RT demonstrated significantly reduced expression of both laminin 5-1-1 and CD49f. When bound, CD49f inhibits phosphorylation of focal adhesion kinase (FAK) resulting in maintenance of pluripotent stem cells. Analysis of tumor-associated HSPCs demonstrated significantly higher levels of phosphorylated FAK following tumor exposure to RT. Since RT induces HSPC differentiation in to M2-macophages, this observation suggests that tumor-associated HSPC functionality is maintained through the interaction between laminin 5-1-1- and CD49f. The identification of single HSPCs that differentiated in to M2-macrophages and which expressed phosphorylated FAK supports this conclusion. Taken together, this data province's evidence of a mechanism wherein RT disrupts laminin 5-1-1/CD49f interactions releasing tumor-associated HSPCs from maintenance signals. Concomitantly, RT induces the production of CSF-1 within the TME, which induces these “released” HSPCs to differentiate into tumor supportive M2-macrophages and promote tumor regrowth post-RT. Overall, these studies define a new understanding of tumor biology in which HSPCs are significant mediators of tumor response to RT. These findings also identify new cellular and molecular pathways to target in the treatment of NSCLC to achieve more optimal clinical outcomes.

Nanoparticles targeting hematopoietic stem and progenitor cells: Multimodal carriers for the treatment of hematological diseases.

Modern-day hematopoietic stem cell (HSC) therapies, such as gene therapy, modify autologous HSCs prior to re-infusion into myelo-conditioned patients and hold great promise for treatment of hematological disorders. While this approach has been successful in numerous clinical trials, it relies on transplantation of ex vivo modified patient HSCs, which presents several limitations. It is a costly and time-consuming procedure, which includes only few patients so far, and ex vivo culturing negatively impacts on the viability and stem cell-properties of HSCs. If viral vectors are used, this carries the additional risk of insertional mutagenesis. A therapy delivered to HSCs in vivo, with minimal disturbance of the HSC niche, could offer great opportunities for novel treatments that aim to reverse disease symptoms for hematopoietic disorders and could bring safe, effective and affordable genetic therapies to all parts of the world. However, substantial unmet needs exist with respect to the in vivo delivery of therapeutics to HSCs. In the last decade, in particular with the development of gene editing technologies such as CRISPR/Cas9, nanoparticles (NPs) have become an emerging platform to facilitate the manipulation of cells and organs. By employing surface modification strategies, different types of NPs can be designed to target specific tissues and cell types in vivo. HSCs are particularly difficult to target due to the lack of unique cell surface markers that can be utilized for cell-specific delivery of therapeutics, and their shielded localization in the bone marrow (BM). Recent advances in NP technology and genetic engineering have resulted in the development of advanced nanocarriers that can deliver therapeutics and imaging agents to hematopoietic stem- and progenitor cells (HSPCs) in the BM niche. In this review we provide a comprehensive overview of NP-based approaches targeting HSPCs to control and monitor HSPC activity in vitro and in vivo, and we discuss the potential of NPs for the treatment of malignant and non-malignant hematological disorders, with a specific focus on the delivery of gene editing tools.

Pp90RSK Isoforms Play Distinct Roles during Hematopoiesis

Ribosomal S6 Kinases (RSKs) include a family of serine/threonine kinases that regulate cell proliferation and survival. In humans, four RSK isoforms (RSK1-4) have been identified. Although they are 73-80% conserved in sequence homology, RSK isoforms are reported to have distinct functions. RSKs phosphorylate cyclic AMP response element binding protein (CREB) which is a regulator of hematopoietic proliferation and differentiation. RSK1 is hyperactivated in AML and is therefore a potential therapeutic target for acute myeloid leukemia (AML). Therefore, inhibition of RSK requires understanding of its role during normal hematopoiesis. Furthermore, the role of RSK isoforms during hematopoiesis and leukemogenesis has not been well characterized. To study the expression of RSK isoforms in hematopoiesis, we cultured human cord blood CD34+ (CBCD34+) hematopoietic stem and progenitor cells (HSPCs) in differentiating media containing SCF, Flt3L, TOP, IL-3, IL-6, GM-CSF, and EPO and performed qPCR with mRNA of the RSK isoforms at different stages of maturation up to 14 days. RPS6KA1 (RSK1) was expressed throughout hematopoiesis, but RPS6KA3 (RSK2) and RPS6KA2 (RSK3) showed higher expression by 2-fold (p<0.05) in the earlier stages of normal hematopoiesis. Moreover, RPS6KA3 is highly expressed in CD38-CD34+ HSCs compared with other progenitor populations (LMPP, MPP, CMP, GMP, and MEP) isolated from human cord blood. RPS6KA2 expression was higher in LMPP by 7-fold (p<0.05) and CMP by 3-fold (p<0.05) compared with HSCs but was barely detectable in MEPs. RPS6KA1 was ubiquitously expressed in all progenitor populations. These results suggest that RSK2 and RSK3 have distinct functions during early myelopoiesis. To study the requirement of RSK isoforms during hematopoiesis, we knocked down RSK expression by transducing lentivirus expressing isoform-specific shRNAs or scramble controls into human CBCD34+ cells. On day 5 following transduction, cells were sorted and plated in methylcellulose media and assessed for the colony-forming activity of RSK knockdown in HSPCs. While RSK3 knockdown increased BFU-E colonies by 1.5-fold (p<0.05) compared with control cells, RSK1 knockdown decreased BFU-E and CFU-E erythroid colonies by 2-fold (p<0.05) but increased CFU-GM colonies by 2-fold (p<0.05) compared with control cells. However, RSK2 knockdown did not show any significant effects on colony-forming activity of HSPCs. We confirmed the isoform-specific effect of RSKs in normal hematopoiesis using liquid culture assays. We collected transduced cells at day 8, 11, and 14 after transduction and analyzed cell populations by flow cytometry. Similar to colony formation assay results, RSK3 knockdown increased the CD71+ erythroid population by 1.5-fold (p<0.001), but suppressed production of the CD14+ monocyte population. RSK1 knockdown enhanced production of the CD11b+/CD14+ myeloid population, but inhibited the CD71+ erythroid production by 1.5-fold (p<0.001) compared with control HSPCs. There was no significant change in blood cell differentiation in RSK2 knockdown cells. These data demonstrate that RSK1 and RSK3 exert opposite functions during erythroid and myeloid differentiation of HSPCs, suggesting a novel role for RSK isoforms as a determinant of early fate decisions of HSCs. Disclosures No relevant conflicts of interest to declare.

Exercise finds its niche

Heart Disease Regular physical activity is associated with a lower rate of death from heart disease, but the underlying mechanisms are not fully understood. Frodermann et al. examined the effect of exercise on cardiovascular inflammation, a known risk factor for atherosclerosis, by studying mice that voluntarily ran for long distances on exercise wheels. They found that these physically active mice had fewer inflammatory cells (leukocytes) than sedentary mice, an effect they traced to diminished activity of hematopoietic stem and progenitor cells (HSPCs). The lower activity of HSPCs was due at least in part to exercise-induced reduction in the levels of leptin, a hormone produced by fat tissue that regulates cells within the hematopoietic bone marrow niche. Nat. Med. 25 , 1761 (2019).

Interferon gamma inhibits the differentiation of mouse adult liver and bone marrow hematopoietic stem cells by inhibiting the activation of notch signaling

BackgroundThe paradigm of hematopoietic stem and progenitor cells (HSPCs) has become accepted ever since the discovery of adult mouse liver hematopoietic stem cells and their multipotent characteristics that give rise to all blood cells. However, differences between bone marrow (BM) and liver hematopoietic stem cells and the hematopoietic microenvironment remain poorly understood. In addition, the regulation of the liver hematopoietic system remains unknown.MethodsClone formation assays were used to confirm that the proliferation of adult mouse liver and bone marrow HSPCs. Model mice with different interferon gamma (IFN-γ) levels and a co-culture system were used to detect the differentiation of liver HSPCs. The γ-secretase inhibitor (GSI) and the JAK/STAT inhibitor ruxolitinib and cell culture assays were used to explore the molecular mechanism by which IFN-γ impairs HSPC proliferation and differentiation.ResultsThe colony-forming activity of liver and bone marrow HSPCs was inhibited by IFN-γ. Model mice with different IFN-γ levels showed that the differentiation of liver HSPCs was impaired by IFN-γ. Using a co-culture system comprising liver HSPCs, we found that IFN-γ inhibited the development of liver hematopoietic stem cells into γδT cells. We then demonstrated that IFN-γ might impair liver HSPC differentiation by inhibiting the activation of the notch signaling via the JAK/STAT signaling pathway.ConclusionsIFN-γ inhibited the proliferation of liver-derived HSPCs. IFN-γ also impaired the differentiation of long-term hematopoietic stem cells (LT-HSCs) into short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MPPs) and the process from LSK (Lineage−Sca-1+c-Kit+) cells to γδT cells. Importantly, we proposed that IFN-γ might inhibit the activation of notch signaling through the JAK/STAT signaling pathway and thus impair the differentiation process of mouse adult liver and BM hematopoietic stem cells.

Oxidant Signaling Mediated by Nox2 in Neutrophils Promotes Regenerative Myelopoiesis and Tissue Recovery following Ischemic Damage.

Ischemic tissue damage activates hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM)-generating myeloid cells, and persistent HSPC activity may drive chronic inflammation and impair tissue recovery. Although increased reactive oxygen species in the BM regulate HSPC functions, their roles in myelopoiesis of activated HSPCs and subsequent tissue recovery during ischemic damage are not well understood. In this paper, we report that deletion of Nox2 NADPH oxidase in mice results in persistent elevations in BM HSPC activity and levels of inflammatory monocytes/macrophages in BM and ischemic tissue in a model of hindlimb ischemia. Ischemic tissue damage induces oxidants in BM such as elevations of hydrogen peroxide and oxidized phospholipids, which activate redox-sensitive Lyn kinase in a Nox2-dependent manner. Moreover, during tissue recovery after ischemic injury, this Nox2-ROS-Lyn kinase axis is induced by Nox2 in neutrophils that home to the BM, which inhibits HSPC activity and inflammatory monocyte generation and promotes tissue regeneration after ischemic damage. Thus, oxidant signaling in the BM mediated by Nox2 in neutrophils regulates myelopoiesis of HSPCs to promote regeneration of damaged tissue.

Dynamics of genetically engineered hematopoietic stem and progenitor cells after autologous transplantation in humans.

Hematopoietic stem and progenitor cells (HSPC) are endowed with the role of generating and maintaining lifelong the extremely diverse pool of blood cells1. Clinically, transplantation of human HSPC from an allogeneic healthy donor or infusion of autologous gene-corrected HSPC can effectively replenish defective blood cell production caused by congenital or acquired disorders2-9. However, due to methodological and ethical constraints that have limited the study of human HSPC primarily to in vitro assays10 or xenotransplantation models11,12, the in vivo activity of HSPC has to date remained relatively unexplored in humans13-16. Here we report a comprehensive study of the frequencies, dynamics and output of seven HSPC subtypes in humans that was performed by tracking 148,093 individual clones in six patients treated with lentiviral gene therapy using autologous HSPC transplantation and followed for up to 5 years. We discovered that primitive multipotent progenitor and hematopoietic stem cell (HSC) populations have distinct roles during the initial reconstitution after transplant, compared with subsequent steady-state phases. Furthermore, we showed that a fraction of in vitro-activated HSC are resilient and undergo a defined delayed activation period upon transplant. Finally, our data support the concept that early lymphoid-biased progenitors might be capable of long-term survival, such that they can be maintained independently of their continuous production from HSC. Overall, this study provides comprehensive data on HSPC dynamics after autologous transplantation and gene therapy in humans.

Daily Onset of Light and Darkness Differentially Controls Hematopoietic Stem Cell Differentiation and Maintenance

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

Expansion and Maintenance of Hematopoietic Stem and Progenitor Cells in Course of Long-Term Inhibition of CXCR4/CXCL12 Signaling

During the past two decades peripheral blood stem cells have become the favored graft source for HSCT with 80 % of allogeneic and almost 100 % of autologous HSCT performed with mobilized blood. The critical role of the interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 for retention and migration of hematopoietic stem and progenitor cells (HSPC) has been well established. Interference with CXCR4/CXCL12 signalling iscurrentlybeing exploited as a strategy to mobilize HSPC indirectly with the most clinically relevant mobilizing agent to date, G-CSF as well as directly with the bicyclam CXCR4 antagonist Plerixafor (AMD3100).In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of CXCR4/CXCL12 axis within the HSPC compartment were investigated in healthy C57BL/6 mice using the non-peptidic small molecule CXCR4 antagonists Plerixafor and ALT1188 along with the Protein-EpitopeMimeticsInhibitor POL5551.Up to 12-14 fold higher mobilization efficiency was achieved by applying the antagonists via two weeks of continuous infusion (up to 8-10x104 CFU-C and LSK/ml) as compared to bolus treatment (4-6x103 CFU-C and LSK/ml) or 5-day course of G-CSF (3-6x103 CFU-C/ml).Despite dramatic increase in numbers of circulating HSPC, the BM HSPC pool dis not decrease; in fact it expanded up to 2-4-fold compared to steady state reservoir (sham-operated control mice) as measured by immunophenotypical (LSK SLAM) and functional (e.g. serial competitive transplantation) properties of the cells. Thus, in contrast to genetically CXCR4 ablatedHSPC, the reversible long-term blockade of the receptor did not diminish the long-term repopulating capacity of HSPC.Cell cycle analysis showed a 2-3-fold increase in cycling activity of BM HSPC: only 10-20% of LSK and 30-40 % of LSK SLAM cells were found to be quiescent (in G0 phase of the cell cycle) after two weeks of CXCR4 antagonist infusion versus 50-60 % of LSK and 70 % of LSK SLAM found in G0 under homeostatic conditions. This increased proliferation was very similar to the one induced transiently at day 3 G-CSF treatmentand would conceivably explain the sustained mobilization without concomitant depletion of the BM HSPC pool. Profiling of differentially treated BM HSC (LSK SLAM) via microarray analysis did not reveal substantial effects of CXCR4 inhibitor infusion on the expression signature.Ofnote, major cytological changes typically associated with G-CSF induced mobilization, e.g. depletion of bone lining osteoblast lineage cells and macrophages, were not detected in continuous infusion of POL5551 exposed BM suggesting limitedeffects within the BM niche compartment. Moreover analysis of the BM HSPC after different washout periods at the end of continuous infusion treatment revealed a rapid (within 1-3 days after discontinuation of infusion) reestablishment of steady state HSPC numbers in the BM.Our data suggest that prolonged pharmacologic blockade of the CXCR4/CXCL12 axis using multiple small molecule inhibitorsrepresents an approach thatreleasesHSPCwith efficiency superiorto any other knownmobilization strategybut also may serve as an effective method induce cell cycling and thus expand BM HSPCs. [Display omitted] DisclosuresLevesque:GlycoMimetics: Equity Ownership.

Histone Deacetylase Inhibitors As Enhancers of Human Hematopoietic Stem Cell Activity

IntroductionThe identification of compounds which increase the number but also keep or enhance the activity of hematopoietic stem and progenitor cells (HSPCs) could improve the clinical outcome after autologous and allogeneic hematopoietic stem cell transplantation (HSCT). So far, most attempts to increase HSPC numbers ex vivo have been unsuccessful because of either inadequate cell numbers and/or loss of engraftment capacity and HSPC quality during expansion. Executing drug discovery screens in vertebrate systems is generally expensive, technically challenging and time consuming. Therefore, the zebrafish represents a versatile vertebrate model allowing HSPC regulation and development studies during embryogenesis and adulthood.MethodsWe used a semi-automated chemical screen to identify modulators of HSPC activity by transgenic (cmyb:EGFP) zebrafish embryos. Verification of identified histone deacetylase (HDAC) inhibitor candidates was carried out in vitro using human CD34+ HSPCs which were isolated from apharesis samples of healthy donors after mobilization with G-CSF by anti-CD34 coupled magnetic beads. The influence of HDAC inhibitors on HSPC phenotype, gene expression pattern as well as adhesion and migration capacity was analyzed after 5 days of treatment either in single or in co-culture with bone marrow-derived mesenchymal stromal cells (MSCs).ResultsThe HDAC inhibitors valproic acid (VPA), resminostat and entinostat were shown to significantly amplify the number of hematopoietic precursors in a chemical in vivo zebrafish embryo screen (Arulmozhivarman et al. 2016). Treatment of human CD34+ HSPCs with these compounds in vitro resulted in a significantly increased percentage of CD34+CD90+ cells up to 60% compared to controls which showed only 2% of double positive cells as well as in 3-fold higher CD34+ and about 12-fold higher CD34+CD90+ absolute cell numbers. CD34 is a well-known surface marker for human immature HSPCs and in combination with CD90 it defines a potentially pluripotent subpopulation. In a co-culture setting, we found that VPA treated cells showed 2 to 3-fold higher attachment capacity on MSCs compared to the control cells. This finding led us to quantify the adhesive capacity of cells using static adhesion assay and atomic force microscopy based single-cell force spectroscopy (AFM-SCFS). Interestingly, detachment forces of VPA treated HSPCs were 3 times increased on MSCs compared to control cells and a similar phenotype was observed by static adhesion assay. Accordingly, the chemokine-mediated migration of VPA treated HSPCs towards SDF-1/CXCL12 was inhibited. To reveal underlying downstream molecules and mechanisms mediating the modified cellular characteristics, a whole genome expression array was carried out for HSPCs treated with VPA in comparison to untreated controls. Amongst a panel of regulated genes, the melanoma cell adhesion molecule (MCAM/CD146), Notch 3 and its downstream effector Hes-1 as well as the SDF-1 receptor CXCR-4 were found to be significantly changed. Whereas the decreased expression of CXCR4 correlates with the inhibited migration potential of VPA-treated HSPCs and Notch-3/Hes-1 have a known role in normal and malignant hematopoiesis (Gu et al. 2016), the induced expression of MCAM on HSPCs was not described so far. The result was confirmed by flow cytometry which revealed a 40% MCAM-positive cell population when treated with VPA, whereas the control showed only negative cells. Additionally, significant higher transcript levels were detected for MCAM by quantitative real-time PCR in VPA expanded cells. Recently, we described a role of MCAM in MSCs for the hematopoietic support (Stopp et al. 2013). The inducible expression in HSPCs may reflect homotypic interactions which preserve a more immature subpopulation with high stem cell activity.ConclusionWe describe for the first time the ability of the HDAC inhibitors VPA, resminostat and entinostat to efficiently expand CD34+ HSPCs ex vivo especially supporting a CD34+CD90+ subpopulation with potentially high stem cell activity. Moreover, a potential role of MCAM in this context may offer new perspectives of the HSPC expansion ex vivo for the improvement of HSCT. DisclosuresNo relevant conflicts of interest to declare.

In Vivo Chemical Screen in Zebrafish Embryos Identifies Regulators of Hematopoiesis Using a Semiautomated Imaging Assay

Hematopoietic stem and progenitor cells (HSPCs) generate all cell types of the blood and are crucial for homeostasis of all blood lineages in vertebrates. Hematopoietic stem cell transplantation (HSCT) is a rapidly evolving technique that offers potential cure for hematologic cancers, such as leukemia or lymphoma. HSCT may be autologous or allogenic. Successful HSCT depends critically on the abundance of engraftment-competent HSPCs, which are currently difficult to obtain in large numbers. Therefore, finding compounds that enhance either the number or the activity of HSPCs could improve prognosis for patients undergoing HSCT and is of great clinical interest. We developed a semiautomated screening method for whole zebrafish larvae using conventional liquid handling equipment and confocal microscopy. Applying this pipeline, we screened 550 compounds in triplicate for proliferation of HSPCs in vivo and identified several modulators of hematopoietic stem cell activity. One identified hit was valproic acid (VPA), which was further validated as a compound that expands and maintains the population of HSPCs isolated from human peripheral blood ex vivo. In summary, our in vivo zebrafish imaging screen identified several potential drug candidates with clinical relevance and could easily be further expanded to screen more compounds.

Metabolism Regulates Cellular Functions of Bone Marrow-Derived Cells used for Cardiac Therapy.

Administration of bone marrow-derived mononuclear cells (BMC) may increase cardiac function after myocardial ischemia. However, the functional capacity of BMC derived from chronic heart failure (CHF) patients is significantly impaired. As modulation of the energy metabolism allows cells to match the divergent demands of the environment, we examined the regulation of energy metabolism in BMC from patients and healthy controls (HC). The glycolytic capacity of CHF-derived BMC is reduced compared to HC, whereas BMC of metabolically activated bone marrow after acute myocardial infarction reveal increased metabolism. The correlation of metabolic pathways with the functional activity of cells indicates an influence of metabolism on cell function. Reducing glycolysis without profoundly affecting ATP-production reversibly reduces invasion as well as colony forming capacity and abolishes proliferation of CD34(+) CD38(-) lin(-) hematopoietic stem and progenitor cells (HSPC). Ex vivo inhibition of glycolysis further reduced the pro-angiogenic activity of transplanted cells in a hind limb ischemia model in vivo. In contrast, inhibition of respiration, without affecting total ATP production, leads to a compensatory increase in glycolytic capacity correlating with increased colony forming capacity. Isolated CD34(+) , CXCR4(+) , and CD14(+) cells showed higher glycolytic activity compared to their negative counterparts. Metabolic activity was profoundly modulated by the composition of media used to store or culture BMC. This study provides first evidence that metabolic alterations influence the functional activity of human HSPC and BMC independent of ATP production. Changing the balance between respiration and glycolysis might be useful to improve patient-derived cells for clinical cardiac cell therapy. Stem Cells 2016;34:2236-2248.

Conditioned Medium Represents a Useful Solution to Increase the Expansion of Multipotent Progenitors with Strong Platelet Engraftment Activity

Delayed neutrophil and platelet engraftment is a significant issue of cord blood (CB) transplantation. Ex vivo expansion of CB hematopoietic stem and progenitor cells (HSPC) before infusion has been shown to accelerate hematopoietic recovery in patients. Recently, we reported that serum free medium (SFM) conditioned with osteoblasts derived from human bone marrow (BM) mesenchymal stromal cells, referred as M-OST CM, was superior to SFM or MSC CM for the expansion of CB CD34+ cells, and that HSPC expanded in M-OST CM provided better platelet engraftment. Since large number of expanded cells were transplanted in the original study, it was not possible to estimate the increased expansion of HSPC with short-term (ST) and long-term (LT) thrombopoietic and BM engraftment activities. The objectives herein were to investigate these shortcomings using limit dilution analysis (LDA) in transplantation assay and to investigate the cellular mechanisms at play.M-OST CM was prepared by conditioning SFM with immature M-OST overnight. CB CD34+ cells were expanded in M-OST CM or in SFM (defined as control) for 7 days with SCF, FL and TPO. CB cell expansion was significantly greater in M-OST CM cultures vs. SFM control (2.4 ±0.9 fold, mean ± SD, n=4, p=0.01). LDA transplantation assays were done by infusing the progeny of 500-8000 CD34+ cells in NSG mice. First, we compared the ST (< 31 days) and LT (˃ 100 days) thrombopoietic activities of expanded HSPC by measuring circulating human platelets (hPLT). The threshold for hPLT engraftment was set above the mean background level measured in control mice + 1SD. The median ST levels of hPLT in M-OST mice tended to be greater (2.5-fold, p˃0.05) in M-OST recipients (21 mice/condition, n=2). The frequency of ST hPLT HSPC estimated by LDA was 3.4 ±0.2 fold higher in M-OST CM cultures though the difference vs. control was not significant (p=0.11). LT hPLT levels were significantly greater in M-OST recipients (median 33 vs. 8 hPLT/uL blood, p=0.0027). Consistent with this, the frequency of HSPC with LT hPLT engraftment was increased in M-OST CM cultures (3.5±0.8 fold, p<0.04). Considering the differences in cell expansion, the net expansion of HSPC with ST and LT hPLT engraftment were raised by 5.5 ±1.7 and 6.0 ±3.4 fold in M-OST CM cultures vs. control (n=2). Next, LT human BM engraftment was analyzed at week 16. Preliminary results (13 mice/condition) suggest that the frequency of LT Scid repopulating cells (SRC) was increased by 27% in the M-OST CM culture vs. SFM control (frequency of 1/2878 vs. 1/3626 of day 0 starting cell).Next, we set to determine how M-OST CM increases the thrombopoietic activity of expanded CB HSPC. First, cytometry analysis (CD34, CD38, CD45RA, CD90 and CD123) revealed that M-OST CM preferentially increased the expansion of common myeloid progenitors (CMP, 8-fold, p=0.2, n=3), megakaryocyte-erythroid progenitors (MEP, 7-fold, p=0.02) and granulocyte-macrophage progenitors (GMP, 9-fold, p=0.02) vs. SFM control. Expansion of HSC-enriched cells was unchanged while that of multipotent progenitors (MPP) was reduced 2-fold (p<0.05). We set to confirm these results by culturing purified primary CB HSPC subsets in M-OST CM or SFM; M-OST CM induced greater expansions of MEP (3-fold), GMP (˃10-fold) while expansion in MPP cultures was greater with SFM control (1.5-fold). No growth was noted with the HSC and CMP cultures likely due to low sort yields.To complement these findings, we measured the expansion of myeloid CFU progenitors and long term culture-initiating cells (LTC-IC) by LDA. The total number of CFU was increased 2.4-fold (<0.02, n=4) by M-OST CM due mostly to increased expansion of CFU-G/GM colonies (2-fold, p<0.05) and BFU-E (2-fold, p=0.05). M-OST CM also sustained a 3.4-fold increase in LTC-IC expansion vs. SFM culture, though this finding remains to be confirmed in ongoing experiments.Finally, we investigated the effect of M-OST CM on the chemotaxis of HSPC toward SDF-1 since we previously reported increased expression of its receptor CXCR4 on CB cells in M-OST CM cultures. M-OST CM HSPC showed a modest 15% increase in migration vs. SFM control (n=4, p=0.10).In conclusion, our results demonstrate that the ST and LT hPLT engraftment activities of ex vivo expanded CB HSPC can be increased 5-6 folds by the use of M-OST CM due to the expansion of immature CB HSPC subsets including perhaps LT SRC. Whether M-OST CM can also modulate the homing activity of HSPC remains unclear. DisclosuresNo relevant conflicts of interest to declare.

The Mir-193 Family Antagonizes Stem Cell Pathways and Is a Potent Tumor Suppressor in Childhood and Adult Acute Myeloid Leukemia

MicroRNAs (miRNAs) are essential for maintenance and differentiation of normal hematopoietic cells and their dysregulation is strongly implicated in leukemias. In order to identify tumor suppressor miRNAs in the context of hematological malignancies, we performed two complementary miRNA expression screenings in normal hematopoiesis as well as in childhood and adult acute myeloid leukemias (AML). We reasoned that tumor suppressor miRNAs are upregulated in mature myeloid cells, as compared to normal hematopoietic stem and progenitor cells (HSPCs), and downregulated in AML.Based on this screening strategy, we identified the miR-193 family members as potent suppressors of HSPC activity and AML growth. During normal hematopoiesis mmu-miR-193a-3p is exclusively expressed in mature myeloid cells and absent in normal HSPCs. Accordingly, in a cohort of 165 pediatric AML patients hsa-miR-193b-3p was broadly repressed throughout the cytogenetically characterized subgroups. In addition, in a cohort of 43 adult AML patients, its homolog hsa-miR-193a-3p was significantly upregulated in APL cases (p=0.0025, n=7) compared to bone marrow from healthy donors (n=5).To assess the impact of the miR-193 family members on AML maintenance and development, we lentivirally expressed miR-193a/b in the MLL-rearranged cell lines ML2 and THP1, which induced monocytic differentiation in concert with calcitriol treatment, measured by CD11b/CD14 expression (p=0.024). Consistently, enforced miR-193-expression led to a significant growth disadvantage in ML2 and THP1 cells (p=<0.001 and p=0.02, respectively) as well as to reduced colony formation (p=0.008) in methylcellulose-based colony-forming unit (CFU) assays. Noteworthy, these effects were not restricted to MLL-rearranged AML cell lines only, but were also evident in six other AML cell lines representing the most common AML subgroups, such as t(8;21) and t(15;17). Beyond the growth-suppressive and differentiation-inductive effect of miR-193 in human AML cell lines, overexpression of miR-193a caused a significant decrease of proliferation in murine bone marrow cells immortalized in vitro by retroviral expression of Hoxa9 or Hoxa9 and Meis1 (p=0.019 and p=0.008, respectively).Based on these findings in AML, we further investigated the impact of the miR-193 family on normal hematopoiesis. We retrovirally expressed miR-193a in 32D cells treated with granulocyte-colony stimulating factor (G-CSF), which resulted in a strong induction of myeloid differentiation already after day 2 (p=0.006) as assessed by CD11b/Gr-1 surface marker expression. We lentivirally transduced mouse lineage negative (Lin-) HSPCs and transplanted them into irradiated isogenic recipients. Bleedings performed on weeks 4, 8 and 11, as well as the examination of the bone marrow on week 11, showed a severe competitive disadvantage of miR-193-transduced cells (week 11: 2% GFP+ miR-193- vs. 25% GFP+ miR-NSC-transduced cells). These results were further refined using highly purified ESLAM (CD45+ EPCR+ CD48− CD150+) HSCs which failed to reconstitute hematopoiesis when overexpressing miR-193a, indicated by the absence of miR-193a/GFP+ cells at week 8 post transplantation. These observations might be explained by a potent G1 cell cycle arrest in HSPCs when overexpressing miR-193a/b (4-fold decrease in the S phase population) and induction of apoptosis.Our results in normal and malignant hematopoiesis suggested that the miR-193 family acts globally through targeting relevant stem cell pathways. To validate this hypothesis we quantified the knockdown of ten predicted miR-193 target genes. qRT-PCR analysis confirmed the down regulation of KIT, KRAS, SOS2 (key components of the MAPK signaling pathway) and CCND1, a CDK regulator of G1/S phase transition. We propose a dual regulatory platform where firstly, miR-193 targets CCND1 and controls the cell cycle kinetics of stem cells. Secondly, miR-193 interferes with the KIT proto-oncogene and the RAS pathway thereby inhibiting crucial pro-proliferation and anti-apoptotic signaling cascades.Taken together, we identified the miR-193 family as a pan-tumor suppressor in childhood and adult AML. Its anti-leukemic effect is mediated by targeting the stem cell KIT/SOS2/RAS/RAF axis. DisclosuresNo relevant conflicts of interest to declare.

Induction of DNA Damage Response and Repair Pathways in HSPCs Following Exposure to AML Exosomes

Exosomes are extracellular vesicles that function in cell-cell communication by trafficking protein and RNA species to bystander cells. While exosomes are produced by all cell types, those released by cancer cells have come to the forefront of investigation for their potential to modulate the tumorigenic niche. We recently reported that exosomes released from acute myelogenous leukemia (AML) cells impact the phenotype and function of stromal and hematopoietic stem and progenitor cells (HSPC) found in the bone marrow (Huan et al. Cancer Res. 2013). As part of these studies, we observed a decrease in clonogenic potential of murine HSPCs exposed to exosomes isolated from the Molm-14 AML cell line in vitro and in xenograft transplantation studies. To determine if this observation was more widely applicable, we next exposed murine c-kit-selected HSPCs to exosomes isolated from both primary patient samples and the HL-60 AML cell line in vitro. Strikingly, exposure to primary AML patient and HL-60 exosomes produced a significant reduction in colony formation; on average, only 7.3% as many colonies formed in exposed conditions compared to controls. We also previously showed that exosomes traffic a complex mixture of protein and RNA to bystander cells. A recent report demonstrated increased DNA damage in mammary epithelial cells due to elevated reactive oxygen species (ROS) following exposure to exosomes derived from multiple breast cancer cell lines (Dutta et al. PLOS One 2014). A similar mechanism has been shown to restrict the replicative capacity of human HSPCs. Here, we hypothesized that exosome transfer might elicit a DNA damage response in murine HSPCs, contributing to the decreased ability of exposed cells to form colonies. When we performed 48 hours of in vitro HL-60 exosome exposure of c-kit enriched progenitor cells, we found a statistically significant upregulation of genes involved in DNA damage sensing as well as homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways compared to unexposed controls. Immunofluorescence analysis on exosome-exposed cells also revealed an increase in the formation of γH2AX foci in cells exposed to HL-60 exosomes, indicating an increase in DNA damage burden. Next, we tested if increased ROS might account for DNA damage and the resulting progenitor frequency. Using a flow cytometric analysis of ROS (DCF-DA) revealed a clear upward shift in median fluorescence intensity of exosome-treated c-kit+ cells compared to untreated controls. To further confirm the involvement of ROS, we treated exosome-exposed cells with the antioxidant NAC. While this did not result in a substantial reduction in ROS levels as measured by flow cytometry, analysis of the transcriptional DNA damage response revealed a dose-response pharmacological rescue of HR and NHEJ pathway gene expression. Our work in aggregate suggests that AML exosomes have a direct suppressive effect on HSPCs that involves, at least in part, gains in ROS that promote DNA damage accumulation and genomic instability. We propose a model whereby the paracrine trafficking of exosomes plays an active role in the erosion of HSPC activity in the AML niche that leads to characteristic cytopenias even at low leukemic burden. DisclosuresNo relevant conflicts of interest to declare.

Mitochondrial respiration defects modulate differentiation but not proliferation of hematopoietic stem and progenitor cells

Mitochondrial energy production is involved in various cellular processes. Here we show that ATP content is significantly increased in lineage-restricted progenitor cells compared with hematopoietic stem and progenitor cells (HSPCs) or more differentiated cells. Transplantation analysis using a mouse model of mitochondrial disease revealed that mitochondrial respiration defects resulted in a significant decrease in the total number and repopulating activity of bone marrow cells, although the number of HSPCs increased. The proliferative activity of HSPCs and lineage-restricted progenitor cells was not impaired by reduction of ATP content and there seems to be no associated increase in reactive oxygen species levels and apoptosis. Our findings indicate that mitochondrial respiration defects modulate HSPC commitment/differentiation into lineage-restricted progenitor cells.

Aldehyde Dehydrogenase (ALDH) Activity Resolves Neuroblastoma (NB) Cells and Hematopoietic Stem and Progenitor Cells (HPC) in Graft Material: Potential Method for Clinical Purging.

High dose, myeloablative chemotherapy followed by autologous HPC transplantation is used clinically to treat NB in children. The presence of NB cells in transplant material adversely affects outcome. Grafts have been purged by using monoclonal antibodies to select CD34+ HPC for transplantation or to remove tumor cells expressing NB surface antigens from graft material. We are exploring the feasibility of purging NB autografts by flow sorting HPC and NB cells based on differential expression of ALDH. Cell populations with high levels of ALDH activity and low side scatter (ALDHbrSSClo cells) can be identified in mobilized peripheral blood (PBSC), bone marrow (BM), and umbilical cord blood using the ALDEFLUOR™ [StemCo Biomedical, Inc.] method for flow cytometric measurement of cellular ALDH activity. ALDHbrSSClo cells isolated by flow sorting are highly enriched for HPC activity. We report here that none of three established NB cell lines (SK-N-SH, SK-N-BE(2), and IMR-32) express detectable ALDH activity in the ALDEFLUOR™ assay. Furthermore, when any of these NB lines was labeled with the stable, red fluorescent intracellular marker SNARF™ [Molecular Probes, Inc.] and then mixed with BM or PBSC, NB were readily resolved from the green fluorescent ALDHbrSSClo cells. In mixtures containing equal numbers of tumor cells and PBSC or BM cells 0–6 events were detected in the ALDHbrSSClo region out of a total of 3-10 x 105 events in the SSC vs. FSC gate. Even when mixtures containing 9 tumor cells to 1 leukocyte were used, fewer than 21 SNARF-labeled NB were detected in the ALDHbrSSClo region out of a total of 2-9 x 105events in the SSC vs. FSC gate. Additional studies were done by sorting mixtures of SNARF-labeled NB cells and PBSC or BM and measuring tumor burden post-sort. In a representative study, pre-sort NB content of 30% was reduced to 0.04% when care was taken to eliminate NB-HPC doublets by using pulse width discrimination; without this precaution, NB contamination in the sorted cells was 1.3%. RNA was prepared from pre- and post-sorted cells, and real time PCR analysis was performed using probes to NB gene products tyrosine hydroxylase, GD2 synthase and PRAME. PCR signals were readily detected in the starting sample and were reduced by >1000-fold in the sorted ALDHbrSSClo cells. To extend these findings with NB cell lines to clinical material, a bone marrow sample was obtained from a NB patient following appropriate institutional consent procedures and was analyzed by multiparameter flow analysis using ALDEFLUOR™ and a monoclonal antibody to GD2 synthase, an antigen strongly expressed by NB cells. Approximately 60% of the cells in this BM sample expressed GD2 synthase, but only 0.03% of the NB cells were detected among ALDHbrSSClo events. RNA was prepared from 100,000 flow-sorted ALDHbrSSClo cells, and real-time PCR analysis was performed. NB gene products were readily detected in the donor BMs, but no signal was detected in the sorted cell preparations. The NB burden in the flow-sorted ALDHbrSSClo population was reduced >100-fold compared to the unpurged, heavily contaminated BM. We are extending this analysis to maximize purging obtainable with clinical material. As ALDEFLUOR™ does not impair any tested functional characteristics of HPC, flow sorting based ALDH activity may provide a new method to purge autografts of NB cells for clinical transplantation.