Frequency Of Long-term Culture-initiating Cells Research Articles

Ferritinophagy Is a Druggable Vulnerability of Quiescent Leukemic Stem Cells

Acute myeloid leukemia (AML) poses significant challenges in terms of prognosis and treatment options. Leukemic stem cells (LSCs) contribute to therapy resistance, relapse and adverse outcomes. This study aimed to explore one of the major features of LSCs (e.g. quiescence) and its underlying molecular mechanisms, as well as identify potential targets for therapeutic intervention. Using in vivo cell labeling in patient-derived xenograft (PDX) assays, we identified a quiescent cell subpopulation with high LSC potential. Transcriptomics experiments conducted separately on quiescent and cycling cell subsets revealed gene signatures characteristic of quiescent (Q_AML_UP) and cycling cells (C_AML_UP). Using single-cell RNA sequencing, we identified clusters of quiescent leukemic cell that were highly enriched in gene signatures related to LSCs (Figure 1A). Through a machine learning approach, we identified a gene set highly specific of quiescent cells (named the QS35 gene signature) that emerged as a significant prognostic factor for poor outcomes in independent cohorts of AML patients (Figure 1B). Next, we aimed to identify new vulnerabilities of LSCs by investigating the quiescent subset of leukemic cells. Transcriptomics and proteomics approaches on quiescent vs cycling cells revealed enhanced autophagy within the quiescent subset. Additionally, the transferrin receptor TFRC and ferritin, two key regulators of iron metabolism, exhibited lower expression in the quiescent subpopulations. As intracellular iron availability is tightly regulated by ferritinophagy, a specific form of autophagy leading to the degradation of ferritin, we investigated the coordinated regulation of autophagy and iron metabolism in the quiescent LSC subset. Intriguingly, the intracellular iron pool was lower in quiescent LSCs, particularly in the CD34+CD38- compartment compared to more mature leukemic cells. Moreover, autophagy inhibition selectively induced cytotoxicity in the CD34+CD38- compartment and reduced the frequency of long-term culture-initiating cells (LTC-IC), and these effects were rescued by exogenous iron supply, underscoring the critical role of autophagy in iron bioavailability within the quiescent LSC subpopulation. To target ferritinophagy in this compartment, we depleted NCOA4, the main carrier in ferritinophagy responsible for delivering ferritin-bound iron to the autophagosome, using shRNAs in primary AML samples. Interestingly, NCOA4 was found more expressed in quiescent compared to cycling AML cells. In ex vivo assays, NCOA4 downregulation significantly reduced the frequency of LTC-IC and inhibited the clonogenic capacities of leukemic cells. Furthermore, NCOA4 invalidation abrogated leukemia initiation and self-renewal in serial transplantation experiments in vivo, particularly within the quiescent LSC population (Figure 2A). Additionally, treatment with a novel small molecule inhibitor named compound 9a that disrupts the NCOA4-ferritin interaction, depleted intracellular iron in primary AML cells, reduced their clonogenic potential and LTC-IC frequency ex vivo, and induced cell death specifically within the quiescent CD34+CD38- subset. Importantly, the viability of LSCs treated with compound 9a was rescued by the addition of iron, demonstrating that the cytotoxic effects of this compound were dependent on iron depletion. Notably, compound 9a exhibited minimal toxicity to normal hematopoietic cells ex vivo. In PDX models, in vivo short-term treatment with compound 9a decreased tumor burden and induced cell death within the CD34+CD38- LSC population (Figure 2B). Mechanistically, genetic of chemical inhibition of NCOA4 led to the accumulation of dysfunctional mitochondria, and to the generation of reactive oxygen species especially within the LSCs population. Furthermore, inhibition of NCOA4 by compound 9a decreased mitophagy, strongly suggesting that NCOA4 coordinates ferritinophagy and mitophagy pathways to increase intracellular iron, thereby specifically promoting the survival of LSCs. These findings highlight the critical vulnerability of quiescent LSCs to ferritinophagy. Thus, NCOA4 inhibition represents an innovative therapeutic approach for AML patients.

Combined influence of biophysical and biochemical cues on maintenance and proliferation of hematopoietic stem cells.

Homeostasis of hematopoietic stem and progenitor cells (HSPC) is controlled by a combination of biochemical and biophysical environmental cues in the bone marrow (BM) niche, where a tight balance of quiescence and proliferation of HSPC is maintained. Specifically, alongside soluble factors and extracellular matrix (ECM) proteins, spatial confinement and ECM stiffness have been recognized to be critical for regulation of HSPC fate. Here we employ a modular, glycosaminoglycan (GAG)-based biohybrid hydrogel system to balance proliferation of human HSPC and maintenance of quiescent hematopoietic stem cells (HSC) through simultaneous regulation of exogenous biochemical and biophysical cues. Our results demonstrate that HSPC respond to increased spatial confinement with lowered proliferation and cell cycling, which results in higher frequency of quiescent LTC-IC (long-term culture initiating cells), while GAG-rich 3D environments further support maintenance of the cells.

CITED2 Cooperates with Low PU.1 and DNMT3A to Maintain Self-Renewal in Hematopoietic Stem Cells

CITED2 has a conserved role in the maintenance of normal hematopoiesis. We have recently shown that ~70% of acute myeloid leukemia (AML) patients display enhanced CITED2 expression levels. Interfering with CITED2 expression is detrimental for leukemia maintenance in vitro and in vivo, demonstrating that CITED2 is critically important for the survival of leukemic stem cells (LSCs). Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) led to significantly better human engraftment in transplanted NSG mice, consistent with the maintenance of very primitive lin- CD34+ CD38- CD90+ CD45RA- HSCs within the bone marrow 28 weeks after transplantation. Although the CITED2-engrafted mice displayed enlarged spleens, blood development appeared normal, as measured through myeloid, B and T cell staining. This indicates that CITED2 as a single hit is not sufficient to transform human CD34+ cells.CITED2 expression frequently coincides with low expression of the myeloid transcription factor PU.1, suggesting that combined effects, rather than single events are important during AML development. To investigate this, we combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and studied hematopoietic development. CITED2 increased the percentage of immature CD34+ CD38- cells 5-fold, which was not further increased by the additional downregulation of PU.1. However, functional analysis through limiting dilution LTC-iC assays indicated that combining PU.1 down-, with CITED2 upregulation led to a synergistic 8.5-fold increase in LTC-iC frequency, whereas only changing PU.1 or CITED2 induced a respective 1.4 to 3-fold change in HSC frequency. To more stringently assess self-renewal, we cultured transduced cells for 4 weeks on MS5 cells under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently performed CFC assays. Whereas after 4 weeks all groups displayed similar colony numbers, secondary and tertiary replatings demonstrated that self-renewal could only be maintained for more than 10 weeks when CITED2 upregulation was combined with PU.1 downregulation. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+ CD38- HSCs, as replating of CD34+ CD38+ progenitor-derived colonies did not yield new CFCs.In order to investigate the underlying mechanisms, we performed transcriptome analysis on human HSCPs after knockdown of PU.1, overexpression of CITED2 or the combination of both. PU.1Low-CITED2High cells displayed a gene expression pattern different from the PU.1Low or CITED2High only cells, suggesting that the two events have synergistic effects. Some genes, like HLX and SF3B1 have been shown to cause or are mutated in AML, demonstrating that the synergistic changes are related to AML. When comparing the differentially regulated genes in the PU.1Low -CITED2High cells to the gene expression in the Hemaexplorer database, a similar pattern was observed, when compared between AML and normal cells.In order to investigate the effects of the PU.1low CITED2high combination on AML development, we resorted to a PU.1-dependent mouse model of AML development. CITED2 expression in BM cells from PU.1KD/KD mice (in which deletion of an Upstream Regulatory Element leads to an 80% downregulation of PU.1), led to a steady increase of GFP+ cells over time as compared to control cells and demonstrated a dramatic expansion of Gr-1+ Mac-1+ cells, a hallmark of AML in these mice. This suggests that CITED2 contributes to a faster progression towards AML upon lowering of PU.1.To identify if our model corresponds to AMLs with a specific subset of mutations, we clustered publically available AML data (TCGA), based on the gene expression changes in the PU.1Low -CITED2High cells. The majority of AMLs clustered together in 2 groups, in which FLT3, p53 and DNMT3A mutations were most prevalent. FLT3 mutations, through its activation of STAT5, are consistent with high CITED2 expression, whereas p53 mutations are consistent with our data indicating that CITED2 loss regulates HSCs in a p53-dependent manner. The presence of DNMT3A mutations suggests that DNA methylation changes collaborate with high CITED2 and low PU.1 during leukemogenesis. This is currently under investigation.In summary, our data imply that CITED2, low PU.1 and potentially changes in DNA methylation all contribute to maintenance of self-renewal and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Exhaustion in Myeloid Lineage and Very Early Defect in HSPC Pool: An Embryonic Origin of Fanconi Haematological Disorders

Fanconi anemia (FA) is a genetic disorder due to mutations in one of the sixteen FANC genes involved in DNA repair. Many FA patients develop bone marrow failure (BMF) during childhood, and FA strongly predisposes to myelodysplasia syndrome and/or acute myeloid leukaemia. The pathogenesis of the BMF remains uncompletely understood. Low hematopoietic progenitor cell (HPCs) counts observed early in life and preceeding the onset of blood cytopenia in patients led we, and other, to hypothesize that the hematopoietic development might be abnormal in the FA embryo. Indeed, unlike adult hematopoietic stem cells (HSCs) which are quiescent in the BM niche, during embryonic life HSCs are in active proliferation in sites of expansion such as fetal liver and placenta, where they get amplified and acquire properties of adult HSC .We hypothesized that in FA, the FA defect in response to the replicative stress could impair the expension of the HSC pool.In order to investigate this hypothesis, we carried out studies in Fancg-/- knock out mice and in human FA fetuses obtained with informed consent from medical abortion.In Fancg-/- mice, FACS analysis revealed a 1,5- to 3-fold deficiency in hematopoietic stem and progenitor cells (HSPC) very early during embryonic development (i.e 11.5 days of gestation - E11.5) in fetal liver (FL) and placenta (Pl) (p <0.001). In both organs, this defect persists during the whole period of amplification (until E14.5 for FL and E12.5 for Pl). In vitro clonogenic assays also demonstrated a 2- fold defect in granulocyte, erythrocyte and macrophage (GEM) progenitors both in Fancg-/- FL or Pl compared to WT (p <0.001), and 4 to 5- fold defect in more immature mixed GEM progenitors in FL (p <0.001). LTC-IC frequency of the HSC-enriched Lineage- Sca1+ AA4.1+ population (LSA) of E14.5 Fancg-/- FL comforted this later result, since it was 5-fold lower than for WT.In vivo long-term hematopoietic reconstitution (LTR) assays confirmed a deficit of the HSC enriched LSA population of E14.5 Fancg-/- FL. Indeed, although the percentage of mice reconstituted was as good as that obtained with the same number of WT LSA, the CD45 Ly5.2 chimerism was reduced (49±20% vs 84±4% for 1000 LSA injected, and 56±12% vs 87±2% for 5000 LSA). Interestingly, bone marrow analysis of mice reconstituted with Fancg-/- LSA 22 weeks after injection showed a level of CD45 Ly5.2 chimerism 3-fold lower than that found in blood, spleen and thymus, as well as a very low chimerism for myeloid GEM lineages, contrasting with a high chimerism for B and T lymphoid lineages. Moreover, we were able to demonstrate that this deficit is already present at E12.5, both in Fancg-/- FL and Pl. Indeed, no mice reconstituted with 3.105 total Fancg-/- fetal liver cells, while 100% injected with the same number of WT FL cells got reconstituted with a chimerism of 59,5±5%. For Pl, when 500 000 cells were injected, reconstitution was observed in only 1 out of 3 mice for Fancg-/- (29% chimerism), and in 3 out of 3 mice for WT (88±4% chimerism).In human FA FL of 14 weeks of gestation, we also observed a 4-fold defect of HSPC with a total lack of in vitro amplification compared to control, in agreement with the mice data.Taken together, these data demonstrate that a profound deficit of HSCs and progenitors cells is present since the earlier stages of embryonic development in FA. In addition, using organotypic cultures of E11 aortas, we could show that this defect of amplification is already present in HSCs emerging from Fancg-/- aorta, which showed a 2-fold lower rate of amplification compared to WT. More importantly, our results show for the first time exhaustion in myeloid lineage of FA, in agreement with what is observed in children with FA disease. Altogether, our work suggests a role of the FA pathway during the development of the hematopoietic system leading to a deficit of amplification of HSC. Comparison of FA HSC transcriptome with that of control HSC in FL and Pl is in progress. It should allow to identify the key pathways involved in the embryonic HSC amplification that are deregulated in FA, and hopefully getting more insights in the pathogenesis of the BMF and leukemogenesis in FA patients. DisclosuresNo relevant conflicts of interest to declare.

EphB4 Expressing Stromal Cells Exhibit an Enhanced Capacity for Hematopoietic Stem Cell Maintenance.

The tyrosine kinase receptor, EphB4, mediates cross-talk between stromal and hematopoietic populations during bone remodeling, fracture repair and arthritis, through its interactions with the ligand, ephrin-B2. This study demonstrated that transgenic EphB4 mice (EphB4 Tg), over-expressing EphB4 under the control of collagen type-1 promoter, exhibited higher frequencies of osteogenic cells and hematopoietic stem/progenitor cells (HSC), correlating with a higher frequency of long-term culture-initiating cells (LTC-IC), compared with wild type (WT) mice. EphB4 Tg stromal feeder layers displayed a greater capacity to support LTC-IC in vitro, where blocking EphB4/ephrin-B2 interactions decreased LTC-IC output. Similarly, short hairpin RNA-mediated EphB4 knockdown in human bone marrow stromal cells reduced their ability to support high ephrin-B2 expressing CD34(+) HSC in LTC-IC cultures. Notably, irradiated EphB4 Tg mouse recipients displayed enhanced bone marrow reconstitution capacity and enhanced homing efficiency of transplanted donor hematopoietic stem/progenitor cells relative to WT controls. Studies examining the expression of hematopoietic supportive factors produced by stromal cells indicated that CXCL12, Angiopoietin-1, IL-6, FLT-3 ligand, and osteopontin expression were more highly expressed in EphB4 Tg stromal cells compared with WT controls. These findings indicate that EphB4 facilitates stromal-mediated support of hematopoiesis, and constitute a novel component of the HSC niche.

Functional Inhibition of Mesenchymal Stem and Progenitor Cells (MSPC) Significantly Contributes to Hematopoietic Insufficiency with Acute Myeloid Leukemia (AML)

Pancytopenia is the most prominent clinical finding in patients with acute myeloid leukemia (AML) and represents a major cause for morbidity and mortality. So far, the underlying mechanisms leading to hematopoietic insufficiency in AML are poorly understood and therefore often mechanistically summarized as marrow replacement by infiltrating leukemic cells. Mesenchymal stem and progenitor cells (MSPC) are integral components of the bone marrow (BM) microenvironment and play an indispensable role for the regulation of normal hematopoiesis. Two AML mouse models have recently shown, that expansion of the leukemic clone leads to numeric changes and functional disturbances of niche components such as MSPC and osteoblasts, resulting in insufficient hematopoietic support (Hanoun et al. 2014; Frisch et al. 2012). As the knowledge about MSPC in human AML is limited so far, we conducted a detailed analysis of AML-derived MSPC in order to elucidate their contribution to hematopoietic failure.For this purpose we investigated the molecular and functional properties of BM-derived MSPC of 46 patients with AML covering all relevant subtypes according to WHO classification at diagnosis and/or during course of disease and compared them with MSPC functions of healthy controls. Hematopoietic insufficiency in the 31 patients at diagnosis was mirrored by 76% of the patients having bi- or pancytopenia and by median ANC of 1183/μl, median hemoglobin of 9.0 g/dl, and median platelet count of 67.000/μl. MSPC of these newly diagnosed patients exhibited significantly impaired growth capacities as shown by an altered morphology, reduced CFU-F activity, a lower number of passages and cumulative population doublings. While adipogenic differentiation potential was not affected, osteogenic differentiation potential of AML-derived MSPC was significantly reduced as indicated by cytochemical stainings, reduced Osterix and Osteocalcin (OC) mRNA levels as well as OC serum levels. Furthermore we detected altered mRNA and/or protein expression of key molecules involved in the regulation of hematopoietic stem and progenitor cells (HSPC), namely SCF, Angiopoietin-1, Jagged1 and Osteopontin. Functionally, this translated into a significantly diminished ability of AML-derived MSPC to support healthy CD34+ HSPC in LTC-IC assays.This insufficient stromal support was reversible and correlated with disease status, as LTC-IC frequency returned to normal values in patients in remission, but remained low in patients with refractory disease. Along with this we also observed a significant increase of OC serum levels in patients, who achieved complete remission. These data clearly suggests a direct causal relationship between the presence of leukemic cells and MSPC functionality. In further support of this idea we observed reduced proliferation and osteogenic differentiation of healthy MSPC following cultivation in conditioned media (CM) of 4 AML cell lines (THP-1, HL-60, MV4-11, MOLM-13). A comparable inhibitory effect of AML cells on healthy MSPC growth was seen in transwell-assays arguing in favor for a cell-contact independent mechanism.In summary, our data show that AML-derived MSPC are structurally and functionally altered resulting in an insufficient stromal support for normal hematopoiesis in AML. The correlation between clinical remission status and stromal support function together with the finding, that healthy MSC can adopt an AML-like phenotype when exposed to AML-CM suggest an instructive role of the leukemic precursor cells. DisclosuresDührsen:Celgene: Honoraria, Research Funding. Gattermann:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Germing:Novartis: Research Funding; Celgene: Honoraria, Research Funding; AMGEN: Research Funding; Janssen-Cilag: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria. Kobbe:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medac: Other; Astellas: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Neovii: Other.

Decreased PU.1 and Enhanced CITED2 Cooperate To Maintain Self-Renewal In Hematopoietic Stem/Progenitors

The transcriptional co-activator CITED2 has a conserved role in the maintenance of normal adult hematopoiesis. We have shown before that CD34+ cells from a subset of acute myeloid leukemia (AML) patients display enhanced CITED2 expression and that interfering with this expression is detrimental for leukemia maintenance. Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) resulted in increased proliferation and skewed myelo-erythroid differentiation in vitro. Long-Term Culture-Initiating Cell assays (LTC-IC) revealed a 5-fold increase in the number of Cobblestone Area Forming Cells (CAFCs), as a result of an increase in the number of phenotypically defined CD34+CD38- HSCs. CFC frequencies were also enhanced 5-fold upon CITED2 overexpression. To further substantiate these observations in vivo, we transplanted CITED2-transduced CD34+ cells into NSG mice. CD34+ cells with increased CITED2 expression displayed a >10x higher engraftment at week 12, as compared to control cells, confirming the higher frequency of CD34+CD38- HSCs, while myelo-lymphoid differentiation of these cells was comparable to control transplanted cells. Till date we have not observed leukemia development in these transplanted mice, suggesting that CITED2 as a single hit is not sufficient to transform human CB CD34+ cells.We recently identified the myeloid transcription factor PU.1 as a strong negative regulator of CITED2 and enhanced CITED2 expression in AML samples correlates with low PU.1 expression. We therefore investigated whether high CITED2 and low PU.1 expression would collaborate in maintaining self-renewal of HSCs. We combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and performed LTC-IC cultures on MS5 stroma. These experiments revealed that combined loss of PU.1 and enhanced CITED2 expression was sufficient to induce a strong proliferative advantage compared to control cells. Furthermore, a 3-fold increase of progenitor numbers was observed in CFC assays. While overexpression of CITED2 alone was not sufficient to allow 2nd CFC formation, additional downregulation of PU.1 now led to colony formation upon serial replating. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+CD38- HSCs, as replating of CD34+CD38+ progenitor cells did not yield CFCs. This suggests that the combined loss of PU.1 and enhanced CITED2 expression is sufficient to maintain self-renewal properties of HSC, but this combination is not sufficient to reinforce self-renewal in committed progenitor cells.To more stringently assess self-renewal, cells were first cultured for 4 weeks on MS5 under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently plated into CFC assays, followed by secondary and tertiary replating experiments. Only PU.1Low-CITED2High cells were able to form CFCs after 10 weeks of culture, indicating that this combination indeed preserves self-renewal. Current experiments focus on the in vivo engraftment and self-renewal properties of these PU.1Low-CITED2High cells. Preliminary data indicate that these PU.1Low-CITED2High cells contribute ∼3-fold more to the myeloid lineage at week 12, compared to control and CITED2 only cells, and AML development is currently being investigated in these mice.Together, these data suggest that CITED2 is sufficient to increase LTC-IC and CFC frequencies, to skew myeloid differentiation, and to enhance engraftment of CB CD34+ cells in xenograft mice. Furthermore, CITED2 overexpression together with reduced PU.1 levels is necessary to maintain stem cell self-renewal. Disclosures:No relevant conflicts of interest to declare.

Imatinib and Nilotinib Inhibit Hematopoietic Progenitor Cell Growth, but Do Not Prevent Adhesion, Migration and Engraftment of Human Cord Blood CD34+ Cells

BackgroundThe availability of tyrosine kinase inhibitors (TKIs) has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to inhibit the tyrosine kinase of the stem cell factor receptor, c-Kit. Nilotinib is 30 times more potent than imatinib towards BCR-ABL in vitro. Studies in healthy volunteers and patients with chronic myelogenous leukemia or gastrointestinal stromal tumors have shown that therapeutic doses of nilotinib deliver drug levels similar to those of imatinib. The aim of this study was to compare the inhibitory effects of imatinib and nilotinib on proliferation, differentiation, adhesion, migration and engraftment capacities of human cord blood CD34+ cells.Design and MethodsAfter a 48-hour cell culture with or without TKIs, CFC, LTC-IC, migration, adhesion and cell cycle analysis were performed. In a second time, the impact of these TKIs on engraftment was assessed in a xenotransplantation model using NOD/SCID/IL-2Rγ (null) mice.ResultsTKIs did not affect LTC-IC frequencies despite in vitro inhibition of CFC formation due to inhibition of CD34+ cell cycle entry. Adhesion of CD34+ cells to retronectin was reduced in the presence of either imatinib or nilotinib but only at high concentrations. Migration through a SDF-1α gradient was not changed by cell culture in the presence of TKIs. Finally, bone marrow cellularity and human chimerism were not affected by daily doses of imatinib and nilotinib in a xenogenic transplantation model. No significant difference was seen between TKIs given the equivalent affinity of imatinib and nilotinib for KIT.ConclusionsThese data suggest that combining non-myeloablative conditioning regimen with TKIs starting the day of the transplantation could be safe.

A Possible Role for Wnt3a in Enhancing StemRegenin1-Mediated Expansion of Hematopoietic Stem Cells

AbstractAbstract 4130Insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT) may be improved by the administration of ex vivo expanded UCB-derived hematopoietic stem cells (HSC). Although culturing HSC with cytokines such as SCF, Flt3L and TPO results in robust proliferation, it is accompanied with extensive differentiation and loss of self renewal capacity. Inhibition of the Aryl hydrocarbon Receptor (AhR) leads to potent expansion of hematopoietic progenitor cells (HPC) (Boitano et al. Science 2010). Reportedly, Wnt3a inhibits differentiation in different types of stem cells including hematopoietic stem cells(Reya et al. Nature 2003, Ten Berge et al. Nature Cell Biology 2011). Here, we evaluated possible additive or synergistic effects of combining the AhR antagonist StemRegenin1 (SR1) with recombinant purified Wnt3a to expand hematopoietic stem cells ex vivo.UCB-derived CD34-selected cells were cultured in serum-free Glycostem Basal Growth Medium (GBGM) supplemented with the early-acting growth factors SCF, Flt3L and TPO (SFT medium) with or without the addition of SR1 and Wnt3a. Cell number, viability and subset composition within the CD34+ cells were measured using flowcytometry. The multilineage differentiation potential and self renewal capacity of expanded CD34+HPC were evaluated in stroma-supported long-term culture-initiating cell (LTC-IC) assays.Culturing CD34+ cells in SFT medium resulted in a mean 10.2-fold increase in CD34+ cells after 1 week of culture (n=3). Addition of SR1 to the SFT-medium resulted in a 16-fold increase of CD34+ input cells within 7 days, while on the other hand, addition of Wnt3a alone to the SFT-medium resulted in a 7-fold increase in CD34+ cells. However, combining SR1 and Wnt3a in the SFT medium resulted in a 20-fold expansion of CD34+ cells compared to input. The early additive effect of Wnt3a on SFT+SR1-induced expansion of CD34+ cells, however, disappeared upon prolonged culture up to 2–3 weeks.Approximately 3–10% of UCB-derived CD34+ cells could be characterized as phenotypic HSC, as was defined by CD34+CD38lowCD45RAlowCD90+ cells. After culture, we sorted different CD34+-populations to evaluate their functional capacity. Evaluation of LTC-IC frequencies yielded a frequency of 1/23 LTC-IC in the phenotypic HSC-subset (CD34+CD38lowCD45RAlowCD90+) after 28 days of culture in SFT medium with SR1. However, no LTC-IC appeared to be present in the multipotent progenitor subset (MPP, CD34+CD38lowCD45RAlowCD90low). These results indicate that phenotypic HSC maintain their functional LTC-IC capacity after expansion culture.Collectively, our results confirm that SR1 expands HSC with preservation of self-renewal capacity and ability to differentiate into various hematopoietic lineages. In addition, we show that Wnt3a initially enhances SFT+SR1-driven expansion of CD34+ HPC, but reduces the increase in number of CD34+ cells at later stages of culture. These data may suggest that the period of expansion needed for clinical application may be shortened by combining SR1 and Wnt3a. Disclosures:Spanholtz:Glycostem Therapeutics: Employment. Groenewegen:Glycostem Therapeutics: Equity Ownership.

Effective Development of Hematopoietic Niche Cells From hESCs: hESC-Derived Stromal Cells Support LTC-ICs Better Than Bone Marrow-Derived Stromal Cells

Abstract 4804Quantification of putative hematopoietic stem cells (HSCs) in human bone marrow or cord blood is typically assessed by its potential to mediate long-term, multilineage engraftment when transplanted into immunodeficient murine recipients. However, in vitro surrogate assays are extremely attractive due to their relative ease of implementation, low cost and improved throughput of results. The long-term culture initiating cell (LTC-IC) assay serves this purpose by quantifying the ability of putative HSCs in a given population to be cultured for an extended period, typically 5 weeks. Previously, we have derived both mesenchymal stromal cells (MSCs) and endothelial cells (ECs) from human embryonic stem cells (hESCs). MSCs and ECs are important components of the osteogenic and vascular hematopoietic niches and hESC-derived stromal cells can provide autologous supporting cell populations for putative hESC-derived HSCs. Therefore, we evaluated if hESC-derived MSCs (hESC-MSCs) and hESC-derived ECs (hESC-ECs) would have improved ability to support LTC-IC activity of UCB and hESC-derived hematopoietic cells compared to MSCs isolated from human bone marrow (BM-MSCs) and a murine stromal line, M2-10B4. In the present study, we utilized LTC-IC culture of both hESC-derived CD34+CD45+ populations and UCB 34+ cells to test their LTC-IC potential on hESC-MSCs, hESC-ECs and a combination of the two hESC-derived populations. LTC-IC frequency and maintenance of input populations was assessed by survival/proliferation and preservation of colony forming cell (CFC) progenitors over 5 weeks. Initial studies showed all stromal layers were able to support short-term (3 week) culture of CFCs from CD34+ UCB. Total cell proliferation was found to be greatest with the hESC-derived stroma at 3 weeks. Additionally, hESC-MSCs and hESC-ECs yielded CFC comparable to M2-10B4 and significantly more than BM-MSCs. Co-culture of CD34+ UCB with a 1:1 mixture of hESC-MSCs and hESC-ECs yielded similar proliferation and CFC results to either stroma alone. After 5 weeks of culture to quantify LTC-ICs, both of the hESC-derived cells again supported expansion of UCB total cell numbers better than BM-MSCs and similar to M2-10B4. LTC-IC yield at 5 weeks was 85 ± 29 and 74 ± 12 per 103 input cells for hESC-MSC and hESC-EC, respectively, as compared to 15 ± 12 for BM-MSCs and 104 ± 56 for M2-10B4 co-culture. Combining the hESC-derived MSCs and ECs produced an LTC-IC yield of 54 ± 4. Next, we tested the ability of hESC-derived CD34+CD45+ cells to produce CFCs and LTC-ICs using co-culture with hESC-MSCs and M2-10B4. In short-term (2-3 week) assays, hESC-derived CD34+CD45+ cells declined in number to about 30% of the starting population on both stroma. However, between 3–5 weeks, cell number remained fairly constant on M2-10B4 while continuing to decrease on hESC-MSC. Interestingly, CFC yield for hESC-derived CD34+CD45+ cells at 2 weeks was 45 ± 43 per 103 input cells on M2-10B4, while co-culture with hESC-MSCs regularly failed to produce any CFC at this time. Between 3–5 weeks of culture no CFC or LTC-ICs were produced from hESC CD34+CD45+ using any of the stromal cells. Similar results were seen for hESC-derived CD34+CD43+ populations. Together, these results have two key findings. First, hESC-derived ECs and MSCs are effective in supporting LTC-ICs from UCB, and do so better than human BM-MSCs. Second, these same stromal cells do not support survival of hESC-derived LTC-ICs. In this manner, these results closely mimic studies that show limited long-term hematopoietic engraftment of hESC-derived cells when transplanted into immunodeficient mice. Therefore, this system can now be used to aid in identification of both hematopoietic niche elements and specific cell populations that mediate development and support of putative HSCs derived from hESCs and iPSCs. Disclosures:No relevant conflicts of interest to declare.

Side population cells in highly enriched CD34-positive cells from peripheral blood progenitor cells identify an immature subtype of hematopoietic progenitor cells but do not predict time to engraftment in patients treated with high-dose therapy

A Hoechst 33342 dye efflux assay can be used to define a population of immature hematopoietic progenitor cells (HPC) that are called side population (SP) cells. Previously, SP cells examined from bone marrow (BM) and peripheral blood progenitor cells (PBPC) were found to be predominantly CD34 negative. In this study, we show that the level of CD34+ cells within the SP fraction increases from 2% in BM to 15% in mobilized PBPC. Furthermore, SP cells are found in highly enriched CD34+ cells from both BM and PBPC, and these cells define an immature phenotype of HPC. We also observed a higher level of CD133+ cells within the SPCD34+ cell population. Moreover, the frequency of long-term culture-initiating cells (LTC-IC) was markedly increased in SPCD34+ cells. To further investigate whether variations in the level of SP cells in the CD34+ cell fraction influenced short-term engraftment, we studied 20 patients with Hodgkin lymphoma that were autotransplanted with highly enriched CD34+ cells from PB. The percentage of SP cells in the PBCD34+ cell fraction was highly variable, ranging from 0.3 to 22%. No correlation was found between the content of SP cells in the autotransplanted CD34+ cells and time to short-term engraftment. SPCD34+ cells in PBPC define an immature phenotype of HPC with increased numbers of LTC-IC, and they are more frequently found in PBPC than in BM. The number of SP cells does not predict time to engraftment.

KRASG12V Enhances Proliferation and Initiates Myelomonocytic Differentiation in Human Stem/Progenitor Cells via Intrinsic and Extrinsic Pathways

In human hematopoietic malignancies, RAS mutations are frequently observed. Yet, little is known about signal transduction pathways that mediate KRAS-induced phenotypes in human CD34(+) stem/progenitor cells. When cultured on bone marrow stroma, we observed that KRAS(G12V)-transduced cord blood (CB) CD34(+) cells displayed a strong proliferative advantage over control cells, which coincided with increased early cobblestone (CAFC) formation and induction of myelomonocytic differentiation. However, the KRAS(G12V)-induced proliferative advantage was transient. By week three no progenitors remained in KRAS(G12V)-transduced cultures and cells were all terminally differentiated into monocytes/macrophages. In line with these results, LTC-IC frequencies were strongly reduced. Both the ERK and p38 MAPK pathways, but not JNK, were activated by KRAS(G12V) and we observed that proliferation and CAFC formation were mediated via ERK, while differentiation was predominantly mediated via p38. Interestingly, we observed that KRAS(G12V)-induced proliferation and CAFC formation, but not differentiation, were largely mediated via secreted factors, since these phenotypes could be recapitulated by treating non-transduced cells with conditioned medium harvested from KRAS(G12V)-transduced cultures. Multiplex cytokine arrays and genome-wide gene expression profiling were performed to gain further insight into the mechanisms by which oncogenic KRAS(G12V) can contribute to the process of leukemic transformation. Thus, angiopoietin-like 6 (ANGPTL6) was identified as an important factor in the KRAS(G12V) secretome that enhanced proliferation of human CB CD34(+) cells.

Bcl-Xl Mitigates p53 Activity in AML1-ETO-Expressing Human Hematopoietic Cells.

Abstract 387A subset of acute myeloid leukemia (AML) is caused by chromosomal translocation (8;21), resulting in the expression of the AML1-ETO (AE) fusion protein. We have recently shown that AE represses DNA damage repair genes, resulting in increased DNA damage and upregulation of p53. These findings suggest that AE contributes to leukemogenesis by promoting additional mutations. However, it remains undefined how AE cells survive in the context of increased p53. In the current study, we showed that Bcl-xL is upregulated upon AE expression. Bcl-xL knockdown resulted in growth disadvantage and increased apoptosis in AE cells, but not in normal CD34+ cells. On the other hand, Bcl-xL overexpression in AE cells leads to an expansion of colony-forming cells and an increase in the frequency of long-term culture-initiating cells (LTC-IC). Interestingly, similar results were obtained when a Bcl-xL mutant (Bcl-xLG138A) that is defective in BH3 binding is overexpressed in AE cells, suggesting that the additional cytoprotection is not contributed by pro-apoptotic protein binding. Instead, published evidence on the p53 sequestering function of Bcl-xL prompted us to speculate that AE expression may lead to the establishment of a new balance between pro-apoptotic signals conveyed by activated p53 and the pro-survival characteristic of Bcl-xL. Indeed, using confocal microscopy, we observed colocalization of Bcl-xL and p53 in CD34+ AE cells. In addition, we also observed an increase in p53 target gene expression in AE cells following Bcl-xL knockdown, implicating interplay between Bcl-xL levels and p53 activity. Strikingly, examination of the two critical apoptosis-inducing p53 downstream targets, Bim and Puma, revealed that, whereas Bim is upregluated in AE cells, Puma is downregulated upon AE expression. AE ablation in an established cell line induces Puma expression. A chromatin immunoprecipitation assay revealed that AE binds to the first intron of the Puma gene. These data suggest that AE directly represses Puma gene expression, thereby preventing Puma-induced dissociation of p53 from Bcl-xL. Taken together, our in vitro findings demonstrated that the interplay among p53, Bcl-xL and Puma in response to DNA damage in normal cellular physiology is disrupted in the presence of AE to ensure cell survival. To recapitulate the critical role of Bcl-xL in AE expressing cells in an in vivo xenotransplantation assay, we transduced scrambled and Bcl-xL shRNA into AE/N-RASG12D cells, followed by transplantation into the bone marrow cavity of immunodeficient mice. We found that Bcl-xL knockdown is associated with a significant reduction in the long-term engraftment capability of AE/N-RASG12D cells. In conclusion, our data indicate that AE expressing human hematopoietic cells depend on Bcl-xL for their survival and growth. Disclosures:No relevant conflicts of interest to declare.

Overexpression of Oncogenic KRAS G12V in Human Stem and Progenitor Cells Enhances Proliferation and Initiates Monocytic Differentiation Via Intrinsic and Extrinsic Pathways.

Abstract 3975Poster Board III-911In human hematopoietic malignancies, Ras mutations are frequently present in monocytic and T-cell leukemias. In this study we investigated KRAS G12V-induced phenotypes in human stem and progenitor cells and identified signal transduction pathways that are involved. Using a retroviral expression system, KRAS G12V was introduced to human CD34+ cord blood (CB) cells and proliferation, differentiation and stem cell/progenitor frequencies were evaluated. Overexpression of constitutively active KRAS G12V induced a strong increase in cell expansion over 5-fold in MS5 bone marrow stromal cocultures as well as in cytokine-driven liquid cultures, which coincided with increased early cobblestone formation and induction of monocytic differentiation. Erythroid progenitors were greatly reduced by introduction of KRAS G12V and Q-PCR analysis revealed that expression of PU.1 was increased in conjunction with reduced GATA1 expression in KRAS G12V cells. Progenitor frequencies were increased 6-fold in KRAS-transduced cells within 1 week after plating on MS5. By week three progenitors were exhausted and KRAS-transduced cells were terminally differentiated into monocytes/macrophages. These results were in line with the strong reduction in LTC-IC frequencies at week 5, indicating that also the stem cell pool was exhausted. Intriguingly, when KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, we observed that the non-transduced cells also displayed a strong growth advantage, coinciding with enhanced early cobblestone formation. Furthermore, the addition of conditioned medium from KRAS G12V-transduced cells grown on MS5 to non-transduced CB cells induced a strong growth advantage and formation of early CAFCs. These observations indicate that, besides intrinsic pathways, secreted factor(s) play an important role in the phenotypes induced by KRAS G12V in human CB CD34+ cells. Current studies include mass-spectroscopy analysis of the secretome of KRAS G12V-transduced CB CD34+ cells to identify the factor(s) that are involved.In order to elucidate signal transduction pathways that mediate KRAS G12V-induced phenotypes, Western-blot analysis was performed. These experiments revealed an increase in phospho-ERK1/2, phospho-p38 and phospho- STAT5 (Y694) levels in KRAS-transduced cells, whereas phospho-JNK was not induced and phospho-C/EBPa (S21) levels were slightly reduced. Induction of STAT5 Y649 phosphorylation by KRAS G12V was confirmed by intracellular phosphoFACS analysis, whereby both in HSCs as well as in more committed MPPs KRAS-induced phosphorylation of STAT5 was observed. KRAS-transduced cells did not show GM-CSF hypersensitivity in any measured cell population upon activation. Inhibition of the ERK/MAPK pathway using the MEK inhibitor U0126 resulted in strongly reduced expansion in MS5 cocultures, whereby both intrinsically induced proliferation as well as proliferation induced via secreted factor(s) were impaired. KRAS G12V-induced monocytic differentiation was not significantly affected by MEK inhibition. While inhibition of the JNK pathway hardly affected proliferation and differentiation of KRAS G12V cells, inhibition of the p38 pathway using SB203580 inhibitor impaired both proliferation and differentiation. When KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, inhibition of p38 predominantly affected the transduced cells but not the non-transduced cells, suggesting that the p38 pathway particularly mediates intrinsic phenotypes imposed by KRAS G12V.In conclusion, we show that overexpression of oncogenic KRAS G12V in human CD34+ cells enhances proliferation and initiates monocytic differentiation via intrinsic and extrinsic pathways. Disclosures:No relevant conflicts of interest to declare.

Identification of Human Hematopoietic Stem Cell-Specific STAT5 Target Genes Involved in Self-Renewal and Transformation.

Abstract 568The transcription factor STAT5 fulfils an essential role in self-renewal of both mouse and human HSCs and persistent activation of STAT5 contributes to leukemic transformation. In patients with acute myeloid leukemia, increased STAT5 activity has been observed in over 60% of the cases. Yet, little is known about mechanisms that are involved. To gain further insight into these processes we studied whether STAT5-imposed long-term self-renewal is exclusively restricted to HSCs, or whether long-term self-renewal can also be imposed on progenitor cells. Human cord blood (CB) cells were transduced with control and STAT5-ER retroviral vectors allowing the induction of STAT5 activity by treatment of cells with 4-hydroxytamoxifen (4-OHT). Four populations were isolated: hematopoietic stem cells (HSC, CD34+CD38low), common myeloid progenitors (CMP, CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP, CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors (MEP, CD34+CD38+CD123-CD45RA-). MS5 bone marrow stromal cocultures were initiated and STAT5 activity was induced by 4-OHT. In HSCs, STAT5 overexpression induced a long-term proliferative advantage as well as a significant increase in cobblestone formation. This coincided with elevated levels of Colony Forming Cells (CFCs) that were maintained over 5 weeks. In contrast, STAT5 was unable to induce cobblestone formation in progenitor cocultures and only a transient STAT5-induced increase in cell numbers was observed. CFC numbers dropped significantly after 2 weeks and progenitor initiated cultures could not be maintained longer than 3 weeks regardless of STAT5 activity. Myelopoiesis was blocked and an increase in erythroid differentiation in STAT5-ER-transduced HSC, CMP, and MEP populations was observed, while the differentiation potential of the GMP remained unaffected.Next, we aimed to identify HSC-specific STAT5 target genes by performing microarray analysis on HSC, CMP, GMP and MEP populations transduced with our STAT5-ER vectors. To limit STAT5 mediated effects on erythropoiesis GATA1 was downmodulated in STAT5-transduced CB cells by a lentiviral RNAi approach, which completely abrogated erythropoiesis but maintained enhanced HSC self-renewal. Microarrays were performed on GATA1 downmodulated STAT5-transduced CB cells and controls, and these data sets were compared to the HSC-specific STAT5 target gene lists. This combined approach resulted in the identification of 36 GATA1-independent STAT5 target genes in the HSC population. One of the identified genes was HIF2a. The involvement of HIF2a in STAT5 phenotypes was studied functionally by using a lentiviral HIF2a RNAi approach in STAT5 transduced CB cells. These studies revealed that expansion of STAT5/HIF2a RNAi-transduced cells on MS5 bone marrow stromal cocultures was reduced, coinciding with reduced CFC and LTC-IC frequencies, while differentiation was not affected.In summary, our data show that hematopoietic stem cells, but not progenitors are the exclusive target for STAT5-induced long-term self-renewal. Furthermore, we show that HIF2a is a novel STAT5 target gene which plays an important role in STAT5-induced stem cell phenotypes. Disclosures:No relevant conflicts of interest to declare.

Lenalidomide Modulates the Phenotype and Function of Human Mesenchymal Stromal Cells From Healthy Donors and MDS Patients.

Abstract 3816Poster Board III-752 IntroductionRecent studies in patients with MDS have clearly demonstrated the clinical efficacy of lenalidomide. However, its exact mechanisms of action have not been elucidated yet. Myelosuppression is the most common adverse event and seems to be dependent on dose as well MDS subtype, being rather infrequent in patients other than del5q. The aim of this study was to investigate whether lenalidomide affects the bone marrow microenvironment. Therefore, we analyzed in-vitro characteristics of isolated mesenchymal stromal cells (MSCs) from MDS patients and from healthy controls. MethodsBone marrow samples were collected from healthy donors (n=5) and patients with MDS (del5q MDS n=3, RA n=2, RAEB1/2 n=3). MSCs were isolated according to the standard adhesion protocol and cultured in the presence or absence of lenalidomide. ResultsLenalidomide treatment of MSCs caused no morphological changes but proliferation was slightly increased. Typical surface molecules as CD73, CD90, CD105 and CD166 were expressed in MSCs from MDS patients at comparable levels to healthy controls. Lenalidomide treatment caused an upregulation of CD29 by 17.8 ± 4.4% and of CD73 by 24 ± 5.7% (mean fluorescence intensity). Investigating the cytokine production, we found lower IL-8 mRNA and protein levels in MSCs from MDS patients (mean in MDS MSC: 138.1 pg/ml vs. mean in healthy MSC: 1177 pg/ml). Interestingly, the IL-8 production can be increased by approximately 40% under lenalidomide treatment. MDS MSCs retained the capacity for adipogenic and osteogenic differentiation as well as their supportive function towards hematopoietic cells in long term culture-initiating assays (LTC-IC). However, the LTC-IC frequency was lower on MSC which had been preincubated with lenalidomide compared to controls. Lenalidomide also slightly accelerated osteogenic differentiation because mineralization started as early as on day 5 with lenalidomide whereas in the control cells first calcium deposits were visible after 7 days. Other samples showed augmented lipid vacuoles after adipogenic differentiation under lenalidomide treatment. ConclusionIn conclusion, lenalidomide modulates the phenotype of MSC and leads to an increase of their IL-8 secretion by a yet unknown mechanism. Whether these in-vitro effects are associated with the clinical efficacy of this compound in patients with MDS remains to be investigated. Disclosures:Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Comparative in-Vitro Evaluation of the Myeloid Toxicity of Pentostatin and the Novel PNP-Inhibitor Forodesine.

Abstract 3766Poster Board III-702Inhibitors of the purine metabolism show promising results in the treatment of lymphatic malignancies due to their suppressive effects on lymphogenesis. Their first representative, Pentostatin (Pento), an inhibitor of the deoxyadenosine deaminase, has been in clinical use for several decades. However, early clinical trials with higher dose ranges of the drug reported unforseen severe myelotoxic effects. Recently, Forodesine (Foro), a novel inhibitor of the nucleoside phosphorylase (PNP) has been introduced and is currently deployed in clinical phase I/II trials for the treatment of acute lymphatic leukemia (ALL). In order to systematically evaluate the myelotoxic effects of Pento and Foro, we have now examined their influence on the proliferation and differentiation of primitive and lineage committed hematopoietic progenitor cells (HPCs). In vitro dose/effect-curves for Foro, Pento, and Cytarabine (AraC) were generated for the leukemic cell line jurkat by 48 hours of co-incubation with the compounds. Adequate cytotoxic effects, measured in the XTT assay and by flow cytometric analysis, were observed in clinically relevant dose ranges. For the following studies, an equivalent IC60 dose of each chemotherapeutic agent was selected and CD34+ HPCs from either bone marrow, mobilized peripheral blood, or umbilical cord blood were incubated with the compounds for 48 hours. Subsequently, the rate of vital cells was determined by flow cytometry after stainig with Annexin-V and Propidium Iodide. Compared to the untreated control, the lowest amount of vital CD34+ cells was found in AraC-treated samples (30%); Foro and Pento yielded more vital cells (66% vs 61%). The combination of Foro and Pento unexpectedly had the least toxic effect on CD34+ cells (72%; n=5; p<0.05). Cells from those primary cultures were harvested and short- and long term in vitro assays for colony forming units were performed to evaluate the compounds' toxicity on primitive and lineage committed HPCs. The frequency of primitive myeloic progenitors (LTC-IC) was 2.3% in the untreated samples and diminished after treatment with AraC (1.2%) and Pento (1.9%) but surprisingly significantly increased after Foro-treatment (2.7%); the combination of Foro and Pento resulted in a LTC-IC frequency of 2.3% (p<0.01; n=5) suggesting that Foro may have attenuated the myelotoxicity of Pento. Similar effects of Foro were also observed in the short term colony forming assays where Foro seemed to have a protective effect on multipotent GEMM-progenitors: colony count increased 1.3-fold in comparison to the control; AraC yielded only 0.1-fold, Pento 0.8-fold and the combination of Pento and Foro reached 0.9-fold of the control (p<0.05; n=15). In summary, the novel PNP-inhibitor Forodesine has not only proven to have a low in vitro toxicity on lineage committed HPCs but, surprisingly, the frequency of primitive myeloic progenitors (LTC-IC) increased; clinical studies should therefore be performed to evaluate whether Forodesine, while adding to the therapeutic efficiency, may attenuate adverse effects in combination with other chemotherapeutic agents, such as Pentostatin. Disclosures:Schubert:Mundipharma Int. LTD: Research Funding.

Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis

AbstractHigh expression of BMI1 in acute myeloid leukemia (AML) cells is associated with an unfavorable prognosis. Therefore, the effects of down-modulation of BMI1 in normal and leukemic CD34+ AML cells were studied using a lentiviral RNA interference approach. We demonstrate that down-modulation of BMI1 in cord blood CD34+ cells impaired long-term expansion and progenitor-forming capacity, both in cytokine-driven liquid cultures as well as in bone marrow stromal cocultures. In addition, long-term culture-initiating cell frequencies were dramatically decreased upon knockdown of BMI1, indicating an impaired maintenance of stem and progenitor cells. The reduced progenitor and stem cell frequencies were associated with increased expression of p14ARF and p16INK4A and enhanced apoptosis, which coincided with increased levels of intracellular reactive oxygen species and reduced FOXO3A expression. In AML CD34+ cells, down-modulation of BMI1 impaired long-term expansion, whereby self-renewal capacity was lost, as determined by the loss of replating capacity of the cultures. These phenotypes were also associated with increased expression levels of p14ARF and p16INK4A. Together our data indicate that BMI1 expression is required for maintenance and self-renewal of normal and leukemic stem and progenitor cells, and that expression of BMI1 protects cells against oxidative stress.

Do Microenvironmental Factors Play Important Role in Supporting the Maintenance of Leukemic Stem Cells of Chronic Myelogenous Leukemia?

Background : In the era of tyrosine kinase inhibitor(TKI) for chronic myelogenou leukemia(CML), it is becoming more important to develop the way to get rid of the leukemic stem cells(LSC), as there is frequent relapse when TKI therapy is stopped even when molecular remission is achieved. Some researchers reported macrophages and Wnt signaling in bone marrow microenvironment might play significant role in the development and the progression of CML.Methods : We isolated CD34+CD38− LSC from bone marrow cells from 8 patients with CML chronic phase and also CD34+CD38− normal hematopoietic stem cell from 7 normal controls using magnetic cell separation system. CD34+CD38− cells were cultured on stromal cells for 5 weeks and transfered to methylcellulose media for long-term culture initiating cell(LTC-IC) assay. We checked whether there is a difference in LTC-IC capacity of LSC when cultured on leukemic or normal stromal feeder cells. CD45− stromal supporting cells were established by 3-week culture after 12Gy irradiation of CD34− bone marrow cells. We studied the expression of VCAM, ICAM, E-selectin in stromal cells and the presence of BCR-ABL transcript. Wnt and β-catenin, frizzled, Notch1 were analyzed in the LSCs. CD14+ cells were isolated and added in LTC-IC assay whether the presence of leukemic or normal CD14+ cells would make a change in the maintenance of LSCs.Results : The number of CD34+CD38− cells in CML was in the range of 1.3 × 103 to 9.2 × 104, that we could do only two sets of 2 × 2 LTC-IC assay that statistical analysis was impossible. LTC-IC frequency of LSCs was about 5-times higher compared to that of normal stem cells irrespective of the kind of stromal cells used. CD14+ cell co-culture did not changed significantly the LTC-IC frequency both in normal and LSCs. Wnt 2B, 5A and 10B were highly expressed in LSCs irrespective of the stromal cells. β-catenin was also strongly expressed in LSCs compared to normal stem cells. Notch1 was expressed only in CD34+CD38− LSCs but not in normal stem cells. The kind of stromal cells or the addition of CD14+ cells in LTC-IC assay did not affected the Notch1 expression in LSCs.Conclusion : CML CD34+CD38− LSCs seem to have more LTC-ICs and wnt and notch pathway may play important role in the maintenance of LSCs that could be therapeutic targets.

The Neuropeptides Orexin a and B Have An Impact on Functional Properties of Human CD34+ Stem and Progenitor Cells.

Orexin receptors play a role in regulation of sleep-wake-rhythm, food intake and energy homeostasis and they were long thought to be exclusively expressed in the nervous system. During the last years orexin receptors are being identified in a growing number of peripheral tissues. We have earlier detected orexin receptor 1 and 2 expression on human CD34+ blood stem and progenitor cells. Still, the sources of their physiological ligands, the peptides orexin A and B, seem to be restricted to the central nerve system to this date. The main downstream signaling pathways of the orexin receptors include Ca2+-dependent signaling associated with activation of mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (ERK1/2) pathways. In an attempt to investigate if the receptors are functionally active in CD34+ stem and progenitor cells, we used live cell calcium imaging and stimulated purified CD34+ stem and progenitor cells with orexin A and B. Upon stimulation a massive intracellular calcium release was seen which could not been detected using cells preincubated with the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or the selective OX1R-Antagonist SB334867 and CD34 negative cells. Additionally, upon stimulation with orexin A and B we found ERK (1/2) activation at a maximum 3 hours following incubation with orexin A whereas no effect was seen after stimulation with orexin B. To investigate a potential impact on the functional properties of human CD34+ cells we performed proliferation and apoptosis assays, migration and adhesion assays as well as colony forming and long-term culture assays. So far, no effects of orexin stimulation on the proliferation and apoptosis of CD34+ cells were apparent. Remarkably, stimulation with orexin A and B led to a significantly higher proportion of early pluripotent hematopoietic progenitor (CFU-GEMM) colonies and a significant reduction of erythroid precursors BFU-E (burst forming unit erythrocyte) and CFU-E (colony forming unit erythrocyte). A more immature phenotype of orexin-stimulated CD34+ cells is also reflected by array-based gene expression profiling. Long-term culture assays revealed a significantly higher frequency of LTC-IC (long-term-culture initiating cells) indicating also a more immature phenotype of orexin-stimulated cells and a greater repopulating capacity. The selective orexin receptor antagonist SB-334867 abrogated these effects. No differences could be observed regarding the migration towards SDF-1 with and without stimulation with orexin A and B. Still, orexin A and B led to a decrease in the adhesive capacity of CD34+ stem and progenitor cells to fibronectin coated dishes. Since orexin receptors are coupled to inhibitory G-proteins (Gi/q) and stimulatory G-proteins (Gs) dependent on the tissue, we incubated CD34+ cells with the selective inhibitor of Gi – proteins pertussis toxin concurrently to stimulation with orexins and observed no differences in the adhesive capacity of CD34+ cells compared to the unstimulated controls suggesting coupling of the orexin receptor 1 and 2 to Gi – proteins rather than Gs-proteins in CD34+ cells. Given this functional impact of the orexin system on CD34+ cells, we asked if orexins are secreted locally in the bone marrow or autocrine by CD34+ cells or if they are humorally transported to the bone marrow cavity. Using ELISA we did not find autocrine production of orexin by CD34+ cells whereas orexin could be detected in the serum obtained by bone marrow biopsies and peripheral blood pointing rather towards a humoral delivery of orexins to CD34+ cells. Taken together, our findings indicate a functional role of the orexin system in CD34+ stem and progenitor cells.