Competitive Repopulation Assay Research Articles

TET2 Is Essential for Survival in Mice, and Decreased TET2 Expression Enlarges HSC Compartment and Alters Cell Differentiation

Abstract 2471Loss-of-fuction mutations of Ten-Eleven-Translocation-2 (TET2) was found in a variety of myeloid malignancies. But little is known about the function of TET2 in normal hematopoiesis and in the pathogenesis of myeloid malignancies. To study the function of TET2 in hematopoiesis in vivo, we conducted detailed analyses on hematopoiesis of TET2 gene trap mice (Tang et al. 2008), in which gene trap vector was inserted into exon 2 of TET2.Homozygous (trap/trap) mice were born at the proportion with Mendelian expectations, but died at a high rate by 2 days after birth. The fraction of trap/trap mice at P0 and P3 was 33% and 12%, respectively. TET2 mRNA expression level in fetal liver cells (FLs) at embryonic day 14.5 (E14.5) was approximately 60% in trap/wt mice and 20% in trap/trap mice compared to wild-type (WT) mice. Homozygous TET2 gene trap mice revealed to be TET2 low expression mice (TET2low).In E14.5 TET2low FLs, cellularity was comparable to WT FLs. The proportion of lineage-marker−Mac-1+Sca-1+ cells containing hematopoietic stem cells (HSCs) and multipotent progenitors was higher in TET2low FLs compared to WT FLs (0.43±0.05% vs 0.18±0.01%, p<0.01)). In colony forming assay, CFU-GM was significantly increased in number in TET2low FLs compared to WT FLs (104±18 vs 83±16, p=0.03).We next transplanted FLs from WT or TET2low mice into lethally irradiated recipient mice, and the hematological parameters were examined. Twenty weeks after transplantation, there were no significant differences in blood counts between two groups. The difference in peripheral blood (PB) was observed in the proportion of Gr-1+/Mac1+ cells and monocytes. The proportion of Gr-1+/Mac1+ cells and monocytes of WBCs in recipient mice transplanted with WT FLs was 20.7±1.6% and 11±1%, respectively, and that in recipient mice transplanted with TET2low FLs was 29.7±3.4% and 16±6%, respectively (p<0.05). In BM compartments including LSK, there were no significant differences between two groups. TET2low BM cells presented a functional increase in colony forming progenitors in vitro. The number of CFU-GM in 2 × 104 BM cells from recipient mice transplanted with WT FLs and TET2 FLs was 130±6 and 199±9, respectively (p<0.01).Recipient mice transplanted with TET2low FLs showed splenomegaly and extramedullary hematopoiesis. The spleen weights in recipient mice transplanted with WT FLs and TET2low FLs was 0.076±0.003g and 0.092±0.005g, respectively (p=0.02). The proportion of LSK, CD41+, and CD71+TER119− cells was higher in recipient mice transplanted with TET2low FLs compared to those with WT FLs. The number of CFU-GM and CFU-MEP was also higher in TET2low group.To determine whether TET2 low expression affects the HSC function in vivo, we performed a competitive repopulation assay by transplanting 1 × 106 CD45.2+ FLs from WT or TET2low mice along with 1 × 106 CD45.1+ WT BM cells (BMs), into lethally irradiated CD45.1+ recipient mice (1st recipient mice). After 12 weeks, 1 × 106 BMs from 1st recipient mice were serially transplanted into irradiated recipient mice (2nd recipient mice). The contribution of CD45.2+ cells in total and differential lineage of WBC was analyzed. The proportion of CD45.2+ in total WBC from 1st recipient mice transplanted with WT and TET2low FLs was 75±2.6% and 88±1.1%, respectively. In the 2nd recipient mice, the contribution of TET2 low cells was still high as 89±1.1%, although that from WT cells decreased to 54±4.5%. In both myeloid and lymphoid lineages, the contribution of TET2low cells was higher compared to that of WT cells. The proportion of CD45.2+ cells in Gr-1+/Mac1+ cells, B220+ cells, and CD4+/CD8+ cells in 2nd recipient mice transplanted with WT BMs was 49±6.5%, 72±4.7%, and 41±3.3%, respectively, and that in 2nd recipient mice transplanted with TWT2low BMs was 92±0.6%, 97±0.7%, and 64±2.7%, respectively.These data indicate that (1) TET2 is essential for survival in mice, (2) the decreased TET2 expression resulted in the enlargement of HSCs, and cell-autonomous growth and competitive advantage in hematopoietic cells, (3) the decreased TET2 expression altered the cell differentiation skewing toward myeloid/monocytic lineages. TET2 controls the hematopoietic homeostasis. Disclosures:No relevant conflicts of interest to declare.

Dynamic Expression of Id2 Determines Cell Fate Decisions in Multipotent Hematopoietic Cells

Abstract 2369Inhibitor of DNA binding protein-2 (Id2) is a member of the helix-loop-helix family of transcriptional regulators that is required for the normal development of natural killer (NK) cells, dendritic cells, B cells and erythrocytes. However, little is known about the expression and function of Id2 in hematopoietic stem and progenitor cells (HSPC). Recent evidence suggests that cell fate is resolved in HSPC by regulating the levels transcription factor expression. Therefore, to determine if Id2 functions in cell fate decisions, and in which progenitor populations these decisions are made, we generated an Id2-EYFP reporter mouse model and validated that EYFP accurately reflected Id2 expression. Using this model we mapped Id2 expression levels in purified HSPC and their differentiated progeny. Id2 is highly expressed in differentiated neutrophils, dendritic cells and NK cells, but is suppressed during erythroid development. Id2 expression is dynamically regulated during lymphoid development, with high levels of Id2 expression in lymphoid progenitors that are down regulated during the early stages of T cell (DN2-DN4) and B cell (Pre-B) differentiation. Id2 is then up-regulated and highly expressed in CD4+CD8+ and single positive CD4 and CD8 thymocytes, suggesting that Id2 may have novel functions in differentiated T cells and in neutrophils. Id2 is expressed in HSC and multi-potent progenitors (MPP), is decreased in common myeloid progenitors (CMP), and further decreased in myeloid/erythroid progenitors (MEP), but increased in granulocyte/macrophage progenitors (GMP). We also observed a range of Id2 expression within purified HSPC, suggesting that the levels of Id2 expression in purified HSC, MPP and CMP may correlate with potential of these progenitors for myeloid, erythroid and lymphoid development. To investigate this, CMP Id2-hi and CMP-Id2-low expressing cells were sorted and evaluated for differentiation potential in vitro. CMP-Id2-hi cells showed greatly increased myeloid developmental potential compared to the CMP-Id2-low cells as indicated by flow cytometry and growth in soft agar. Gene expression profiles confirmed these results and showed that the CMP-Id2-hi cell expression profile correlated with the expression profile of GMP, while the CMP-Id2-lo cells showed an expression profile that resembled MEP. Based on these observations we anticipate that MPP-Id2-hi and MPP-Id2-lo cells will show differences in myeloid and lymphoid potential. The expression of Id2 in primitive HSPC suggested that Id2 may be required for the maintenance and fate of HSC. Using the Id2−/− mouse model, we discovered that Id2−/− bone marrow cells have impaired ability to rescue mice after serial transplantation, suggesting that Id2 is required for HSC self-renewal. Competitive repopulation assays showed that Id2−/− HSC have diminished repopulation potential. Thus, these data suggest that Id2 is required for the self-renewal of HSC, and that changes in the levels of Id2 expression are associated with cell fate determination of HSPC. Disclosures:No relevant conflicts of interest to declare.

JAK2-V617F Expressing Stem Cells Display a Competitive Advantage At Low Limiting Dilution and Are Capable of Initiating MPN Phenotype

Abstract 615JAK2-V617F is the most common genetic alteration in myeloproliferative neoplasms (MPN). Forced expression of JAK2-V617F in animal models induced a MPN phenotype. However, MPN in these models was initiated by polyclonal expansion of multiple hematopoietic progenitor and stem cells. In contrast, patients with MPN display clonal hematopoiesis. Frequently, additional somatic mutations precede the acquisition of JAK2-V617F, suggesting that a pre-JAK2 mutation could be required for clonal expansion. Compatible with this hypothesis, in one JAK2-V617F knockin model the MPN phenotype was not transplantable and the stem cells showed signs of exhaustion. To address the question of whether JAK2-V617F alone is sufficient to initiate MPN we performed competitive repopulation assays and serial transplantations in a transgenic JAK2-V617F mouse model.Our transgenic mice express the human JAK2-V617F under the endogenous JAK2 promoter and display a polycythemia vera (PV) or essential thrombocythemia (ET) phenotype. The transgene can be activated by Cre recombinase and the donors were induced 5–6 weeks prior to transplantation. To allow monitoring of the reconstitution, a GFP expressing transgenic strain was used as the source of competitor cells. Bone marrow transplantations were performed into lethally irradiated mice in the inbred BL/6 background. A total of 2×106 bone marrow cells per recipient were transplanted with ratios of mutant (V617F) to wild type (WT) set as 1:1, 1:10 or 1:100. Results:At 1:1 and 1:10 ratios, the mice displayed a PV phenotype beginning at 4 weeks post transplantation (Hb ∼200-220 g/L and neutrophil count of 5–20×109 /L) that remained stable for ∼26 weeks. An increase in platelet counts (4-6×1012/L) followed at later time points. V617F cells rapidly outcompeted WT cells reaching >95% chimerisms in peripheral blood. The MPN phenotype was transplantable into secondary and tertiary recipients and no signs of exhaustion were observed. An increase in the frequencies of lineage-negative, Sca-1+ c-kit+ (LSK) cell numbers in bone marrow was noted (4.1% in V617F vs 1.2% for WT, p<0.05). To assess their cycling rate, LSK cells were sorted, labeled with carboxyfluorescein succinimidyl ester and transplanted into non-conditioned recipients. After 8 weeks, the number of LSK cells was 62-fold increased and V617F LSK cells outcompeted host cells (52% vs. 3% for WT, p<0.01). The number of slow-dividing (0-2 divisions) LSK cells was substantially reduced in the V617F transplanted mice (0.095% vs. 7.54% for WT, p<0.01). Thus, V617F increases the cycling of LSK cells and provides a competitive advantage.To allow detecting low numbers of donor cells in 1:100 transplantations, the GFP transgene was bred into the V617F background and a mixture of 2×104 V617F/GFP bone marrow cells plus 1.98×106 WT competitors was used. Based on the expected frequency of 3 long term hematopoietic stem cells (HSC) per 105 bone marrow cells, the recipients should receive on average 0.6 GFP-positive long term HSC. In mice transplanted with V617F/GFP and WT competitors at 1:100, we detected reconstitution defined as >0.1% GFP positive cells in 12 of 40 recipients (28%) that persisted beyond 20 weeks, whereas in mice transplanted with WT/GFP and WT competitors, only 5/40 (12%) showed reconstitution. The relative contribution in the WT group ranged from 0.1 – 3%, whereas in the V617F group we observed chimerism between 5 –100%. Four of the 12 mice that reconstituted with V617F/GFP cells developed a MPN phenotype (3PV, 1ET). Terminal workup of 3 mice (2PV and 1ET) at 29 weeks revealed that mice with erythrocytosis had enlarged spleens (∼150mg vs. 65mg for WT) and increased erythropoiesis in spleen histology. In contrast the mouse with ET phenotype showed only a slight increase in spleen size (90mg vs. 65mg for WT) and no increase in spleen erythropoiesis. Conclusions:Our results show that JAK2-V617F positive cells were able to rapidly outcompete WT cells. The MPN phenotype was transplantable into secondary and tertiary recipients. At low limiting dilution (1:100), V617F cells displayed > 2-fold higher engraftment rate (28%) than WT cells (12%) and a higher contribution to hematopoiesis (5-100% chimerism vs. 0.1–3%). Since 4/12 mice (30%) reconstituted with V617F cells also developed a MPN phenotype, our data demonstrate than JAK2-V617F as the sole genetic alteration can initiate MPN under conditions resembling monoclonal disease. Disclosures:No relevant conflicts of interest to declare.

Differential Effects of HOXB4 and NUP98-HOXA10hd on Hematopoietic Repopulating Cells in a Nonhuman Primate Model

Expansion of hematopoietic stem cells (HSCs) is beneficial in settings where HSC numbers are limited, such as cord blood transplantation. The human homeobox transcription factor HOXB4 has been shown to enhance stem cell expansion in several experimental models. We have shown previously that HOXB4 overexpression in monkey CD34(+) cells has a dramatic effect on expansion and engraftment of short-term repopulating cells. Here, we wished to compare the effects of HOXB4 and another candidate gene, NUP98-HOXA10hd (NA10hd). We used a competitive repopulation assay in pigtailed macaques to study engraftment of CD34(+) cells modified with gammaretroviral HOXB4YFP or NA10hdGFP. We found that HOXB4YFP contributed more to early hematopoiesis (<30 days), whereas NA10hdGFP contributed more to later hematopoiesis. In each case, we observed two distinct peaks in engraftment of NA10hd-transduced cells, one within 20 days post transplant and another after 5-6 months. Analysis of CD14(+), CD3(+), and CD20(+) subsets confirmed that higher percentages of cells of each lineage were derived from NA10hdGFP(+) progenitors than from HOXB4YFP(+) progenitors. In conclusion, we show that HOXB4 and NA10hd both have a significant impact on hematopoietic reconstitution; however, these effects are differential and therefore may offer complementary strategies for HSC expansion.

Hematopoietic Stem Cells Survive Circulation Arrest and Reconstitute Hematopoiesis in Myeloablated Mice

Hematopoietic stem and progenitor cells (HSPC) for bone marrow transplantation are currently obtained directly from living voluntary donors or from cord blood units. However, a suitable donor is not always found. Because HSPC are known for their relative resistance to hypoxia, using an experimental murine model, we explored cadaveric bone marrow (BM) as their alternative source. After donor mice were sacrificed, BM was left in intact femurs at 37°C, 20°C, or 4°C under ischemic conditions, resulting in combined oxygen and metabolic substrate shortage and the accumulation of metabolic waste products. BM cells were harvested after a set time period ranging from 0 to 48 hours. To determine the impact of delayed harvesting on the transplantability of HSPC, a competitive repopulation assay using a murine Ly5.1/Ly5.2 congenic model in 2 different settings was used: after submyeloablative (6 Gy) or myeloablative (9 Gy) total-body irradiation, Ly5.2 hosts received cadaveric Ly5.1 cells or a mixture of cadaveric Ly5.1 cells and fresh Ly5.2 cells in a 1:1 ratio. Chimerism resulting from cadaveric donor cells, followed up to 6 months after transplantation, proved that the long-term repopulation ability of HSPC was fully preserved for 2 hours, 6 hours, and 12 hours at 37°C, 20°C, and 4°C of ischemia, respectively. A colony-forming unit-spleen (CFU-S) clonogenic assay revealed a higher sensitivity of proliferating hematopoietic progenitors to ischemia compared to repopulating cells (STRC and LTRC). Flow cytometry analysis of apoptosis in cadaveric BM demonstrated that the LSK (Lin(low)Sca-1(+)c-Kit(+)) subpopulation, enriched in HSPC, contained less apoptotic and dead cells than the BM as a whole. Furthermore, the number of LSK SLAM (CD150(+)CD48(-)) and LSK SP (side population) cells (fractions highly enriched in hematopoietic stem cells) decreased in parallel with BM transplantability. As well as cadaveric BM cells, we also tested the transplantability and survival of BM cells after storage in a suspension in vitro without specific hematopoietic growth factors. HSPC did not display any decrease in transplantability after 2 days of storage at 37°C or 4 days at 4°C. A higher sensitivity of progenitors to unfavorable conditions was observed again using CFU-S and granulocyte macrophage-colony forming cell (GM-CFC) assays, especially at 37°C. This paper shows that HSPC survive the cessation of circulation for a considerable time and maintain their engraftment potential. This time is significantly extended with in vitro storage compared to the cadaveric BM.

Guanine nucleotide exchange factor Vav1 regulates perivascular homing and bone marrow retention of hematopoietic stem and progenitor cells

Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depend on their ability to respond to extracellular signals from the bone marrow microenvironment, but the critical intracellular pathways integrating these signals remain poorly understood. Furthermore, recent studies provide contradictory evidence of the roles of vascular versus osteoblastic niche components in HSPC function. To address these questions and to dissect the complex upstream regulation of Rac GTPase activity in HSPC, we investigated the role of the hematopoietic-specific guanine nucleotide exchange factor Vav1 in HSPC localization and engraftment. Using intravital microscopy assays, we demonstrated that transplanted Vav1(-/-) HSPC showed impaired early localization near nestin(+) perivascular mesenchymal stem cells; only 6.25% of Vav1(-/-) HSPC versus 45.8% of wild-type HSPC were located less than 30 μm from a nestin(+) cell. Abnormal perivascular localization correlated with decreased retention of Vav1(-/-) HSPC in the bone marrow (44-60% reduction at 48 h posttransplant, compared with wild-type) and a very significant defect in short- and long-term engraftment in competitive and noncompetitive repopulation assays (<1.5% chimerism of Vav1(-/-) cells vs. 53-63% for wild-type cells). The engraftment defect of Vav1(-/-) HSPC was not related to alterations in proliferation, survival, or integrin-mediated adhesion. However, Vav1(-/-) HSPC showed impaired responses to SDF1α, including reduced in vitro migration in time-lapse microscopy assays, decreased circadian and pharmacologically induced mobilization in vivo, and dysregulated Rac/Cdc42 activation. These data suggest that Vav1 activity is required specifically for SDF1α-dependent perivascular homing of HSPC and suggest a critical role for this localization in retention and subsequent engraftment.

Mesenchymal Stem Cells Secreting Angiopoietin-Like-5 Support Efficient Expansion of Human Hematopoietic Stem Cells Without Compromising Their Repopulating Potential

Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34(+) CD133(+) cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded ~60-fold by limiting dilution assay in NOD-scid Il2rg(-/-) (NSG) mice. The cultured CD34(+) CD133(+) cells had similar engraftment potential to uncultured CD34(+) CD133(+) cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34(+) CD133(+) cells maintain both short-term and long-term HSC activities. To our knowledge, this ~60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.

Hemp, an mbt domain-containing protein, plays essential roles in hematopoietic stem cell function and skeletal formation

To clarify the molecular pathways governing hematopoietic stem cell (HSC) development, we screened a fetal liver (FL) HSC cDNA library and identified a unique gene, hematopoietic expressed mammalian polycomb (hemp), encoding a protein with a zinc-finger domain and four malignant brain tumor (mbt) repeats. To investigate its biological role, we generated mice lacking Hemp (hemp(-/-)). Hemp(-/-) mice exhibited a variety of skeletal malformations and died soon after birth. In the FL, hemp was preferentially expressed in the HSC and early progenitor cell fractions, and analyses of fetal hematopoiesis revealed that the number of FL mononuclear cells, including HSCs, was reduced markedly in hemp(-/-) embryos, especially during early development. In addition, colony-forming and competitive repopulation assays demonstrated that the proliferative and reconstitution abilities of hemp(-/-) FL HSCs were significantly impaired. Microarray analysis revealed alterations in the expression levels of several genes implicated in hematopoietic development and differentiation in hemp(-/-) FL HSCs. These results demonstrate that Hemp, an mbt-containing protein, plays essential roles in HSC function and skeletal formation. It is also hypothesized that Hemp might be involved in certain congenital diseases, such as Klippel-Feil anomaly.

Role of Cohesin-Resolving Protease, Separase, In Hematopoiesis.

AbstractAbstract 1593 Introduction:Cohesin is an evolutionarily conserved protein complex that forms during the replication of sister chromatids. It is a multi-protein complex that consists of four proteins, Smc1, Smc3, Rad21, and Scc3. Resolution of sister chromatid cohesion at the onset of anaphase depends on Separase, an endopeptidase that separates sister chromatids by cleaving cohesion Rad21. A recent study suggests a new role of Cohesin proteins in gene expression and development with implications in hematopoiesis. Our data indicates that cohesin-resolving protease Separase may play a critical role in hematopoiesis. HYPOTHESIS: We hypothesize that Separase plays a role in hematopoiesis by increasing the quantity of hematopoietic stem cells (HSC). METHODS: Our experimental approach was to isolate murine long-term HSC from WT mice and mice with one mutated copy of Separase (i.e. Separase heterozygotes). In addition, in vivo competitive long term repopulation assays were used assess the function of HSC in Separase heterozyotes. RESULTS: Separase heterozygote have increased HSC numbers (p<0.05) as compared to WT mice. In addition, an improved engraftment in a competitive repopulation assay (p < 0.001) was seen in the Separase heterozyotes. Analysis of the engrafted cells demonstrated no difference between the wild type and Separase heterozygote animals, indicating the increased engraftment may be due to unique features in the primitive hematopoietic stem cells. CONCLUSION: Investigation of the mechanism for improved HSC engraftment in Separase heterozygote mice will significantly contribute to our understanding of marrow engraftment and function. Elucidating the mechanisms of hematopoietic dysregulation will provide insights into the development of life-threatening disorders such as leukemia and, in the setting of bone marrow transplant, engraftment failure. Disclosures:No relevant conflicts of interest to declare.

Self-Renewal and BM Niche Occupancy Defects In NHEJ Deficient HSCs.

AbstractAbstract 1455Maintenance of hematopoietic stem cells (HSC) requires proper interaction between HSC and the bone marrow niche. DNA repair proteins, especially proteins involved in nonhomologous end joining (NHEJ), are critical for HSC maintenance. We hypothesize that NHEJ deficient HSCs have a BM niche occupancy defect.NHEJ pathway is the major mechanism for double strand break repair in mammalian cells, several proteins participate in the process. Ku70 recognizes and binds to the DNA DSB ends with Ku80, recruits DNA activated catalytic polypeptide (Prkdc) to form DNA dependent protein kinase complex, and further recruits Artemis to process the ends and DNA ligase 4 to rejoin the broken ends. NHEJ deficient cells are hypersensitive to irradiation, and NHEJ deficient mice display SCID phenotypes.We have previous shown that Ku70 deficient HSCs are defective in repopulation as well as BM niche occupancy. Prkdc is another key factor in the NHEJ pathway, and mice homologous for a spontaneous nonsense mutation (Prkdcscid, commonly referred to as scid) are characterized by an absence of functional T cells and B cells due to the inability of V(D)J recombination. Though scid mice has been widely used for human HSCs engraftment studies and the nonobese diabetic/severe combined immune deficiency (NOD/scid) xenotransplantation model is now the “gold standard” for assaying human HSC activity, the function and BM niche occupancy capacity of HSCs of scid mice origin has not been characterized.scid mice show comparable HSC frequency and similar apoptosis rates compared to HSC from WT mice. To assess scid HSC function in vivo, single and competitive serial transplantation was performed. BM from scid mice were able to reconstitute the myeloid lineage (Mac1+) but not lymphoid lineages (CD3+ or B220+) in primary recipients due to the deficiency in V(D)J recombination. Serial transplantation was hampered by a high frequency of donor-derived thymic lymphomas in primary recipients. Competitive repopulation assays between WT and scid BM cells showed that when scid BM were mixed with WT competitor BM at a 1:1 ratio, scid BM cells were completely outcompeted by the WT BM cells.To investigate the BM niche occupancy ability of scid HSCs, we used the hematopoiesis niche occupancy assay, in which 5×106 WT congenic BM cells were transplanted into WT and scid recipients without any recipient conditioning and evaluated for blood and marrow cell origins at 16–24 weeks. Remarkably, transplanted WT BM made a long-term multi-lineage (Mac1+, CD3+ and B220+) contribution to hematopoiesis as well as HSC engraftment in scid recipients, (18.4+ 3.8% Mac1+, all the CD3+, B220+ cells, and 8.2+ 2.6% of HSC are donor derived). By contrast, less than 1% stem cell engraftment occurred in WT recipients as expected. Thus, have a long term niche occupancy defect impacting hematopoiesis.NHEJ is involved in both DNA double strand break repair and V(D)J recombination in lymphocytopoiesis. To determine whether lymphocytopoiesis is required for HSC function and BM niche occupancy, RAG1 deficient mice were used in the competitive repopulation assay and the hematopoiesis niche occupancy assay. Our results showed that RAG1-/- HSCs competed with WT HSC did not have defects in competitive repopulation or BM niche occupancy.These data demonstrate that Prkdc, a key component of NHEJ, is required for HSC function. Prkdc deficiency in the HSC compartment results in loss of competitive repopulation ability and loss of long term BM niche occupancy. Together with our previous results obtained from Ku70-/- mice, we conclude that NHEJ is critical for HSC self-renewal and BM niche occupancy, a function independent of its role in lymphogenesis. These data also explains the nature of the conducive marrow niche environment of scid mice for xenotransplantation. Disclosures:No relevant conflicts of interest to declare.

S1P1 Agonists for Use as Adjunct Mobilizing Agents

AbstractAbstract 826Harvesting hematopoietic stem cells (HSC) mobilised into peripheral blood (PB) for transplantation is mediated through bone marrow (BM) retentive and egress factors. Factors that retain hematopoietic stem cells (HSC) in the BM are well defined, with CXCL12 and VCAM1 playing major roles. However, the factors involved in the egress of HSC from the BM into the peripheral blood (PB) are currently uncharacterised. Sphingosine-1-Phosphate (S1P) is a lymphoid organ egress factor for lymphocytes, mediated through the S1P1 receptor, which is also expressed on HSC. We hypothesised that S1P mediates the egress of HSC out of the BM and into the PB.Our laboratory used a number of different mouse models with various S1P levels or S1P receptor expression to elucidate the role of the S1P gradient between the BM and PB. Sphingosine kinase-1 knock-out (SK1KO) mice were utilized for their reduced PB S1P levels. A sphingosine lyase inhibitor 4′deoxypyridoxine (DOP) was used to increase BM S1P levels. Mice treated with FTY720 for 14h had suppressed S1P1 expression and an S1P1 conditional knock-out mouse was also generated by our group. Animals were also treated with S1P receptor agonists such as SEW2871. Mobilisation experiments, competitive repopulation assays and chemotaxis assays were performed utilizing the various models.Plasma from SK1KO mice had a reduced capacity to induce migration in haematopoietic progenitor cells (HPC), confirming the chemokine activity of S1P. Consistent with this, AMD3100 induced mobilization was inhibited in SK1KO mice and DOP treated mice, demonstrating the role of an S1P gradient in HPC mobilization. Mice treated with FTY720 significantly inhibited AMD3100, although not G-CSF, mediated mobilisation of HPC in mice. No HPC accumulation was detected in secondary lymphoid organs such as lymph nodes or spleen. Most importantly, FTY720 treatment reduced the number of transplantable HSC in the blood following AMD3100-mediated mobilisation using a competitive repopulation assay. Our laboratory also generated an S1P1 conditional knock-out mouse. When mobilised with AMD3100, these S1P1 knock-out animals displayed a marked reduction in HPC mobilisation compared to wild-type animals. Finally, the S1P1 agonist SEW2871 increased HPC mobilisation synergistically, by approximately 2 fold when combined with AMD3100, but not G-CSF.S1P supports the egress of HSC from the BM into the PB following inhibition of the CXCL12/CXCR4 axis. S1P1 conditional knock-out mice display a significantly reduced mobilising capacity. S1P receptor agonist, SEW2871, acts synergistically with AMD3100 to increase HPC mobilisation in vivo, raising the possibility that such a combination may increase the efficiency of HSC collection for transplantation purposes. Disclosures:No relevant conflicts of interest to declare.

Features of Myeloproliferative Neoplasms and Clonal Advantage of Hematopoietic Stem Cells In Mice Expressing An Hmga2 cDNA Lacking the 3′UTR.

AbstractAbstract 1161Overexpression of high mobility group AT-hook 2 (HMGA2) has been identified in patients with paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndromes, and myeloproliferative neoplasms (MPN). Overexpression is often associated with truncation or deletions of the 3′ untranslated region (UTR) of HMGA2 and is regulated by the binding of let-7-family micro RNAs to the 3′UTR of HMGA2 mRNA. PNH is an acquired hemolytic anemia caused by clonal expansion of a hematopoietic cell (PNH cell) with a somatic mutation in the X-linked PIGA gene occurring in the background of bone marrow (BM) failure. The PIGA gene is essential in the synthesis of GPI-floats, and therefore progeny cells of this clone lack all GPI-linked proteins. However, the nature of the clonal growth advantage of a PNH cell is unclear. Here, to clarify the role of expression of 3′UTR-truncated HMGA2 in hematopoiesis and clonal expansion of hematopoietic cells, we generated transgenic mice (ΔHmga2 mice) expressing an Hmga2 cDNA with a truncation which removes six of the seven let-7-complementary sequences in the 3′UTR, which is similar to the truncations of HMGA2 found in two patients with PNH (Inoue et al., Blood, 2006). Expression of Hmga2 mRNA and protein were both significantly higher in BM, spleen, and thymus of ΔHmga2 mice compared to wild-type (WT) mice, with overexpression being more prominent in protein rather than mRNA. Unexpectedly, ΔHmga2 mice showed proliferative hematopoiesis that mimicked an MPN-like phenotype with increased numbers of peripheral white blood cells, red blood cells, and platelets, and low serum erythropoietin (EPO) values, hypercellular BM, and splenomegaly with extramedullary erythropoiesis compared to WT mice. Furthermore, colony assays of BM cells from ΔHmga2 mice showed significantly higher numbers of burst forming-unit erythroid, colony forming-unit erythroid (CFU-E), and CFU granulocyte-macrophage compared to those from WT mice. Formation of EPO-independent CFU-E was also observed. In competitive repopulation assays after BM transplant (BMT), BM cells from ΔHmga2 mice had a growth advantage over those from WT mice in all lineages. Surprisingly, cells derived from ΔHmga2 BM took over most of hematopoiesis during serial BMT with a ratio of 1:9 donor:recipient cells at the initial BMT, suggesting that overexpression of Hmga2 confers a clonal advantage to hematopoietic cells at the level of the hematopoietic stem cell (HSC). We found significantly higher expression of phosphorylated Stat3 in BM HSC-enriched KLS (c-kit+lineage-Sca-1+) cells and phosphorylated Akt in total BM cells from ΔHmga2 mice compared to WT mice without stimulation by cytokines, suggesting that intrinsic activation of Stat3 and Akt may contribute to proliferative hematopoiesis in ΔHmga2 mice. Microarray analysis of KLS cells and megakaryocyte-erythrocyte progenitor (MEP) cells showed a wide range of differences, in part mimicking MPN. When we bred our mice with circulating PNH cells (Piga- mice) with ΔHmga2 mice, the ΔHmga2+Piga+ cells and ΔHmga2+Piga- cells showed similar growth kinetics, indicating that the lack of GPI-linked proteins did not add an additional clonal advantage to the ΔHmga2+ cells. In summary, our results showed that the overexpression of a 3′UTR-truncated Hmga2 leads to proliferative hematopoiesis with clonal advantage at the level of HSC, which may also explain the effect of HMGA2 expression on clonal expansion in PNH or MPN. Disclosures:Bessler:Alexion Pharmaceutical Inc: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Taligen: Consultancy.

Irradiated Bone Marrow Environment Impairs Hematopoietic Stem Cells by Exposure to Tumor Necrosis Factor a.

AbstractAbstract 3724 Background:Hematopoietic stem cell transplantation (HSCT) represents a curative treatment for various disorders including hematopoietic malignancies. Most HSCT requires cytoreductive conditioning such as total body irradiation (TBI) and/or chemotherapy to ensure engraftment of HSCs by emptying recipients' marrow niches. Our preliminary experiments, however, revealed that TBI could induce local inflammation peaking around days 2–3 within marrow environment. Of note is that conditioning regimens widely used in the current HSCT settings are mostly compatible with HSC exposure to inflammatory storm in post-irradiation bone marrow (BM). Although certain inflammatory cytokines have been shown to affect HSC functions in in vitro, it remains unknown whether in vivo exposure to inflammatory BM environment can alter the characteristics of transplanted HSCs. We therefore sought to investigate what effects irradiated BM environment would have on transplanted donor HSCs using murine systems. Methods and Results:We first tested whether infusion of HSCs at varying timing post TBI would affect transplantation outcomes and HSC functions. To this end, fifty HSCs (CD34-negative/low, cKit+, Sca-1+, lineage marker-negative cells) obtained from Ly5.1-B6 mice were transplanted into lethally irradiated Ly5.2-B6 mice with 1 × 106 competitor Ly5.1/5.2-B6 BM cells at day 0, 3, or 5 post irradiation. No mice survived long-term in the group that received transplants 5 days post irradiation, indicating insufficient HSC engraftment and hematopoietic reconstitution. Although hematopoiesis reconstituted in long term survivors was comparable between another two groups (d0 and d3), we found significant difference in donor HSC ability when tested in a competitive repopulation assay using Ly5.1-KSL cells sorted from primary recipients: Test HSCs obtained from mice transplanted 3 days after irradiation showed poor secondary reconstitution ability, suggesting alteration of pre-engraftment HSC functions depending on transplantation protocols. We then tested whether in vivo exposure of HSCs to irradiated BM environment would have negative effects on HSC functions. Test HSCs (400 cells) from Ly5.1-B6 mice were transplanted into Ly5.2-B6 primary recipients at varying time points (day 0, 1, 2, or 3) after lethal-dose TBI. Approximately 24 h later, BM samples were subjected to a competitive repopulation assay to test secondary reconstitution ability in test HSCs that homed to irradiated BM environment. Consequently, test HSCs that were exposed for ≂f24 h to BM environment at day 2 post TBI showed marked impairment in their long-term reconstitution ability. When “BM-homed” HSCs were enumerated 24 h after infusion, we found modestly impaired HSC homing to BM irradiated 2 days prior to transplantation, compared to BM irradiated on day 0, indicating negative effects on HSCs transplanted during a peak phase of inflammatory storm. Next we examined whether inflammatory cytokines were capable of impairing HSC functions. When tested in liquid culture using purified HSCs, both IFN-g and IL-1 had little effects on colony formation. In contrast, TNF-a inhibited colony formation in a dose-dependent manner. When in vivo HSC exposure to irradiated BM environment was tested using TNF-a KO mice as primary recipients, significant improvement of HSC ability was observed, indicating a major role for this inflammatory cytokine in the HSC-inhibitory effect observed within the irradiated host tissues. We finally sought to test if blocking of TNF-a signaling in HSCs at a peak phase of inflammation could lead to better transplantation outcomes. We utilized the peptide previously shown to block TNFR signaling, and confirmed that pre-incubation of HSCs with this molecule did suppress TNF-a induced reactive oxygen species production in HSCs. Studies are ongoing to test if HSCs transplanted in a peak-phase of TBI-mediated inflammation will benefit from this shielding measure. Conclusion:We demonstrated that inflammatory response in BM environment 2–3 days after irradiation could have a negative effect on donor HSCs regarding both homing efficiency and secondary reconstitution ability. These findings provide important implications for developing the measures that enable HSCs to escape from this inhibitory effect to achieve far more improvement in clinical HSCT outcomes. Disclosures:No relevant conflicts of interest to declare.

Mobilization with Cyclophosphamide Shifts Hematopoiesis From the Bone Marrow to the Spleen In Mouse.

AbstractAbstract 1590 Introduction:Spleen is an active hematopoietic tissue in adult mice. Spleen niches supporting hematopoiesis were not extensively studied yet. Spleen does not contain osteoblastic niches which are considered to be essential for long-term hematopoiesis, and splenic hematopoiesis is assumed to originate mainly from progenitors with short-term repopulating ability. Normal spleen is primarily a lymphoid organ but it also contributes to myelopoiesis by containing approximately 5% of total body multipotent progenitors CFU-S. We have studied the effect of mobilization with cyclophosphamide on trafficking of hematopoietic stem/progenitor cells (HSPC) from the bone marrow to the spleen, and dependence of normal splenic hematopoiesis on continual supply of HSPC from the bone marrow. Method:Murine congenic model C57Bl/6 Ly5.1/Ly5.2, and C57Bl/6 GFP, was used in the experiments. Kinetics of HSPC in the bone marrow and the spleen after a sublethal dose of cyclophosphamide (135 mg/kg) was measured by clonal assays (CFU-GM and CFU-S – progenitors) and by competitive repopulation assay (short- and long-term repopulating cells, STRC and LTRC). Parabiotic pairs were established from control and cyclophosphamide-treated partners in order to demonstrate HSPC exchange between hematopoietic organs. Selective irradiation of either spleen with bone marrow shielded, or irradiation of bone marrow with spleen shielded, was used to measure significance of HSPC migration from the spleen to the bone marrow during hematopoietic regeneration. Selected cytokine expression in the bone marrow and in the spleen was determined by real-time PCR. Result:Bone marrow HSPC rapidly but transiently regenerated from cyclophosphamide damage. Secondary decrease in HSPC between 5 and 7 days after cyclophosphamide in the bone marrow was accompanied with significant reduction of SDF-1 mRNA expression, mobilization of HSPC into peripheral blood and their rapid accumulation in the spleen. Spleen thus transiently became the main hematopoietic organ in the mouse by containing 69% of total body CFU-S, and also contained significantly more LTRC compared to the femur (Fig). [Display omitted] Surprisingly, there was no increase in SDF-1 mRNA expression during homing of HSPC in the spleen. Spleens of parabiotic partners were rapidly colonized by partner cells including HSPC within two weeks. Bone marrow also contained small but significant amount of partner HSPC one and 2 weeks after parabiosis establishment. In pairs made from control and cyclophosphamide treated mice, mobilized HSPC preferentially engrafted in both own and partner spleens. Spleen cellularity and progenitor content was not affected by selective spleen irradiation and was restored from shielded bone marrow 6 days after irradiation. Expansion of extramedullary splenic hematopoiesis after cyclophosphamide occurred even when HSPC were destroyed in the spleen 1 day after cyclophosphamide, i.e. the spleen expansion was dependent on the bone marrow. Irradiation of bone marrow with spleen shielded led to abruption of splenic hematopoiesis in both control and cyclophosphamide treated mice. Conclusion:There is a quantitatively significant trafficking of HSPC by means of the circulation in mice. The splenic hematopoiesis appears to be almost fully dependent on a continuous supply of HSPC from the bone marrow. This trafficking of HSPC can be enhanced by hematopoietic cell mobilization. In mice, damage caused by cyclophosphamide results in a transient, however quantitatively remarkable relocation of HSPC, including LTRC, from the bone marrow to the spleen. This accumulation of HSPC in spleen is not accompanied with increased expression of SDF-1 mRNA. HSPC circulating in peripheral blood also significantly colonize the bone marrow and actively participate in its hematopoiesis. Support: Projects LC06044 and MSM 0021620806 of the Ministry of Education, Youth and Sports of the Czech Republic. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic Stem Cells Survive Warm Ischemia and the Bone Marrow Upregulates a Significant Number of Genes

AbstractAbstract 4694 Background:Quiescent hematopoietic stem cells (HSCs) located in stem cell niches are characterized by a relative resistance to hypoxia. This study is focused primarily on maintainance of the repopulating ability of HSCs in structurally intact BM exposed to anoxia, lack of metabolic substrates and accumulation of metabolic waste products during a period of ischemia at three different temperatures. In the case of a warm ischemia at 37°C, changes in gene expression profile in the whole bone marrow has been also examined. Methods:Murine congenic model C57Bl/6 Ly5.1/Ly5.2 was used in the experiments. Normal mice or mice recovering from a bone marrow damage induced either by cyclophosphamide or a sublethal irradiation were sacrified. Their BM was maintained in intact femurs at 37°C for different time periods up to 6 hours. For normal bone marrow, exposure to ischemia at 20°C and 4°C was also used for up to 20 and 48 hours, respectively. Afterwards, bone marrow cells were harvested and cells corresponding to a half of the femur were transplanted to sublethally (6 Gy) irradiated recipients in a competitive repopulation assay. Resulting chimerism was examined up to 6 months after transplantation to test for STRCs and LTRCs (Short and Long Term Repopulating Cells). Subpopulations of erythropoietic (Ter119+), B-lymphopoietic (B220+), granulo- and monocytopoietic (Gr-1/Mac+), and LSK (Lin-Sca-1+c-Kit+) bone marrow cells were analyzed for dead cells and apoptosis. Total RNA was isolated from bone marrow exposed to warm ischemia ranging 0 to 4 hours and dynamics of changes in its gene expression profile was determined by Illumina MouseRef8 BeadChip. Results:Repopulating ability of ischemic BM was fully preserved for 2 hour of the warm (37°C) ischemia and for 6 hours and 8 hours of 20°C and 4°C ischemia, respectively. There was no difference between STRCs and LTRCs in survival. STRCs and LTRCs from the bone marrow collected 2 days or 5 days after a single dose of cyclophosphamide exposed to warm ischemia showed decreased repopulating ability in comparison with those of normal mice. STRCs significantly prevailed over LTRCs in bone marrow collected 20 days after a sublethal irradiation and showed increased sensitivity to warm ischemia. B220+ cells were the most sensitive cells of the bone marrow to warm ischemia, LSK and Ter119 cells being the most resistant ones. Gene expression profile in bone marrow exposed to warm ischemia changed progressively over time. Despite the highly unfavorable metabolic conditions, hypoxia and lack of energy, a set of overexpressed genes equaled in number the one inhibited. Conclusions:HSCs exposed to warm or cold ischemia maintain their repopulating ability for a considerable time. Bone marrow ischemia activates specific gene expression in paralel with supression of others. Supported by projects LC06044, MSM 0021620806 and the grant SVV-2010-254260507. Disclosures:No relevant conflicts of interest to declare.

Obligatory Asymmetric Cell Division Regulates Self-Renewal In Hematopoietic Progenitor/Stem Cells

AbstractAbstract 571Asymmetric cell division, a proposed mechanism by which hematopoietic progenitor/stem cells (HPSC) maintain a balance between self-renewal and differentiation, has rarely been observed. Here we report the surprising finding that cultured mouse primary HPSC routinely generate pairs of daughter cells with 2 distinct phenotypes after a single round of cell division. Mouse bone marrow cells were cultured on chamber slides in the presence of stem cell factor (SCF). BrdU was added overnight to label dividing cells, and the cells were examined by immunofluorescence microscopy on day 2–4 of culture. In each BrdU+c-Kit+ divided cell doublet, c-Kit was invariably expressed in only 1 of the 2 daughter cells. In contrast, the other daughter cell was negative for c-Kit but positive for the asymmetric cell fate determinant Numb and mature myeloid markers Mac1, Gr1, M-CSFR and F4/80. Similarly, in each BrdU+Sca1+ cell doublet, 1 daughter cell was positive for the stem cell markers Sca1, c-Kit, CD150 and CD201, whereas the other cell was negative for these markers but positive for Numb and the mature myeloid markers. Analysis of 400 such doublets showed that the probability of HPSC undergoing asymmetric division was 99.5% (95% confidence interval 98–100%), indicating that asymmetric division in HPSC is in fact not rare but obligatory. In other model systems, it has been shown that activation of the atypical protein kinase C (aPKC)-Par6-Par3 cell polarity complex and realignment of the microtubule cytoskeleton precede asymmetric cell division. We asked whether similar steps are involved in the asymmetric division of HPSC. We found that c-Kit receptors, upon stimulation by SCF, rapidly capped at an apical pole next to the microtubule-organizing center, followed by redistribution to the same pole of the aPKC-Par6-Par3 complex and microtubule-stabilizing proteins APC, β-catenin, EB1 and IQGAP1. Strikingly, after cell division, the aPKC-Par6-Par3 complex and other polarity markers all partitioned only into the c-Kit+/Sca1+ daughter cell and not the mature daughter cell. The acetylated and detyrosinated forms of stabilized microtubules were also present only in the c-Kit+/Sca1+ cell, as were the Aurora A and Polo-like kinases, 2 mitotic kinases associated with asymmetric cell division. To understand how c-Kit activation triggers downstream polarization events, we studied the role of lipid rafts, cholesterol-enriched microdomains in the cell membrane that serve as organization centers of signaling complexes. These are enriched in phosphatidylinositol 4,5-bisphosphate and annexin 2, putative attachment sites for the aPKC-Par6-Par3 complex. We found that SCF stimulation led to coalescence of lipid raft components at the site of the c-Kit cap, and treatment with a wide range of inhibitors that blocked lipid raft formation abrogated polarization of the aPKC-Par6-Par3 complex and division of the c-Kit+/Sca1+ cells. Because obligatory asymmetric division in cultured HPSC would prevent a net increase in their number, we sought a way to bypass its mechanism. We tested whether inhibition of protein phosphatase 2A (PP2A), a physiological antagonist of aPKC, would enhance aPKC activity and promote self-renewal of HPSC. Treatment of cultured HPSC with okadaic acid or calyculin, 2 well-characterized PP2A inhibitors, increased the percent of c-Kit+/Sca1+ cells undergoing symmetric division from 0% to 23.3% (p<0.001). In addition, small colonies comprised of symmetrically dividing cells uniformly positive for Sca1, c-Kit, CD150 and CD201 were noted in the culture. To functionally characterize the effect of PP2A inhibition, mouse bone marrow cells were cultured in the absence or presence of PP2A inhibitors and transplanted into irradiated congenic mice in a competitive repopulation assay. At 4–8 weeks post-transplant, the donor engraftment rate increased from ∼1 in mice transplanted with untreated cells to >30% in mice transplanted with PP2A inhibitor-treated cells. This dramatic increase indicates that PP2A inhibition can effectively perturb the mechanism of asymmetric cell division and promote the self-renewal of HPSC. In summary, our data showed that obligatory asymmetric cell division works to maintain a strict balance between self-renewal and differentiation in HPSC and pharmacological manipulation of the cell polarity machinery could potentially be used to expand HPSC for clinical use. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic Defects In ACD/TPP1-Deficient Mice Reveal An Essential Role for the Shelterin Complex In Blood-Forming Stem Cell Homeostasis

AbstractAbstract 882Bone marrow failure syndromes can be caused by loss-of-function mutations in telomerase components. Besides the enzymatic activity of telomerase, telomere maintenance requires the protective function of the shelterin protein complex. This complex consists of six proteins that bind to the telomere and prevent an inappropriate DNA damage response from being elicited by exposed telomeric DNA. Within this complex, TPP1, encoded by the gene Acd, plays a central role in complex organization as it links elements that bind the double-stranded portion of the telomere to those that bind the single-stranded overhang. We report an essential function for the shelterin complex and specifically for TPP1 in the maintenance of hematopoietic stem cells (HSCs). Utilizing adrenocortical dysplasia (acd), a spontaneous autosomal recessive mouse mutation causing profoundly hypomorphic Acd expression, we identified phenotypic and functional abnormalities in fetal hematopoietic progenitors. acd mutant Fetal liver HSCs were larger, more granular, and expressed higher levels of the cell surface protein Sca-1, as compared to wild-type counterparts. BrdU incorporation analysis showed an accumulation of Acd-deficient progenitors in the G2M phase of the cell cycle, consistent with G2M arrest. Absolute quantification of fetal liver HSCs revealed a significant reduction in stem cell numbers in acd mutants as compared to control littermates. p53 deficiency rescued the HSC depletion phenotype, but induced a paradoxical exacerbation of the increased progenitor cell size and Sca-1 expression. These findings suggest that p53-dependent and p53-independent elements contribute to the acd HSC phenotype. In competitive repopulation assays, acd HSCs failed to provide tri-lineage hematopoietic reconstitution in lethally irradiated recipients, indicating a profound functional defect. To further study the effects of Acd deficiency in adult HSCs, we generated an Acd allele in which exons 3–8 are flanked by loxP sites, allowing conditional inactivation following Mx1-Cre induction via poly(I:C) administration. Acd inactivation resulted in rapid HSC depletion within 2 weeks after initiation of poly(I:C) injections. To assess whether Acd exerted exclusively cell-autonomous effects in HSCs, we transplanted Acdf/–Mx1-Cre+ HSCs into wild-type recipients. Poly(I:C)-mediated Acd inactivation in these bone marrow chimeric mice resulted in rapid HSC loss and failure of tri-lineage hematopoiesis in animals reconstituted with Acd-deficient bone marrow. These data indicate an exquisite cell-autonomous requirement for Acd/TPP1 in adult mouse HSCs. In view of the extremely rapid HSC loss after Acd inactivation, these observations cannot be explained by telomere shortening. Together, our findings represent the first report of an essential role for Acd/TPP1 and the shelterin complex in HSC maintenance and hematopoietic homeostasis, independently of telomerase function. Disclosures:No relevant conflicts of interest to declare.

Mechanism of Calcium Sensing Receptor Mediated Hematopoietic Stem Cell Lodgment In the Adult Bone Marrow Niche

AbstractAbstract 399The ability of hematopoietic stem cells (HSCs) to maintain an undifferentiated state and undergo self-renewal is partly regulated by external signals originating from the stem cell niche. One receptor expressed on HSCs that is known to be involved in HSC niche biology is the calcium-sensing receptor (CaR). Our previous study using HSCs obtained from the fetal liver of mice deficient in CaR has shown the crucial role of CaR in HSC lodgment and engraftment in the bone marrow (BM), where CaR-/- HSCs lose their ability to lodge in the endosteal surface of the bone, leading to defective engraftment. To further investigate the mechanism of CaR-mediated lodgment of HSCs, we used a pharmacological approach to activate the receptor and assess the in vitro and in vivo effects. Cinacalcet treatment, which acts as a positive allosteric modulator of CaR to increase the sensitivity of the receptor to activation by extracellular Ca2+, leads to a 3-fold increase in primitive hematopoietic cell activity in vitro as assessed by the cobblestone forming cell assay. The increase in activity in vitro does not appear to be an effect of alterations in cell proliferation, cell survival or the expression of cell adhesion molecules such as VLA-4 or L-selectin. Rather, with CaR stimulation, long-term HSCs have an increased adhesion capability to collagen I, a major ECM molecule present predominantly in the BM endosteal region. We also observed that activation of the CaR following Cinacalcet treatment significantly enhances HSC homing to the BM, lodgment at the endosteal surface and in vivo engraftment capabilities as assessed by a competitive repopulation assay. This enhancement of in vivo activity correlates with increased CXCR4 signaling and migration towards an SDF-1alpha stimulus. Signaling through this receptor is known to be important in cell migration, proliferation, survival, and retention of HSCs in the BM following transplantation. Analysis of the CaR on human cells has demonstrated that there is a distinct population of CaR+ cells present in the CD34+ cell population. The frequency of this population varies between approximately 2% on mobilized peripheral blood CD34+ cells and 6% on BM CD34+ cells. Further analysis is being performed to define the identity of this subpopulation of CaR+CD34+ cells. These mechanisms by which the CaR dictates preferential localization of HSCs in the BM endosteal region may provide additional insights for the fundamental interrelationship between the stem cell niche and stem cell fate. These studies also have implications in the area of clinical stem cell transplantation, where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM. Disclosures:No relevant conflicts of interest to declare.

The Scd1 Gene Functions as a Tumor Suppressor In Leukemia Stem Cells

AbstractAbstract 201We have previously shown that the arachidonate 5-lipoxygenase gene (Alox5) functions as a critical regulator of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML) in mice. The Alox5 pathway appears to represent a major molecular network in LSCs. Taking advantage of our DNA microarray analysis for the identification of critical genes regulated by BCR-ABL in LSCs, we identified a small group of candidate genes that likely play tumor suppressor roles in these stem cells, and among them, a gene called stearoyl-CoA desaturase 1 (Scd1), an endoplasmic reticulum enzyme catalyzing the biosynthesis of monounsaturated fatty acids from saturated fatty acids, was shown to have a strong inhibitory effect on survival of LSCs in CML mice. BCR-ABL transduced bone marrow cells from Scd1-/- mice induced CML much faster than BCR-ABL transduced wild type bone marrow cells, and overexpression of Scd1 dramatically delayed CML development. Therefore, we further investigated whether Scd1 suppresses LSCs. FACS analysis showed that the percentages and total numbers of LSCs (GFP+ Lin−c-Kit+Sca-1+) and long-term (GFP+ Lin−c-Kit+Sca-1+ CD34−) or short-term (GFP+ Lin−c-Kit+Sca-1+ CD34+) LSCs in bone marrow of recipients of BCR-ABL transduced Scd1-/- donor bone marrow cells were significantly higher than those in bone marrow of recipients of BCR-ABL transduced wild type donor bone marrow cells, suggesting that Scd1 suppresses LSCs. Next we did a competitive repopulation assay to examine the function of LSCs. LSCs were sorted by FACS from bone marrow of mice with primary CML induced by transplanting BCR-ABL-transduced Scd1-/- (CD45.2) or wild type (CD45.1) bone marrow cells. The sorted CD45.2 and CD45.1 LSCs were mixed in a 1:1 ratio, followed by transplantation into lethally irradiated recipient mice to induce secondary CML. At 8 weeks after transplantation, only less than 10% of GFP+Gr-1+ cells were CD45.1 leukemia cells derived from wild type mice, whereas more than 75%-80% of GFP+Gr-1+ cells in peripheral blood of the mice were CD45.2 leukemia cells derived from Scd1-/- mice. To determine how Scd1 deficiency affects the maintenance of LSCs, we examined the cell cycle and apoptosis of LSCs. We found the percentages of apoptotic LSCs (Annexin V+ cells) were significantly decreased in bone marrow and spleens from Scd1-/- CML mice compared to the wild type group; however, we did not observe significant differences in the cell cycle status of LSCs from bone marrow and spleen, indicating that Scd1 regulates apoptosis but not cell cycle of LSCs.PPARγ agonist rosiglitazone can increase Scd1 expression, and our real time PCR data showed that rosiglitazone significantly induced Scd1 expression in bone marrow cells from CML mice. Therefore, we used PPARγ agonist rosiglitazone to treat these cells, and observed that rosiglitazone treatment dramatically decreased LSCs and that loss of Scd1 partially rescued the effect of PPARγ agonist on LSCs. Further, we investigated the molecular mechanisms that may contribute to the acceleration of CML development resulting from Scd1 deficiency. The decreased apoptosis in LSCs from Scd1-/- CML mice led us to focus on apoptosis-related genes. Real time PCR analysis showed a significant decrease of p53 in Scd1-/- immature leukemia cells as compared with that in wild type immature leukemia cells; however, loss of Scd1 resulted in dramatically and modest increased expression of Bcl-2 and Mcl-1 respectively in immature leukemia cells. We also found that the tumor suppressor gene Pten was significantly downregulated in Scd1-/- LSCs. Together, our results demonstrate a novel tumor suppressor function of Scd1 in LSCs, and provide a rationale for suppressing LSCs by enhancing Scd1 expression. Disclosures:No relevant conflicts of interest to declare.

Evi1 Is a Stem Cell-Specific Regulator of Self-Renewal Capacity In the Definitive Hematopoietic System

AbstractAbstract 838Self-renewal is a defining property of stem cells. Although a number of molecules have been implicated in the regulation of hematopoietic stem cell (HSC) self-renewal, loss of these genes is accompanied with other hematological abnormalities. Thus, it is unclear what will happen with a specific loss of self-renewal capacity of HSCs. Evi1 is an oncogenic transcription factor in myeloid malignancies. Evi1 expression is limited to hematopoietic stem/progenitor fraction, and Evi1 is essential for the maintenance of HSCs, but is dispensable for blood cell lineage commitment. Thus, we hypothesized that Evi1 expression could distinguish hematopoietic stem and progenitor cells, and reduction of Evi1 gene dosage might cause a specific loss of self-renewal activity. First, to elucidate Evi1 expression within the hematopoietic system, we have generated Evi1-IRES-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of Evi1 gene. We found that Evi1 was predominantly expressed in the hematopoietic stem/progenitor fraction (Lin- Sca-1+ c-kit+ (LSK)), but its expression was rapidly extinguished during early stages of lineage commitment. Among the LSK compartment, Evi1 was expressed at the highest level in long-term HSCs (LT-HSCs; Flk2- CD34-, CD48- CD150+, or SP-tip fractions in LSK cells). Next, we hypothesized that Evi1 would have the potential to mark LT-HSCs effectively. To test this, we compared GFP+ and GFP- cells in the LSK fraction, and revealed that GFP+ LSK cells were more immature and quiescent with a higher colony-forming capacity than GFP- LSK cells. In addition, in vivo long-term multilineage repopulating cells were exclusively enriched in the GFP+ LSK fraction. In the embryo, Evi1 was highly expressed in the hematopoietic stem/progenitor fraction; that is, CD34+ c-kit+ cells in embryonic day 10.5 (E10.5) aorta-gonad-mesonephros, CD34+ c-kit+ CD48- cells in E12.5 placenta, and Mac-1+ Sca-1+ Lin- (MSL) CD48- cells in E14.5 fetal liver (FL). In vivo competitive repopulation assay showed that, in the MSL fraction of FL, GFP+ MSL cells exclusively had a long-term multilineage repopulating capacity. These results implied that Evi1 plays a more specific role in HSCs than in other hematopoietic cells. To clarify this, we analyzed heterozygous Evi1 knockout mice (Evi1 +/− mice), as it seems difficult to elucidate the function of a small population of HSCs in Evi1 conditional knockout mice due to the leaky expression of Cre recombinase. We have previously showed that haploinsufficiency of Evi1 leads to decreased numbers of LSK and CD34- LSK cells, and impaired long-term repopulating activity. Here we demonstrated the number of each fraction in Evi1 +/− LSK cells was reduced in proportion to their expression level of Evi1. But, there were no significant differences in the numbers of lymphoid and myeloid progenitors between Evi1 +/+ and Evi1 +/− mice. Evi1 +/− CD34+ LSK cells had an equivalent in vitro colony-forming capacity and day 11 colony-forming unit-spleen activity to Evi1 +/+ CD34+ LSK cells. However, in vivo short-term repopulation assay using CD34+ LSK cells showed that the percentage of donor-derived cells from Evi1 +/− mice was significantly declined at 4 weeks after transplantation. Moreover, Evi1 +/− CD34- LSK cells had a pronouncedly impaired in vivo repopulating capacity. These data suggested that the differentiation capacity of Evi1 +/− HSCs was maintained, but their self-renewal capacity was specifically reduced. Although flow cytometric analysis of cell-cycle status and apoptosis showed no differences in CD34- LSK cells between Evi1+/+ and Evi1 +/− mice, the G0 fraction of Evi1 +/− CD34+ LSK cells was significantly reduced, indicating that these cells might proliferate more rapidly to compensate for the impaired self-renewal capacity of HSCs. In conclusion, we showed that Evi1 is predominantly expressed in HSCs and its expression can mark long-term repopulating HSCs in the fetal and adult hematopoietic system. Moreover, functional loss caused by haploinsufficiency of Evi1 is limited to a defect of self-renewal capacity of HSCs, and the increased cell-cycle progression of CD34+ LSK cells in Evi1 +/− mice seems to be the consequence of the impaired self-renewal capacity. Our data may help to understand the unrevealed effects of loss of self-renewal activity of HSCs and compensative mechanism of their defects. Disclosures:No relevant conflicts of interest to declare.