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Introduction Hematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation. The stem cell fate is tightly connected with the cell cycle, where the major regulator anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks cell cycle regulatory proteins, such as cyclin A and B, for proteasomal degradation and thus controls their activity. Known targets of Cdh1 are involved in regulation of self-renewal and granulopoiesis. This raises the hypothesis that Cdh1 may be a critical mediator of HSC proliferation, self-renewal and differentiation. Methods CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokines. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with CD34 detection. The knockdown (kd) of Cdh1 was achieved by lentiviral delivery of specific shRNA into target cells. Results In vitro cultivation of CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed impressive downregulation of Cdh1 during culture. A high Cdh1 expression in CD34+ cells and lower expression in myeloid cells (CD41a+, CD15+, Glycophorin A+) reflects the situation we found in vivo in bone marrow (BM) subsets. Western blotting also revealed inactivation of Cdh1 by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the Cdk inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. When cultured under self-renewal conditions, CD34+ cells showed diminished proliferation with cells residing in lower generations, whereas during granulopoiesis, cells accumulated within higher generations. These experiments also revealed a more rapid decrease of CD34+ cells in granulopoiesis after three cell divisions in contrast to a moderate decline under self-renewal conditions. We also found a decreased colony-forming ability in cells divided more than twice during granulopoiesis, which correlates with their lower CD34 expression. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual commitment during granulopoiesis. Our current experiments extent these analyses to immunofluorescence of Numb distribution in individual cells to elucidate the impact of Cdh1 on symmetric/asymmetric cell division. We could already show that Cdh1-kd led to expansion of CD34+ HSCs in vitro. To further validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain-/- (NSG) xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of engrafted human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. Further analyses of the contributing subsets to the pool of CD45+ human cells are ongoing. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Conclusions APC/CCdh1 mediates cell cycle entry and proliferation during self-renewal and differentiation in CD34+ HSCs in vitro and improves engraftment capacity in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 1236 Hematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation to all types of blood lineages. These unique processes are regulated by intrinsic and extrinsic signals (e.g. cytokines, cell-cell contacts) and strongly connects stem cell fate with the cell cycle. The ubiquitin-proteasome system regulates spatial and temporal abundance of proteins in the cell. During cell cycle, the anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks proteins for proteasomal degradation and thus controls their activity. Known targets of Cdh1, namely Skp2 and Id2, are involved in regulation of self-renewal and granulopoiesis (Wang et al., Blood 2011; Buitenhuis et al., Blood 2005). This raises the hypothesis that Cdh1 may be a critical upstream regulator of HSC differentiation. The analysis of human bone marrow cell subsets (CD34+, lymphoid and myeloid cells) revealed highest protein level of Cdh1 in CD34+ cells, lower expression in more mature lymphoid subsets (CD3+, CD19+) and only marginal expression in mature myeloid cells (CD41a+, CD11b+). These data suggest that Cdh1 is important to induce differentiation, but dispensable for maintaining the differentiated state. In vitro cultivation of G-CSF mobilized peripheral blood CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed downregulation of Cdh1 during culture compared to d0. Western blots did not only reveal decreasing levels of Cdh1, but also its inactivation by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the cyclin-dependent-kinase inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. To analyze cell division/proliferation and self-renewal versus differentiation more closely, we used the fluorescent dye CFSE as an indicator of cell division in combination with CD34 to indicate the differentiation status. When cultured under self-renewal conditions using SCF, TPO and Flt3-l, CD34+cells showed enhanced proliferation with increased cells in higher generations, whereas using SCF and G-CSF to induce granulopoiesis, cells within lower generations were more prominent. These experiments also revealed a rapid decrease of CD34 expression in granulopoiesis after 3 cell divisions in contrast to a moderate decline under self-renewal conditions. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual cell cycle exit and differentiation under conditions that induce granulopoiesis. To further elucidate the role of Cdh1 for stem/progenitor cell fate, we used a lentiviral knockdown of Cdh1 in CD34+ cells. After 4 days of transduction and cell sorting, the cells were cultivated for 1 week in medium containing SCF, TPO and Flt3-l. Cdh1 depleted cells showed enhanced proliferation compared to the empty vector control and a higher expression of CD34. In colony forming unit (CFU) assays, we observed that CD34+ cells with Cdh1-knockdown were less efficient to differentiate to CFU-G, CFU-M and BFU-E. A higher potential to self-renew was validated by replating of these colonies, where the number with Cdh1-knockdown increased during serial replating. To validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain−/− (NSG) xenotransplant mouse model. The evaluation of engraftment capacity and differentiation potential of human Cdh1 depleted CD34+ cells in this model is ongoing. Our data establish the central cell cycle regulator APC/CCdh1 as a novel regulator of self-renewal and differentiation in CD34+ HSCs. Disclosures: No relevant conflicts of interest to declare.

Abstract 2379 Introduction: The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. This is achieved by ubiquitinylation of various cell cycle regulators to tag them for proteasomal degradation. APC/C in conjunction with its adaptor protein Cdh1 (APC/CCdh1), both stabilizes G1-phase and is involved in the induction of cell cycle arrest and differentiation. Here, we have analyzed the influence of APC/CCdh1 on self-renewal capacity and differentiation potential and kinetics of human hematopoietic stem cells (HSCs). Methods: In order to study the expression levels of Cdh1 among different hematopoietic lineages, we stained mononuclear cells from bone marrow of healthy donors with antibodies against the cell surface markers CD11b, Glycophorin A, CD41a, CD34, CD3 and CD19, and isolated these subsets via cell sorting. We extracted protein from these subsets and performed Western Blot analysis. We established a strong lentiviral Cdh1 knock down (kd) in CD34+ cells and performed colony forming cell (CFC) assays: 1×104 Cdh1-kd-CD34+ cells and CD34+ cells transduced with a control vector (ctrl-CD34+) were plated in MethoCult H4534 and MethoCult H4534 + EPO (1 IU/ml). At 14 days, CFU-GEMM, CFU-GM, CFU-G, CFU-M and BFU-E were scored. CD11b- and Glycophorin A-FACS, FACS analysis of propidium iodide staining and Pappenheim's staining were carried out on individually picked colonies in order to assess differentiation kinetics and cell cycle distribution. Self-renewal capacity of ctrl- and Cdh1-kd-CD34+ cells was examined by performing replating assays (secondary CFCs) with the obtained CFU-GEMM colonies as previously described (Katayama et al., BMT, 1999). Secondary colonies were analyzed after another 14 days of incubation. Results: We observed Cdh1 protein levels to significantly vary among hematopoietic cell subsets: The highest Cdh1 levels were detected in CD34+ cells, lower levels in cells of the lymphoid lineage (CD3+; CD19+) and only marginal expression levels in cells arising from myeloid progenitors (CD11b; Glycophorin A; CD41a). By correlating Cdh1 levels of the individual cell subsets with their cell cycle profiles, we were able to exclude the possibility that this was merely due to differences in cell cycle distribution. Analysis of the CFC assays performed with the lentiviraly infected CD34+ cells showed a considerable decrease of about 40% in the number of BFU-E and 35% in the number of CFU-G- and CFU-M-numbers, when Cdh1-kd-CD34+ cells were plated compared to ctrl-CD34+ cells. Furthermore, we observed an increase of CFU-GEMMs with Cdh1 depletion. The expression levels of the cell surface markers CD11b and Glycophorin A were 10–20% lower among the colonies arisen from Cdh1-kd-CD34+ cells vs. ctrl-CD34+ cells. Upon manual counting of Pappenheim stained preparations, we found the early stages of both erythroid and myeloid differentiation to be more prevalent in the Cdh1-kd colonies. When studying the replating capacity, we observed that Cdh1-depleted cells gave rise to almost twice as many secondary colonies as compared to ctrl-cells. There was no difference with regard to the relative proportions of the colony types. Interestingly, tracking of GFP, which had been used as a reporter-gene for the lentiviral transduction of the ctrl- and Cdh1-kd-cells, showed that it was enhanced in Cdh1-kd-secondary colonies compared to ctrl-secondary colonies. Conclusion: By analyzing human bone marrow subsets we observed that Cdh1 levels diminish from HSCs to mature lymphoid and, to an even greater extent, mature myeloid cells, suggesting that Cdh1 is important to induce differentiation but dispensable for maintaining the differentiated state. Our in vitro results are consistent with an important role of APC/CCdh1 in both myeloid and erythroid differentiation of HSCs. The data suggests that depletion of Cdh1 in HSC interferes with normal differentiation both by decreasing the number of mature lineage progenitors and by delaying individual cell maturation. HSCs deficient in Cdh1 seem to increasingly undergo self-renewal. The stronger the Cdh1-kd the more likely the generation of secondary colonies appeared to be. In vivo models may be particularly helpful to further elucidate these phenomena. Disclosures: No relevant conflicts of interest to declare.