Dissecting Stem Cell Proliferation and Differentiation in Association with the Central Cell Cycle Regulator APC/C^Cdh1

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/C^Cdh1 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/C^Cdh1 as a novel regulator of self-renewal and differentiation in CD34⁺ HSCs.