Dissecting Stem Cell Proliferation and Differentiation In Association With The Central Cell Cycle Regulator APC/CCdh1In Vitro and In Vivo
Authors: Ewerth Daniel , Schmidts Andrea , Kuegelgen Birgit , Wider Dagmar , Schüler Julia , Engelhardt Monika , Waesch Ralph M. 
Published: October 2019

Abstract

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.