APC/C^Cdh1 Regulates Self-Renewal and Differentiation of Hematopoietic Stem Cells

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/C^Cdh1), both stabilizes G1-phase and is involved in the induction of cell cycle arrest and differentiation. Here, we have analyzed the influence of APC/C^Cdh1 on self-renewal capacity and differentiation potential and kinetics of human hematopoietic stem cells (HSCs).

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.

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.

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/C^Cdh1 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.

No relevant conflicts of interest to declare.