Cyclin C Regulates the Quiescence of Human CD34+CD38- Hematopoietic Stem Cells.

Abstract The relative quiescence of adult hematopoietic stem cells (HSCs) at steady state represents an important regulatory mechanism for maintaining their self-renewal and engraftment capacity, as well as their resistance to cytotoxic insults. However, the specific mechanisms regulating the intermittent entry of HSCs into the cell cycle are not well characterized. Here we provide the evidence that cyclin C (CCNC) specifically promotes the G0/G1 transition of human CD34+CD38- HSCs, and thus can significantly affect the loss of HSC self-renewal capacity in in vitro culture. Based on the recently hypothesized specific function of CCNC in G0 exit of human fibroblasts, we have analyzed the effects of CCNC loss on the behavior of human cord blood HSCs. We achieved a highly efficient knockdown of CCNC expression (>90%) using lentiviral shRNA (shCCNC) transduction of freshly isolated human cord blood CD34+ cells, allowing the in vitro assessment of early cell cycle regulation in HSCs. First, we observed a 3-fold increase in the G0 fraction of shCCNC transduced CD34+ cells compared to the empty vector control, based on the Pyronin Y and Hoechst 33342 staining 72h after infection. The depletion of CCNC did not prevent cell cycle progression beyond the G1 entry, as we observed no significant changes in the G1/S/G2-M distribution, indicating that critical CCNC activities may be restricted to the G0 checkpoint. Consistent with the reported enrichment of functional HSCs in the G0 fraction, CCNC knockdown (CCNC KD) cells showed increased activity in all surrogate in vitro assays of stem cell-ness tested: a ∼3 fold increase in CD34+ population after long term culture, a ∼2.5 fold increase in long-term culture initiating cells (LTC-ICs) and a ∼3.5 fold increase in cobblestone area forming cells (CAFCs). In contrast, CFU assays using freshly sorted shCCNC cells (and cells obtained after one-week culture in cytokines) showed only a minimal decrease in total colony number, with no difference in colony composition or morphology, indicating no significant effect on hematopoietic progenitor cell differentiation. However, we did observe a prominent effect on secondary CFUs after 2 and 3 weeks in liquid culture (i.e. using the delta assay), namely a 2-fold and 30-fold increase in shCCNC over control culture respectively, again indicating a specific function of CCNC on the more primitive cells. Consistently, CCNC KD robustly enhanced CD34 expression and secondary CFU maintenance in sorted CD34+CD38- cells (HSCs); both markers of hematopoietic cell immaturity were rapidly lost in CD34+CD38+ cells (i.e. the committed progenitor cells) with no detectable effect of shCCNC transduction. Finally, we have found that these effects of CCNC depletion are likely the result of its initial loss of function, as transient CCNC KD, using siRNA transfection of CD34+ cells, produced similar biological effects as the constitutive lentiviral shCCNC expression. Collectively, these data indicate a cell context-dependent effect of CCNC KD on the initial rate of cell cycle entry by quiescent HSCs and suggest that this approach could be used to preserve their functional capacity in culture, potentially enhancing the ex vivo expansion of HSCs, as well as their use in gene therapy protocols. Transplantation of transduced CD34+ cells into sublethally irradiated immunodeficient mice is now under way to establish the potentially beneficial effects of CCNC KD on the engraftment and repopulating capability of cultured HSCs.