By Maintaining Cells in an Inactive CD34− State RNA-Binding Protein Pum2 Promotes Proliferation and Inhibits Differentiation of Multipotent Hematopoietic Cells.
Authors: Yang Jie , Di Vito Kyle , Nachtman Ronald G. , Jurecic Roland 
Published: April 2020

Abstract
Previously we have reported that over-expression of RNA-binding protein Pum2 supports self-renewal and severely attenuates mutilineage differentiation of murine HSC-like cell line EML. To analyze further the impact of Pum2 on maintenance and differentiation of EML cells, we examined the expression of c-kit, Sca-1, Flk-2 and CD34 HSC markers on wild type EML cells and EML cells over-expressing Pum2 (Pum2-EML cells). Almost all wt EML cells are Sca-1+c-kit+ Flk2−, but exhibit heterogeneous expression of CD34, with 20–40% of cells being CD34−, and 60–80% of cells being CD34+. Similarly, almost all Pum2-EML cells are Sca-1+c-kit+Flk2−, but the vast majority of Pum2-EML cells are CD34− (80–95%). To determine the functional significance of this change in CD34 expression, wt EML and Pum2-EML cells were sorted into CD34+ and CD34− populations (&gt;99% purity), and their differentiation capacity assessed using colony-forming assays. The wt EML and Pum2-EML CD34+ cell population repeatedly contained almost all of the cells capable of mutilineage differentiation in response to cytokines, and forming for example BFU-E, CFU-Meg and CFU-GM colonies. In contrast, the CD34− subpopulation of wt EML and Pum2-EML cells differentiated poorly or not at all. Thus, in terms of the phenotype and cytokine-induced differentiation the CD34− and CD34+ subpopulations of EML cells resemble CD34− and CD34+ subpopulations of HSC-enriched Lin−Sca-1+c-kit+Flk-2− BM cells. Our results also raised the question of developmental relationship between CD34− and CD34+ EML cells. Interestingly, several studies have shown that mouse CD34− HSC become CD34+ after G-CSF mobilization or 5-FU treatment, and that after transplant these “activated” CD34+ HSC revert back to CD34− “inactive” state. These studies have uncovered a putative new link between HSC activation and CD34 expression. In view of these findings we tested whether CD34− and CD34+ EML cell populations represent different developmental stages, where e.g. CD34− cells give rise to CD34+ cells, orwhether CD34 expression is linked to cell “activation”, and EML cells can alternate between CD34− inactive (differentiation inhibited) and CD34+ active (differentiation ready) state.
For that purpose sorted CD34− and CD34+ EML cells (&gt;99% pure) were cultured separately for 3 weeks. An aliquot of cells from these cultures was taken each day and analyzed by flow cytometry for CD34 expression. These experiments have revealed that CD34+ cells started to appear in CD34− cell cultures after 24 hours, and were maintained at different frequencies throughout the culture. Interestingly, we also observed initial slow appearance of CD34− cells in CD34+ cell cultures, however their percentage subsequently increased and was maintained during the culture period. Taken together, our results suggest that Sca-1+ c-kit+ Flk2− CD34− population of EML cells is in “inactive” self-renewal state and differentiates poorly or not at all, whereas the CD34+ population could represent “activated” state at which the cells are ready to undergo differentiation. Furthermore, our results indicate that normally a balance is maintained between CD34− inactive and CD34+ active state of EML cells, andthat increased levels of Pum2 protein tip that balance and maintain the cells in the CD34− inactive state, thus promoting proliferation and inhibiting differentiation, two critical components of self-renewal.