RNA-Binding Protein Pum2 Promotes Self-Renewal and Suppresses Differentiation of Multipotent Hematopoietic Cells by Maintaining Them in Inactive CD34− State.

Over-expression of RNA-binding protein Pum2 supports maintenance and suppresses mutilineage differentiation of murine multipotent HSC/MPP-like cell line EML. The analysis of HSC and MPP markers has revealed that ∼80% of wt EML cells are Sca–1 + c-kit + Flk2 − CD34 + and ∼20% are Sca–1 + c-kit + Flk − CD34 − . In contrast, the majority (80–95%) of EML cells over-expressing Pum2 (Pum2-EML) are Sca–1 + c-kit + Flk − CD34 − . Functional analysis has revealed that purified CD34 + wt EML and Pum2-EML cells reproducibly contained the majority of progenitors capable of mutilineage differentiation in response to cytokines, whereas CD34 − wt EML and Pum2-EML cells differentiated poorly or not at all. The CD34 + wt EML cells can be divided into CD34 low , CD34 medium and CD34 high subpopulations, and the analysis of their progenitor cell content has reproducibly shown that the majority of progenitors reside within CD34 high and CD34 med cells, whereas CD34 low cells contain very small percentage of progenitors. These findings indicated that CD34 + wt EML cells are functionally heterogeneous, and that the overall multilineage differentiation of EML cells strongly correlates with the CD34 expression levels. Since the majority of LTR-HSC reside within LKS Flk2 − CD34 − BM cells, and LKS Flk2 − CD34 + BM cells are highly enriched for STR-HSC and MPPs, we tested whether CD34 − EML cells are more primitive and give rise to CD34 + cells. Multiple experiments have revealed that purified CD34 − and CD34 + wt EML cells can generate each other in culture, suggesting that although the CD34 − EML cells could be more primitive, a subpopulation of CD34 + cells retains the capacity to give rise to CD34 − cells. Identical experiments have revealed that Pum2-EML cells are maintained predominantly as CD34 − cells that generate a small population of CD34 low cells. Further analysis has revealed that among CD34 + wt EML cells the CD34 low cells have the highest capacity to give rise to CD34 − EML cells. Previous studies have shown that mouse LTR-HSC can alternate between CD34 − “inactive” and CD34 + “activated” state, and that the heterogeneous population of LKS CD34 + BM cells contains activated LTR-HSC, STR-HSC and MPPs. Thus, we have proposed a model in which the CD34 − EML cells are more primitive cells in an “inactive” (differentiate inhibited) state that give rise to all CD34 + EML cells. On the other hand, the heterogeneous population of CD34 + EML cells consists of CD34 med/high cells that can readily differentiate into multiple lineages, and CD34 low cells that are in “activated” state and can revert back to the CD34 − state. Cumulatively, these results support the notion that Pum2 protein supports proliferation and suppresses differentiation (two critical components of self-renewal) of multipotent hematopoietic cells by maintaining them in an inactive CD34 − state. To start elucidating molecular differences between wt EML and Pum2-EML cells, and their CD34 + and CD34 − subpopulations, we performed expression analysis using Agilent 44K microarrays. Out of 14,868 expressed genes, 760 exhibited significant differences in expression level between wt EML and Pum2-EML cells, and included Mmp14, Mpp1, Ripk3, Calmodulin 5, DDEF2 (Development and differentiation enhancing factor 2), C/EBPbeta, TXK tyrosine kinase, Egr3, Ermap, Eraf, TNFR1a, Jam4 and others. Among 760 differentially expressed genes, 146 genes exhibited >10-fold higher expression in wt EML cells, and 85 genes exhibited >10-fold higher expression in Pum2-EML cells.