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.