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Global microRNA profiling of normal and Pbx1-null hematopoietic stem cells and progenitors for the identification of new regulators of the balance between self-renewal and differentiation

Final Report Summary - HSC SELF-RENEWAL (Global microRNA profiling of normal and Pbx1-null hematopoietic stem cells and progenitors for the identification of new regulators of the balance between self-renewal and differentiation.)


The ability of balancing self-renewal and multi-potent differentiation is a key hallmark of somatic stem cells; however, the molecular pathways underlying this regulation are not completely understood, including any role for micro-RNAs (miRNAs).
Pbx1 (pre B cell leukemia homeobox 1) is a homeodomain transcription factor that positively regulates hematopoietic stem cells (HSC) quiescence [1]. Its absence in post-natal HSCs causes an excessive proliferation that ultimately leads to their exhaustion, indicating a profound self-renewal defect, and a premature myeloid differentiation at the expenses of the lymphoid one [2]. Cell cycle regulation and differentiation, both abnormal in Pbx1-null HSCs, are two major roles of miRNAs. Therefore, the study of Pbx1-null HSCs provided the unique opportunity to identify miRNAs involved in the maintenance of HSC identity.
We employed Pbx1-conditional knockout mice (and controls) to perform a global miRNA profiling of HSCs and of their immediate downstream progeny named multi-potent progenitors (MPPs), in order to identify new regulators of the balance between self-renewal and differentiation. To address this issue, we performed a TaqMan-based multiplex miRNA profiling of highly purified HSCs and of MPPs not expressing the Flk2 marker. Flk2-negative MPPs share with HSCs the capacity to differentiate towards all blood lineages, however they lack long-term self-renewal capacity, and represent the first maturation step along the hematopoietic hierarchy immediately downstream of the HSC stage. The miRNA profiles of HSCs and Flk2-negative MPPs from Pbx1-null and from littermate control mice were then compared to our previously described microarray mRNA data [1], relative to the same populations, to search for miRNA predicted targets (PT) whose change in expression inversely correlates with those of miRNAs. Unsupervised hierarchical clustering indicates a clear distinction between HSCs and MPPs at the level of miRNA expression, suggesting that miRNAs might regulate the first transition step in the adult hematopoietic development. Within each group, mutant and control cells cluster separately, linking miRNAs to self-renewal impairment. More in detail, SAM (Statistical Analysis of Microarray) analysis showed 48 miRNAs differentially expressed (DE) between Pbx1-null and wt HSCs, among which 84% have anti-correlated PT within the list of DE mRNAs. A similar analysis on wt cells revealed 71 DE miRNAs during the physiological HSC-to-MPP transition, of which 31 are concordantly DE in the Pbx1-null HSCs, in accordance with the hypothesis that miRNAs are involved in HSC self-renewal. Among those 31, by applying very stringent selection criteria we have selected few miRNA candidates. Within this short list the miRNA that is most DE both in Pbx1-null HSCs and in the normal HSC-to-MPP transition is particularly interesting since it is strongly HSC-specific, being quickly down regulated in Flk2-negative MPPs, and not re-expressed further down in the hematopoietic hierarchy. Its level of expression is comparable to that of other miRNAs previously associated to HSCs, such as miR-99b, miR-125, let-7, miR-221 and miR-126. However, it is the only one whose down-regulation occurs already at the first transition from HSCs to Flk2-negative MPPs, whereas the other mentioned miRNAs are still expressed at the progenitors level. We confirmed its HSC-specificity in human CD34+ cells, and found that it is aberrantly expressed in human primary CML samples. We are currently dissecting its role through gain and loss of function experiments, with the aim of studying its mechanism(s) of function and its role in regulating HSC self-renewal. Results obtained so far suggest an in vivo competitive disadvantage of miRNA-overexpressing Lineage- cells over un-transduced cells in long-term transplantation assays, and a self-renewal defect as assessed in secondary recipients. This indicates that a tight regulation of the studied miRNA is crucial to maintain HSC functions. A manuscript describing the findings obtained so far is under preparation. Meanwhile, a paper describing a further dissection of the role of Pbx1 in hematopoietic progenitors entitled “Pbx1 restrains myeloid maturation while preserving lymphoid potential in hematopoietic progenitors” was published in 2013 with the Fellow as first author [2].
Given the potential role of the studied miRNA in regulating mouse and human HSC self-renewal and in CML, this research will have an impact for public health since it will allow to pursue new anti-cancer targeting therapies.
The IRG Grant and the described results allowed the Fellow to obtain a permanent position as a Researcher with the Italian National Research Council, and to establish her Project Leader role within her Institute.


References

1. Ficara F, Murphy MJ, Lin M, Cleary ML: Pbx1 regulates self-renewal of long-term hematopoietic stem cells by maintaining their quiescence. Cell stem cell 2008, 2(5):484-496.
2. Ficara F, Crisafulli L, Lin C, Iwasaki M, Smith KS, Zammataro L, Cleary ML: Pbx1 restrains myeloid maturation while preserving lymphoid potential in hematopoietic progenitors. Journal of cell science 2013, 126(Pt 14):3181-3191.