MicroRNAs (miRNAs) are non-coding RNA molecules that repress translation and stability of target mRNAs. They are key regulators of cell cycle and differentiation in embryonic stem cells, and are differentially expressed among many tumors, but their function in adult stem cells (SC) is not known. Somatic SC are able to self-renew, at the same time generating a differentiated progeny that replenish short-lived cells, to maintain tissue homeostasis. However, how the balance between self-renewal and differentiation is achieved, crucial to avoid SC exhaustion or hyper-proliferation and cancer, has not been fully clarified, including any role for miRNAs. The applicant previously demonstrated that in the absence of the proto-oncogene Pbx1 hematopoietic stem cells (HSCs) display reduced self-renewal, high proliferation rate, a transcriptional profile typical of downstream progeny, and perturbed Tgf-b pathway. In this proposal the applicant will take advantage of the Pbx1-conditional ko mouse model to identify miRNAs involved in the maintenance of self-renewal, through combination of miRNA profiling data of purified HSCs and downstream progenitors from mutant or control mice with mRNA expression data and with miRNA:mRNA target prediction. The role of specific miRNAs regulating self-renewal will then be tested functionally in normal and mutant HSCs, in transplantation assays after over-expression or knock down experiments. Moreover, this study offers the opportunity to identify new players in the Tgf-b pathway, which has been suggested to regulate processing of specific miRNAs in other cell types. Preliminary data indicate that in the absence of Pbx1 several miRNAs are indeed differentially regulated in HSCs, underlying the feasibility of this project. The expected results will have a profound impact on European competitiveness since they address fundamental questions in adult stem cell biology, with potential implications in the fields of regenerative medicine and cancer.