Functional role of Nuclear Fators-I in hematopoietic ontogenesis

Nuclear Factor I (NFI) transcriptional factors constitute a family of four members, NFI-A, B, C and X, known for their positive and negative transcriptional regulatory roles in a cell type and promoter specific context. We previously identified NFI-A as a relevant target of the myeloid regulator microRNA-223, then we found that its levels play a key role in directing hematopoietic progenitors to the erythroid or granulocytic lineage. This prompted us to examine whether the expression of NFI-A and/or other NFIs factors could regulate primitive and definitive hematopoiesis in vivo. To this end we initially studied the expression pattern of NFIs factors in different tissues and stages of embryo development of CD1 mice. Our preliminary results indicate that NFI-A presents the most interesting expression pattern among NFIs factors, being express in hematopoietic tissues earlier and at the highest level during embryo development. In addition, performing colony-forming progenitor assays, we found NFI-A expression in primitive erythroid progenitor and during definitive hematopoietic colonies production, implicating it in having a possible role in primitive and definitive hematopoiesis. To elucidate the role of NFI-A in hematopoiesis we used two different strains of NFI-A-/- mice: B6N31 and B6hyb129mice. Histological examinations of hematopoietic tissues of NFI-A-/- mice showed that Nfi-A disruption results in hypocellularity of hematopoietic compartment together with a marked decrease of M/E ratio. Genes expression analysis performed on B6N31 hematopoietic tissues indicates that NFI-A -/- mice have a delay in the repression of embryonic β-globins and a perinatal decrease in adult globins expression, suggesting an involvement for NFI-A in the control of β-globins switching. In addiction NFI-A -/- hematopoietic tissues presents an up-regulation of NFI-B expression, indicating its possible action as compensator of NFI-A. To investigate about the role of NFI-A in adult hematopoiesis, we performed complete blood counts of peripheral blood from adults B6N31 NFI-A +/- and we observed a decreased MCV, an increased RDW and a decreased MCH compared with adult NFI-A +/+ B6N31 mice, demonstrating an haploinsufficiency of NFI-A factor and an altered hemoglobin synthesis. These data indicates that NFI-A could be involved in the pathogenesis of hematological diseases, further underlying its importance in hematopoietic development

PDE2A is indispensable for mouse liver development and hematopoiesis

Phosphodiesterase 2A (PDE2A) is a cAMP-cGMP hydrolyzing enzyme essential for mouse development and the PDE2A knockout model (PDE2A−/−) is embryonic lethal. Notably, livers of PDE2A−/− embryos at embryonic day 14.5 (E14.5) have extremely reduced size. Morphological, cellular and molecular analyses revealed loss of integrity in the PDE2A−/− liver niche that compromises the hematopoietic function and maturation. Hematopoietic cells isolated from PDE2A−/− livers are instead able to differentiate in in vitro assays, suggesting the absence of blood cell-autonomous defects. Apoptosis was revealed in hepatoblasts and at the endothelial and stromal compartments in livers of PDE2A−/− embryos. The increase of the intracellular cAMP level and of the inducible cAMP early repressor (ICER) in liver of PDE2A−/− embryos might explain the impairment of liver development by downregulating the expression of the anti-apoptotic gene Bcl2. In summary, we propose PDE2A as an essential gene for integrity maintenance of liver niche and the accomplishment of hematopoiesis

Explant-derived human dental pulp stem cells enhance differentiation and proliferation potentials

Numerous stem cell niches are present in the different tissues and organs of the adult human body. Among these tissues, dental pulp, entrapped within the 'sealed niche' of the pulp chamber, is an extremely rich site for collecting stem cells. In this study, we demonstrate that the isolation of human dental pulp stem cells by the explants culture method (hD-DPSCs) allows the recovery of a population of dental mesenchymal stem cells that exhibit an elevated proliferation potential. Moreover, we highlight that hD-DPSCs are not only capable of differentiating into osteoblasts and chondrocytes but are also able to switch their genetic programme when co-cultured with murine myoblasts. High levels of MyoD expression were detected, indicating that muscle-specific genes in dental pulp cells can be turned on through myogenic fusion, confirming thus their multipotency. A perivascular niche may be the potential source of hD-DPSCs, as suggested by the consistent Ca(2+) release from these cells in response to endothelin-1 (ET-1) treatment, which is also able to significantly increase cell proliferation. Moreover, response to ET-1 has been found to be superior in hD-DPSCs than in DPSCs, probably due to the isolation method that promotes release of stem/progenitor cells from perivascular structures. The ability to isolate, expand and direct the differentiation of hD-DPSCs into several lineages, mainly towards myogenesis, offers an opportunity for the study of events associated with cell commitment and differentiation. Therefore, hD-DPSCs display enhanced differentiation abilities when compared to DPSCs, and this might be of relevance for their use in therap

Role of NFIB in normal hematopoiesis and MyeloproliferativeNeoplasms

Introduction: The three-canonical BCR-ABL negative myeloproliferative neoplasms (MPN), Polycythemia Vera (PV), Essential Thrombocytosis (ET) and Primary Myelofibrosis (PMF) are chronic diseases, which share the risk of disease evolution to an acute leukemia. In MPN, the most representative molecular lesion is a substitution of a valine for a phenylalanine (JAK2V617F) in the auto-inhibitory domain of JAK2, resulting in the constitutive expression of the gene.This point mutation has an incidence of about 96% in PV, 65% in PMF and 55% in ET. Microsatellite studies on chromosome 9 show the presence of a uniparental acquired disomy (UPD) of the short arm (9p), where JAK2 is located, as a common defect in MPN (Kralovics et al., ExpHematol. 2002). In a cohort study, all the samples with the 9pUPD were found positive for the JAK2V617F mutation (Klampfl et al, Blood 2011). The Nuclear Factor IB (NFIB) gene is present in the 9pUPD region, where a mutational hot spot takes place. NFIB belongs to the NFI family of CAAT box binding transcription factors, consisting of 4 separate genes (NFIA, -B, -C, -X). NFIA is a post-transcriptional target of myelopoiesis regulator miR-223, which plays a key role in directing the HSC/HPC maturation/differentiation into the erythroid or granulocytic lineages (Fazi et. al. Cell 2005; Starnes et al. Blood 2009). Genomic alterations of NFIA were detected in about 2% of MPN patients (Bernard, Leukemia 2009). The gene expression levels of NFIB are higher in CD34+HSC/HPC isolated from 5 JAK2V617F+PV patients than in normal controls (Berkofsky-Fessler, Clin Cancer Res 2010).However, the role of NFIB in normal and pathological hematopoiesis has not been yet investigated. Methods: DNA, mRNA, and proteins were isolated from human myeloid cell lines (K562, Hel and UKE-1) and buffy coat from peripheral blood (PB) and bone marrow (BM) cells isolated from MPN patients or healthy donors. Gene dosage and gene expression level were measured by qRT-PCR. Statistical analyses were used to calculate differences among and between groups by one way ANOVA and two-way t-test. Results: our preliminary data shows that: i) NFIB locus is amplified and its expression is increased in myeloid cell lines harboring the JAK2V617F; ii) NFIB is barely expressed in mononuclear cells isolated from healthy donors PB (n=23), while its expression increased in PB cells isolated from PV (n=18, p=0.034) and ET patients (n=27, p=0.005), independently from JAK2V617F status; iii) increased gene dosage of NFIB , paralleling JAK2 gene amplification is detected in MPN patients (n=27, p<0,001); iv) gene expression level of NFIB positively correlates with platelets count in ET patients (n=17, p=0.008). Conclusions: our preliminary data show the de-regulation of NFIB expression levels related to MPN, thus suggesting its role in MPN pathogenesis or disease evolution and usage as a marker for MPN diagnosis and/or prognosis