Hematopoietic progenitor cell transplantation can contribute to revascularization of ischemic tissues. Yet, the optimal cell population to be transplanted has yet to be determined. We have compared the therapeutic potential of two subsets of human cord blood CD34+ progenitors, either expressing the VEGF-A receptor 2 (KDR) or not. In serum-free starvation culture, CD34+KDR+ cells reportedly showed greater resistance to apoptosis and ability to release VEGF-A, as compared with CD34+KDR- cells. When injected into the hind muscles in immunodeficient SCIDbg mice subjected to unilateral ischemia, a low number (10(3)) of CD34+KDR+ cells improved limb salvage and hemodynamic recovery better than a larger dosage (10(4)) of CD34+KDR- cells. The neovascularization induced by KDR+ cells was significantly superior to that promoted by KDR- cells. Similarly, endothelial cell apoptosis and interstitial fibrosis were significantly attenuated by KDR+ cells, which differentiated into mature human endothelial cells and also apparently skeletal muscle cells. This study demonstrates that a low number of CD34+KDR+ cells favors reparative neovascularization and possibly myogenesis in limb ischemia, suggesting the potential use of this cell population in regenerative medicine.

MicroRNAs (miRNAs) regulate cell proliferation, differentiation and death during development and postnatal life. The expression level of mature miRNAs results from complex molecular mechanisms, including the transcriptional regulation of their genes. MiR-223 is a hematopoietic-specific miRNA participating in regulatory signaling networks involving lineage-specific transcription factors (TFs). However, the transcriptional mechanisms governing its expression levels and its functional role in lineage fate decision of human hematopoietic progenitors (HPCs) have not yet been clarified. We found that in CD34þ HPCs undergoing unilineage differentiation/maturation, miR-223 is upregulated more than 10-fold during granulopoiesis, 3-fold during monocytopoiesis and maintained at low levels during erythropoiesis. Chromatin immunoprecipitation and promoter luciferase assays showed that the lineage-specific expression level of mature miR-223 is controlled by the coordinated binding of TFs to their DNA-responsive elements located in 'distal' and 'proximal' regulatory regions of the miR-223 gene, differentially regulating the transcription of two primary transcripts (pri-miRs). All this drives myeloid progenitor maturation into specific lineages. Accordingly, modulation of miR-223 activity in CD34þ HPCs and myeloid cell lines significantly affects their differentiation/ maturation into erythroid, granulocytic and monocytic/macrophagic lineages. MiR-223 overexpression increases granulopoiesis and impairs erythroid and monocytic/macrophagic differentiation. Its knockdown, meanwhile, impairs granulopoiesis and facilitates erythropoiesis and monocytic/macrophagic differentiation. Overall, our data reveal that transcriptional pathways acting on the differential regulation of two pri-miR transcripts results in the fine-tuning of a single mature miRNA expression level, which dictates the lineage fate decision of hematopoietic myeloid progenitors.

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.

MicroRNAs (miRNAs) are important regulators of several cellular processes. During hematopoiesis, specific expression signatures have been reported in different blood cell lineages and stages of hematopoietic stem cell (HSC) differentiation. Here we explored the expression of miRNAs in umbilical cord blood stem (HSC) and progenitor cells (HPC) and compared it to unilineage granulocyte and granulo-monocyte differentiation as well as to primary blasts from patients with acute myeloid leukemia (AML). CD34 + CD38- ad CD34 + CD38 +  cells were profiled using a global array consisting of about 2000 miRNAs. An approach combining bioinformatic prediction of miRNA targets with mRNA expression profiling was used to search for putative biologically enriched functions and networks. At least 15 miRNAs to be differentially expressed between HSC and HPC cell population, a cluster of 7 miRNAs are located in the q32 region of human chromosome 14 (miR-377-3p, -136-5p, 376a-3p, 495-3p, 654-3p, 376c-3p and 381-3p) whose expression decreased during the early stages of normal myelopoiesis but were markedly increased in a small set of AML. Interestingly, miR-4739 and -4516, two novel microRNA whose function and targets are presently unknown, showed specific and peculiar expression profile during the hematopoietic stem cells differentiation into unilineages and resulted strongly upregulated in almost all AML subsets. miR-181, -126-5p, -29b-3p and -22-3p resulted dis-regulated in specific leukemias phenotypes. This study provides the first evidence of a miRNA signature in human cord blood stem and progenitor cells with a potential role in hematopoietic stemness properties and possibly in leukemogenesis of specific AML subtypes.

© F e r r a t a S t o r t i F o u n d a t i o ndromes for its deletion on chromosome 5 37 and in the progression of chronic myeloid leukemia. 38 We previously identified the molecular mechanism that regulates the level of CXCR4 protein expression by miR146a targeting 29 (miR-146a/CXCR4) during monocytopoiesis. 39 We also reported that in acute monocytic leukemia (AML-M5), a high CXCR4 protein level is associated with low/absent miR-146a expression. 39 We then showed that enforced miR-146a expression in leukemic cells decreases the level of CXCR4 protein and improves the sensitivity of these cells to drugs. 39CXCR4 is a target gene of HIF-1α and a post-transcriptional target of miR-146a, 9,39 in monocytic cells, known to originate from a common myeloid precursor in the bone marrow giving rise to tissue macrophages and dendritic cells 40 and subject to very different oxygen (O 2 ) levels. 41In this study, we investigated the impact of chronic hypoxia on the miR-146a/CXCR4 regulatory axis during monocytopoiesis and in monocytic leukemic cells. The levels of hypoxia studied were mild (5% O 2 ), such as that present in the sinusoidal cavity of bone marrow, and more severe (1% O 2 ), such as that in hypoxic niches in bone marrow.Altogether, our data demonstrated how the differential expression of HIF-1α and HIF-2α is mediated by O 2 level (mild or severe) and down-regulates CXCR4 expression through a post-transcriptional mechanism mediated by up-regulation of miR-146a in normal monocytic cells and in monocytic leukemia cell lines, expressing a moderate level of CXCR4. However, this mechanism is dysregulated in primary AML-M5 blast cells that fail to decrease CXCR4 expression significantly in response to hypoxia. This dysregulation helps to explain why leukemic blasts express high CXCR4 levels, even in hypoxic conditions, and how they are protected from anti-leukemic drugs through CXCR4 activation mediated by SDF-1α secreted in the hypoxic bone marrow microenvironment. MethodsAdditional information is provided in the Online Supplement. Cell culturesAfter obtaining informed consent, human cord blood was taken from healthy donors and samples of blood were taken from patients with AML. This study was approved by the local ethical committees of the Italian National Institute of Health and the University of Tor Vergata.Cord blood CD34 + hematopoietic progenitor cell purification, unilineage monocytic differentiation and morphological analyses were performed as previously described. 39 Human primary AML-M5 blasts were maintained in culture in vitro in Iscove medium supplemented with 10% fetal calf serum, granulocyte-macrophage colony-stimulating factor (10 ng/mL), stem cell factor (50 ng/mL), and interleukin-3 (10 ng/mL) (PeproTech Inc. Rocky Hill, NJ, USA). RNA and protein samples were prepared as described elsewhere. 39 Leukemic cell lines, U937 and HL-60, were grown in RPMI medium supplemented with 10% fetal calf serum (Gibco, Carlsbad, CA, USA). HypoxiaTo provide a mild or more severe hypoxic environment, acute ...