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// Emiliano Fabiani 1, * , Giulia Falconi 1, * , Nelida Ines Noguera 1, 2 , Ernestina Saulle 3 , Laura Cicconi 1 , Mariadomenica Divona 1 , Cristina Banella 2 , Alessandra Picardi 1 , Anna Maria Cerio 4 , Letizia Boe 5 , Massimo Sanchez 5 , Elvira Pelosi 4 , Ugo Testa 4 , Francesco Lo-Coco 1, 2 and Maria Teresa Voso 1 1 Universita di Roma Tor Vergata, Dipartimento di Biomedicina e Prevenzione, Rome, Italy 2 Fondazione Santa Lucia, Laboratorio di Neuro-Oncoematologia, Rome, Italy 3 Istituto Superiore di Sanita, Centro Nazionale per la Ricerca e la Valutazione Preclinica e Clinica dei Farmaci, Rome, Italy 4 Istituto Superiore di Sanita, Dipartimento di Ematologia ed Oncologia, Rome, Italy 5 Istituto Superiore di Sanita, Grandi Strumentazioni e Core Facilities, Rome, Italy * These authors contributed equally to this work Correspondence to: Maria Teresa Voso, email: voso@med.uniroma2.it Keywords: acute promyelocytic leukemia, forkhead box C1, ATRA, decitabine, epigenetic regulation Received: April 27, 2017 Accepted: August 31, 2017 Published: September 20, 2017 ABSTRACT Forkhead box (FOX) genes encode transcription factors, which regulate embryogenesis and play an important role in hematopoietic differentiation and in mesenchymal niche maintenance. Overexpression of the family member FOXC1 has been reported in solid tumors and acute myeloid leukemia (AML). We studied FOXC1 expression and function in acute promyelocytic leukemia (APL) and normal hematopoietic progenitors. FOXC1 mRNA and protein levels were significantly lower in primary marrow samples from 27 APL patients, as compared to samples obtained from 27 patients with other AML subtypes, and 5 normal CD34+ hematopoietic cells. FOXC1 expression significantly increased in APL samples at the time of remission following consolidation treatment. In cell lines overexpressing PML-RARA, and in the NB4 t(15;17)-positive cell line, FOXC1 expression was lower than in other non-APL cell lines, and increased following treatment with all-trans retinoic acid (ATRA), due to functional binding of ATRA to the FOXC1 promoter region. Reduced FOXC1 expression was also associated to DNA hypermethylation of the +354 to +568 FOXC1 region, both in primary APL, and in NB4 cells. Treatment of NB4 cells with decitabine demethylated FOXC1 and upregulated its expression. Our findings indicate that FOXC1 is consistently repressed in APL due to hypermethylation and the presence of the PML-RARA rearrangement. A potential role of hypomethylating treatment in advanced APL remains to be established.

Circulating endothelial progenitor cells (EPCs) have been suggested as a precious source for generating functionally competent endothelial cells (ECs), candidate for various clinical applications. However, the paucity of these progenitor cells and the technical difficulties for their in vitro growth represent a main limitation to their use. In the present study we hypothesized that the paracrine effects of human umbilical vein endothelial cells (HUVECs) may improve endothelial cell generation from cord blood (CB) EPCs. In line with this hypothesis we showed that HUVEC conditioned medium (CM) or co-culture with HUVECs markedly improved the proliferation and differentiation and delayed the senescence of CB EPCs. The endothelial-promoting effect of CM seems to be related to smaller vesicles including exosomes (sEV/exo) contained in this medium and transferred to CB CD34(+) EPCs: in fact, purified preparations of sEV/exo isolated from CM mimicked the effect of CM to sustain endothelial formation. These observations provided the interesting indication that mature ECs exert a stimulatory effect on endothelial cell differentiation from CD34(+) cells.

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. J. Cell. Physiol. 230: 1770–1780, 2015. © 2014 Wiley Periodicals, Inc.

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. J. Cell. Physiol. 227: 3291–3300, 2012. © 2011 Wiley Periodicals, Inc.

The cellular response to methylation DNA damage was compared in multipotent CD34(+) hematopoietic stem cells and mature CD34(-) cells isolated from cord blood of the same donor. Cytofluorimetric analysis of freshly isolated cord blood cells indicated that both cell types were in the G0/G1 phase of the cell cycle. Quantitative RT-PCR identified a general trend towards high expression of several DNA repair genes in CD34(+) cells compared to their terminally differentiated CD34(-) counterparts. The overexpressed genes included members of the mismatch repair (MMR) (MSH2, MSH6, MLH1, PMS2), base excision repair (AAG, APEX), DNA damage reversal (O(6)-methylguanine DNA methyltransferase) (MGMT), and DNA double strand breaks repair pathways. These differences in gene expression were not apparent in CD34(+) and CD34(-) cells obtained following expansion of CD34(+) cells in a medium containing early acting cytokines. Early progenitor CD34(+) and early precursor CD34(-) cells form the two populations isolated under these experimental conditions, and both contain a significant proportion of cycling cells. The methylating agent N-methyl-N-nitrosourea (MNU) induced similar levels of apoptosis in these cycling CD34(+) and CD34(-) cells. Cytotoxicity required the presence of the MGMT inhibitor O(6)-benzylguanine and the timing of MNU cell death (48 and 72h) was similar in CD34(+) and CD34(-) cells. These data indicate that cycling CD34(+) and CD34(-) cells are equally sensitive to methylation damage. MGMT provides significant protection against MNU toxicity and MGMT and MMR play the expected roles in the MNU sensitivity of these cells.

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 CD34(+)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 CD34(+)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 the triterpene CDDO and its potent derivatives, CDDO-Im and CDDO-Me, are now in phase I/II studies in the treatment of some pathological conditions, their effects on normal hematopoiesis are not known. In the present study we provide evidence that CDDO-Im exerts in vitro a potent inhibitory effect on erythroid cell proliferation and survival and a stimulatory action on megakaryocytic differentiation. The effect of CDDO-Im on erythroid and megakaryocytic differentiation was evaluated both on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid (E) or megakaryocytic (Mk) differentiation and on erythroleukemic cell lines HEL and TF1. The inhibitory effect of CDDO-Im on erythroid cell survival and proliferation is mainly related to a reduced GATA-1 expression. This conclusion is supported by the observation that GATA-1 overexpressing TF1 cells are partially protected from the inhibitory effect of CDDO-Im on cell proliferation and survival. The stimulatory effect of CDDO-Im on normal megakaryopoiesis is seemingly related to upmodulation of GATA2 expression and induction of mitogen-activated protein kinases ERK1/2.

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.