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The interleukin-3 receptor (IL-3R) subunits are overexpressed on acute myeloid leukemia (AML) blasts compared with normal hematopoietic cells and are thus potential targets for novel therapeutic agents. Both fluorescence-activated cell sorter (FACS) analysis and quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) were used to quantify expression of the IL-3Ralpha and beta(c) subunits on AML cells. QRT-PCR for both subunits was most predictive of killing of AML colony-forming cells (AML-CFCs) by diphtheria toxin-IL-3 fusion protein (DT(388)IL3). Among 19 patient samples,the relative level of the IL-3Ralpha was higher than the IL-3Rbeta(c) and highest in CD34(+)CD38(-)CD71(-) cells,enriched for candidate leukemia stem cells,compared with cell fractions depleted of such progenitors. Overall,the amount of IL-3Rbeta(c) subunit did not vary among sorted subpopulations. However,expression of both subunits varied by more than 10-fold among different AML samples for all subpopulations studied. The level of IL-3Rbeta(c) expression versus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (set at 1000) ranged from 0.14 to 13.56 in CD34(+)CD38(-)CD71(-) cells from different samples; this value was correlated (r = .76,P = .05) with the ability of DT(388)IL3 to kill AML progenitors that engraft in beta(2)-microglobin-deficient nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (n = 7). Thus,quantification of IL-3R subunit expression on AML blasts predicts the effectiveness IL-3R-targeted therapy in killing primitive leukemic progenitors. View Publication

An acquired somatic mutation,Jak2V617F,was recently discovered in most patients with polycythemia vera (PV),chronic idiopathic myelofibrosis (CIMF),and essential thrombocythemia (ET). To investigate the role of this mutation in vivo,we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F,but not Jak2wt,resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit,leukocytosis,megakaryocyte hyperplasia,extramedullary hematopoiesis resulting in splenomegaly,and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors,although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity,and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore,we observed strain-specific differences in phenotype,with Balb/c mice demonstrating markedly elevated leukocyte counts,splenomegaly,and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans. View Publication

RATIONALE: Fibrocytes are progenitor cells characterized by the simultaneous expression of mesenchymal,monocyte,and hematopoietic stem cell markers. We previously documented their presence in lungs of patients with idiopathic pulmonary fibrosis. However,the mechanisms involved in their migration,subsequent homing,and local role remain unclear. Matrix metalloproteinases (MMPs) facilitate cell migration and have been implicated in the pathogenesis of pulmonary fibrosis. OBJECTIVES: To evaluate the expression and role of matrix metalloproteinases in human fibrocytes. METHODS: Fibrocytes were purified from CD14(+) monocytes and cultured for 8 days; purity of fibrocyte cultures was 95% or greater as determined by flow cytometry. Conditioned media and total RNA were collected and the expression of MMP-1,MMP-2,MMP-7,MMP-8,and MMP-9 was evaluated by real-time polymerase chain reaction. Protein synthesis was examined using a Multiplex assay,Western blot,fluorescent immunocytochemistry,and confocal microscopy. MMP-2 and MMP-9 enzymatic activities were evaluated by gelatin zymography. Migration was assessed using collagen I-coated Boyden chambers. Stromal cell-derived factor-1α and platelet-derived growth factor-B were used as chemoattractant with or without a specific MMP-8 inhibitor. MEASUREMENTS AND MAIN RESULTS: Fibrocytes showed gene and protein expression of MMP-2,MMP-9,MMP-8,and MMP-7. MMP-2 and MMP-9 enzymatic activities were also demonstrated by gelatin zymography. Likewise,we found colocalization of MMP-8 and MMP-7 with type I collagen in fibrocytes. Fibrocyte migration toward platelet-derived growth factor-B or Stromal cell-derived factor-1α in collagen I-coated Boyden chambers was significantly reduced by a specific MMP-8 inhibitor. CONCLUSIONS: Our findings reveal that fibrocytes express a variety of MMPs and that MMP-8 actively participates in the process of fibrocyte migration. View Publication

Human embryonic stem cells (hESC) are capable to give rise to all cell types in the human body during the normal course of development. Therefore,these cells hold a great promise in regenerative cell replacement based therapeutical approaches. However,some controversy exists in literature concerning the ultimate fate of hESC after exposure to genotoxic agents,in particular,regarding the effect of DNA damaging insults on pluripotency of hESC. To comprehensively address this issue,we performed an analysis of the expression of marker genes,associated with pluripotent state of hESC,such as Oct-4,Nanog,Sox-2,SSEA-4,TERT,TRA-1-60 and TRA-1-81 up to 65h after exposure to ionizing radiation (IR) using flow cytometry,immunocytochemistry and quantitative real-time polymerase chain reaction techniques. We show that irradiation with relatively low doses of gamma-radiation (0.2Gy and 1Gy) does not lead to loss of expression of the pluripotency-associated markers in the surviving hESC. While changes in the levels of expression of some of the pluripotency markers were observed at different time points after IR exposure,these alterations were not persistent,and,in most cases,the expression of the pluripotency-associated markers remained significantly higher than that observed in fully differentiated human fibroblasts,and in hESCs differentiated into definitive endodermal lineage. Our data suggest that exposure of hESC to relatively low doses of IR as a model genotoxic agent does not significantly affect pluripotency of the surviving fraction of hESC. View Publication

Embryonic stem cells (ESCs) exhibit unrestricted and indefinite,but stringently controlled,proliferation,and can differentiate into any lineage in the body. In the current study,we test the hypothesis that expression of ribosomal RNA (rRNA) and ribosomal protein genes (RPGs) contribute to the ability of hESCs to proliferate indefinitely. Consistent with the accelerated growth rate of hESCs,we find that hESC lines H1 and H9 both exhibit significantly higher levels of rRNA when compared to a panel of normal and cancer human cell lines. Although many RPGs are expressed at levels that comparable to other human cell lines,a few RPGs also exhibit higher expression levels. In situ nuclear run-on assays reveal that both nucleoli in hESCs actively transcribe nascent rRNA. Employing genome-wide chromatin immunoprecipitation-deep sequencing and bioinformatics approaches,we discovered that,RPGs are dominantly marked by the activating H3K4me3 histone mark in the G1,M,and G2 phases of the cell cycle. Interestingly,the rDNA repeats are marked by the activating H3K4me3 only in the M phase,and repressive H3K27me3 histone mark in all three cell cycle phases. Bioinformatics analyses also reveal that Myc,a known regulator of cell growth and proliferation,occupies both the rRNA genes and RPGs. Functionally,down-regulation of Myc expression by siRNA results in a concomitant decrease in rRNA levels. Together,our results show that expression of rRNA,which is regulated by the Myc pluripotency transcription factor,and of RPGs in hESCs is associated with the activating H3K4me3 modification. J. Cell. Physiol. 231: 2007-2013,2016. textcopyright 2016 Wiley Periodicals,Inc. View Publication

Granulocyte colony-stimulating factor (G-CSF),the prototypical mobilizing cytokine,induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated,in part,through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)-deficient bone marrow chimeras to show that G-CSF-induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF-induced HSPC mobilization,osteoblast suppression,and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact,demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover,G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally,we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together,these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance,ultimately leading to HSPC mobilization. View Publication

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects,C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover,in K562 cells,RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1,but not of a transcriptional repressor mutant (Gfi-1P2A),inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast,Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together,these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells. View Publication

BACKGROUND: Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood,as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow,detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al,2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens,with particular attention to the expression of ALDH on erythroid precursors. To this aim,we used three kinds of approach: i) multidimensional analytical flow cytometry,detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells,followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. RESULTS: In normal bone marrow,we identified three populations of cells,namely ALDH+CD34+,ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52,0.53 and 0.57,respectively). As compared to ALDH-CD34+ cells,ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells,with brighter expression of CD117 and CD133,accompanied by lower display of CD38 and CD45RA. Of interest,ALDH+CD34- population disclosed a straightforward erythroid commitment,on the basis of three orders of evidences. First of all,ALDH+CD34- cells showed a CD71bright,CD105+,CD45- phenotype. Secondly,induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally,ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). CONCLUSION: Our study,comparing surface antigen expression of ALDH+/CD34+,ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow,clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia. View Publication

Human embryonic stem (hES) cells hold great promise for application of human cell and tissue replacement therapy. However,the overwhelming majority of currently available hES cell lines have been directly or indirectly exposed to materials containing animal-derived components during their derivation,propagation,and cryopreservation. Unlike feeder based cultures,which require the simultaneous growth of feeder and stem cells,resulting in mixed cell populations,stem cells grown on feeder-free systems are easily separated from the surface,presenting a pure population of cells for downstream applications. In this study we have developed a novel method to expand hES cells in xeno-free,feeder-free conditions using two different matrices derived from xeno-free human foreskin fibroblasts (XF-HFFs). Using XF-HFF-derived extracellular matrix,together with 100ng/ml recombinant bFGF supplemented HEScGRO Basal Medium,long term xeno-free expansion of hES cells is possible. Resulting hES cells were subjected to stringent tests and were found to maintain ES cell features,including morphology,pluripotency,stable karyotype,and expression of cell surface markers,for at least 20 passages. Xeno-free culturing practices are essential for the translation of basic hES cell research into the clinic. Therefore,the method presented in this study demonstrates that hES cells can be cultured in complete xeno-free conditions without the loss of pluripotency and furthermore,without the possibility of contamination from exogenous sources. View Publication

Although extracellular nucleotides support a wide range of biologic responses of mature blood cells,little is known about their effect on blood cell progenitor cells. In this study,we assessed whether receptors for extracellular nucleotides (P2 receptors [P2Rs]) are expressed on human hematopoietic stem cells (HSCs),and whether activation by their natural ligands,adenosine triphosphate (ATP) and uridine triphosphate (UTP),induces HSC proliferation in vitro and in vivo. Our results demonstrated that CD34(+) HSCs express functional P2XRs and P2YRs of several subtypes. Furthermore,stimulation of CD34(+) cells with extracellular nucleotides caused a fast release of Ca(2+) from intracellular stores and an increase in ion fluxes across the plasma membrane. Functionally,ATP and,to a higher extent,UTP acted as potent early acting growth factors for HSCs,in vitro,because they strongly enhanced the stimulatory activity of several cytokines on clonogenic CD34(+) and lineage-negative CD34(-) progenitors and expanded more primitive CD34(+)-derived long-term culture-initiating cells. Furthermore,xenogenic transplantation studies showed that short-term preincubation with UTP significantly expanded the number of marrow-repopulating HSCs in nonobese diabetic/severe combined immunodeficiency mice. Our data suggest that extracellular nucleotides may provide a novel and powerful tool to modulate HSC functions. View Publication

Factor induced reprogramming of fibroblasts is an orchestrated but inefficient process. At the epigenetic level,it results in drastic chromatin changes to erase the existing somatic memory" and to establish the pluripotent state. Accordingly� View Publication

The incidence of cardiovascular disease represents a significant and growing health-care challenge to the developed and developing world. The ability of native heart muscle to regenerate in response to myocardial infarct is minimal. Tissue engineering and regenerative medicine approaches represent one promising response to this difficulty. Here,we present methods for the construction of a cell-seeded cardiac patch with the potential to promote regenerative outcomes in heart muscle with damage secondary to myocardial infarct. This method leverages iPS cells and a fibrin-based scaffold to create a simple and commercially viable tissue-engineered cardiac patch. Human-induced pluripotent stem cells (hiPSCs) can,in principle,be differentiated into cells of any lineage. However,most of the protocols used to generate hiPSC-derived endothelial cells (ECs) and cardiomyocytes (CMs) are unsatisfactory because the yield and phenotypic stability of the hiPSC-ECs are low,and the hiPSC-CMs are often purified via selection for expression of a promoter-reporter construct. In this chapter,we describe an hiPSC-EC differentiation protocol that generates large numbers of stable ECs and an hiPSC-CM differentiation protocol that does not require genetic manipulation,single-cell selection,or sorting with fluorescent dyes or other reagents. We also provide a simple but effective method that can be used to combine hiPSC-ECs and hiPSC-CMs with hiPSC-derived smooth muscle cells to engineer a contracting patch of cardiac cells. View Publication