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By retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs),we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here,we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction,human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development,it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary,a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells. View Publication

Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that,unlike steady-state erythropoiesis,erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM),severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In short-term transplantation experiments,donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context,stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system,EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion,and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x),in contrast,was not significantly induced via WT-EpoR,EpoR-HM,or EpoR-H alleles. In Kit+ CD71+ erythroblasts,EpoR-PY343 signals furthermore enhanced SCF growth effects,and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit- CD71+ erythroblasts,oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis,therefore,requires stage-specific EpoR-PY343-Stat5 signals,some of which selectively bolster SCF and oncostatin-M action. View Publication

A first step in hematopoiesis is the specification of the lymphoid and myeloid lineages from multipotent progenitor cells in the bone marrow. Using a conditional ablation strategy in adult mice,we show that this differentiation step requires Patched (Ptch),the cell surface-bound receptor for Hedgehog (Hh). In the absence of Ptch,the development of T- and B-lymphoid lineages is blocked at the level of the common lymphoid progenitor in the bone marrow. Consequently,the generation of peripheral T and B cells is abrogated. Cells of the myeloid lineage develop normally in Ptch mutant mice. Finally,adoptive transfer experiments identified the stromal cell compartment as a critical Ptch-dependent inducer of lymphoid versus myeloid lineage commitment. Our data show that Ptch acts as a master switch for proper diversification of hematopoietic stem cells in the adult organism. View Publication

Animal models of hematopoietic stem cell transplantation have been used to analyze the turnover of bone marrow-derived cells and to demonstrate the critical role of recipient antigen-presenting cells (APC) in graft versus host disease (GVHD). In humans,the phenotype and lineage relationships of myeloid-derived tissue APC remain incompletely understood. It has also been proposed that the risk of acute GVHD,which extends over many months,is related to the protracted survival of certain recipient APC. Human dermis contains three principal subsets of CD45(+)HLA-DR(+) cells: CD1a(+)CD14(-) DC,CD1a(-)CD14(+) DC,and CD1a(-)CD14(+)FXIIIa(+) macrophages. In vitro,each subset has characteristic properties. After transplantation,both CD1a(+) and CD14(+) DC are rapidly depleted and replaced by donor cells,but recipient macrophages can be found in GVHD lesions and may persist for many months. Macrophages isolated from normal dermis secrete proinflammatory cytokines. Although they stimulate little proliferation of naive or memory CD4(+) T cells,macrophages induce cytokine expression in memory CD4(+) T cells and activation and proliferation of CD8(+) T cells. These observations suggest that dermal macrophages and DC are from distinct lineages and that persistent recipient macrophages,although unlikely to initiate alloreactivity,may contribute to GVHD by sustaining the responses of previously activated T cells. View Publication

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors' effort,which,in concert with microRNAs,drives cell fate and responds to promiscuous patterns of gene expression by turning on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNA profiles from human CD34+ hematopoietic progenitor cells and in vitro differentiated erythroblasts,megakaryoblasts,monoblasts and myeloblast precursors that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically upregulated in one single-cell progeny and their putative target genes,which resulted in downregulation. Among the upregulated lineage-enriched microRNAs,hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitor fate,grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates in the regulation of hematopoietic progenitor fate,modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation. View Publication

Although endothelial cells (ECs) have been derived from human pluripotent stem cells (hPSCs),large-scale generation of hPSC-ECs remains challenging and their functions are not well characterized. Here we report a simple and efficient three-stage method that allows generation of approximately 98 and 9500 ECs on day 16 and day 34,respectively,from each human embryonic stem cell (hESC) input. The functional properties of hESC-ECs derived in the presence and absence of a TGF$$-inhibitory molecule SB431542 were characterized and compared with those of human umbilical vein endothelial cells (HUVECs). Confluent monolayers formed by SB431542(+) hESC-ECs,SB431542(-) hESC-ECs,and HUVECs showed similar permeability to 10,000 Da dextran,but these cells exhibited striking differences in forming tube-like structures in 3D fibrin gels. The SB431542(+) hESC-ECs were most potent in forming tube-like structures regardless of whether VEGF and bFGF were present in the medium; less potent SB431542(-) hESC-ECs and HUVECs responded differently to VEGF and bFGF,which significantly enhanced the ability of HUVECs to form tube-like structures but had little impact on SB431542(-) hESC-ECs. This study offers an efficient approach to large-scale hPSC-EC production and suggests that the phenotypes and functions of hPSC-ECs derived under different conditions need to be thoroughly examined before their use in technology development. This article is protected by copyright. All rights reserved. View Publication

Obesity has been suggested to be a low-grade systemic inflammatory state,therefore we studied the interaction between human adipocytes and monocytes via adipose tissue (AT)-derived capillary endothelium. Cells composing the stroma-vascular fraction (SVF) of human ATs were characterized by fluorescence-activated cell sorter (FACS) analysis and two cell subsets (resident macrophages and endothelial cells [ECs]) were isolated using antibody-coupled microbeads. Media conditioned by mature adipocytes maintained in fibrin gels were applied to AT-derived ECs. Thereafter,the expression of endothelial adhesion molecules was analyzed as well as the adhesion and transmigration of human monocytes. FACS analysis showed that 11% of the SVF is composed of CD14(+)/CD31(+) cells,characterized as resident macrophages. A positive correlation was found between the BMI and the percentage of resident macrophages,suggesting that fat tissue growth is associated with a recruitment of blood monocytes. Incubation of AT-derived ECs with adipocyte-conditioned medium resulted in the upregulation of EC adhesion molecules and the increased chemotaxis of blood monocytes,an effect mimicked by recombinant human leptin. These results indicate that adipokines,such as leptin,activate ECs,leading to an enhanced diapedesis of blood monocytes,and suggesting that fat mass growth might be linked to inflammatory processes. View Publication

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

Targeted nucleases,including zinc-finger nucleases (ZFNs),transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However,realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids,the direct delivery of nuclease proteins to cells provides rapid action and fast turnover,leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression,purification and delivery of ZFN proteins,which are intrinsically cell-permeable; TALEN proteins,which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein,whose nucleofection into cells facilitates rapid induction of multiplexed modifications,are described,along with procedures for evaluating nuclease protein activity. Once they are constructed,nuclease proteins can be expressed and purified within 6 d,and they can be used to induce genomic modifications in human cells within 2 d. View Publication

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here,we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34(+)CD31(+) endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable,and endothelial progenitor differentiation was β-catenin dependent. Furthermore,by clonal analysis,we showed that CD34(+)CD31(+)CD117(+)TIE-2(+) endothelial progenitors were multipotent,capable of differentiating into calponin-expressing smooth muscle cells and CD31(+)CD144(+)vWF(+)I-CAM1(+) endothelial cells. These endothelial cells were capable of 20 population doublings,formed tube-like structures,imported acetylated low-density lipoprotein,and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/β-catenin signaling as a primary regulator for generating vascular cells from hPSCs. View Publication

Structural bone allografts are widely used in the clinic to treat critical sized bone defects,despite lacking the osteoinductive characteristics of live autografts. To address this,we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets,which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates,as a tissue-engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets,suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo,allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of X-ray plain radiography,3D microCT,histology,and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation,as well as graft-host osteointegration. Moreover,a large periosteal callus was observed spanning the allografts with MSC sheets,which partially mimics live autograft healing. Finally,biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together,MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair,demonstrating the feasibility of this tissue engineering solution for massive allograft healing. View Publication

Survivin,which is the smallest member of the inhibitor of apoptosis protein (IAP) family,is a chromosomal passenger protein that mediates the spindle assembly checkpoint and cytokinesis,and also functions as an inhibitor of apoptosis. Frequently overexpressed in human cancers and not expressed in most adult tissues,survivin has been proposed as an attractive target for anticancer therapies and,in some cases,has even been touted as a cancer-specific gene. Survivin is,however,expressed in proliferating adult cells,including human hematopoietic stem cells,T-lymphocytes,and erythroid cells throughout their maturation. Therefore,it is unclear how survivin-targeted anticancer therapies would impact steady-state blood development. To address this question,we used a conditional gene-targeting strategy and abolished survivin expression from the hematopoietic compartment of mice. We show that inducible deletion of survivin leads to ablation of the bone marrow,with widespread loss of hematopoietic progenitors and rapid mortality. Surprisingly,heterozygous deletion of survivin causes defects in erythropoiesis in a subset of the animals,with a dramatic reduction in enucleated erythrocytes and the presence of immature megaloblastic erythroblasts. Our studies demonstrate that survivin is essential for steady-state hematopoiesis and survival of the adult,and further,that a high level of survivin expression is critical for proper erythroid differentiation. View Publication