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Killer immunoglobulin-like receptors (KIR) bind self-major histocompatibility complex class I molecules,allowing natural killer (NK) cells to recognize aberrant cells that have down-regulated class I. NK cells express variable numbers and combinations of highly homologous clonally restricted KIR genes,but uniformly express KIR2DL4. We show that NK clones express both 2DL4 alleles and either one or both alleles of the clonally restricted KIR 3DL1 and 3DL2 genes. Despite allele-independent expression,3DL1 alleles differed in the core promoter by only one or two nucleotides. Allele-specific 3DL1 gene expression correlated with promoter and 5' gene DNA hypomethylation in NK cells in vitro and in vivo. The DNA methylase inhibitor,5-aza-2'-deoxycytidine,induced KIR DNA hypomethylation and heterogeneous expression of multiple KIR genes. Thus,NK cells use DNA methylation to maintain clonally restricted expression of highly homologous KIR genes and alleles. View Publication

It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular,the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory,the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel,provides a robust model of human development and in the future,may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally,we demonstrate that following continued cell culture,stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore,also offer an in vitro model of disease. View Publication

BACKGROUND: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However,the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.backslashnbackslashnRESULTS: In this study,we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN,respectively. However,TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5,a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.backslashnbackslashnCONCLUSIONS: TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology. View Publication

Our previous studies have demonstrated that a previously unrecognized role of CFTR in hematopoiesis and acute leukemia. Here,we show that CFTR inhibitor CFTR-inh172 possesses ability to inhibit human T-cell acute lymphoblastic leukemia cells. In detail,CFTR-inh172 inhibited cell proliferation,promoted apoptosis and arrested the cell cycle in human T-cell acute lymphoblastic leukemia cell CCRF-CEM,JURKAT and MOLT-4. Furthermore,transcriptome analysis reveals that CFTR-inh172 induces significant alteration of gene expression related to apoptosis and proliferation. These findings demonstrate the potential of CFTR inhibitor CFTR-inh172 in human T-cell acute lymphoblastic leukemia treatment. View Publication

The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease. View Publication

Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs,this class of cell is remarkably diverse,comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models,but less attention has been paid to human RGCs. Thus,efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics,confirming the combinatorial expression of molecular markers associated with these subtypes,and also provided insight into more subtype-specific markers. Thus,the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs. View Publication

A limited number of accessible and representative models of human trophoblast cells currently exist for the study of placentation. Current stem cell models involve either a transition through a naïve stem cell state or precise dynamic control of multiple growth factors and small-molecule cues. Here,we demonstrated that a simple five-day treatment of human induced pluripotent stem cells with two small molecules,retinoic acid (RA) and Wnt agonist CHIR 99021 (CHIR),resulted in rapid,synergistic upregulation of CDX2. Transcriptomic analysis of RA + CHIR-treated cells showed high similarity to primary trophectoderm cells. Multipotency was verified via further differentiation towards cells with syncytiotrophoblast or extravillous trophoblast features. RA + CHIR-treated cells were also assessed for the established criteria defining a trophoblast cell model,and they possess all the features necessary to be considered valid. Collectively,our data demonstrate a facile,scalable method for generating functional trophoblast-like cells in vitro to better understand the placenta. View Publication

Hematopoiesis during development is a dynamic process,with many factors involved in the emergence and regulation of hematopoietic stem cells (HSCs) and progenitor cells. Whereas previous studies have focused on developmental signaling and transcription factors in embryonic hematopoiesis,the role of well-known adult hematopoietic cytokines in the embryonic hematopoietic system has been largely unexplored. The cytokine interleukin-1 (IL-1),best known for its proinflammatory properties,has radioprotective effects on adult bone marrow HSCs,induces HSC mobilization,and increases HSC proliferation and/or differentiation. Here we examine IL-1 and its possible role in regulating hematopoiesis in the midgestation mouse embryo. We show that IL-1,IL-1 receptors (IL-1Rs),and signaling mediators are expressed in the aorta-gonad-mesonephros (AGM) region during the time when HSCs emerge in this site. IL-1 signaling is functional in the AGM,and the IL-1RI is expressed ventrally in the aortic subregion by some hematopoietic,endothelial,and mesenchymal cells. In vivo analyses of IL-1RI-deficient embryos show an increased myeloid differentiation,concomitant with a slight decrease in AGM HSC activity. Our results suggest that IL-1 is an important homeostatic regulator at the earliest time of HSC development,acting to limit the differentiation of some HSCs along the myeloid lineage. View Publication

BACKGROUND: The testing of cord blood (CB) progenitor and stem cell units for transplantation suitability involves enumeration of total nucleated cells before freezing. CD34+ cell counts may also be a means of determining suitability. Studies have been conducted to evaluate how specific storage conditions influence cell counts. STUDY DESIGN AND METHODS: CB units were processed by hydroxyethyl starch volume reduction. Cryopreserved-thawed samples were diluted 1:3 without washing. CD34+ cells were measured with three commercially available assay methods. In specific studies,apoptosis-indicating reagents were included. CB units were analyzed for nucleated cells,aldehyde dehydrogenase-containing cells,and progenitor colonies. RESULTS: CD34+ cell levels and nucleated cells were retained during storage in test tubes at 1 to 6 degrees C for 3 days. Cryopreserved-thawed samples showed a reduction in CD34+ cells relative to prefreeze levels with the largest decrease with the Stem-Kit (Beckman Coulter) restricted gating procedure. Prefreeze samples contained minimal numbers of presumed apoptotic cells detected with 7-aminoactinomycin D or SYTO16,but after cryopreservation-thawing there was an increase. Nucleated cell levels determined with a hematology analyzer or flow cytometry were reduced after thawing. Cryopreservation-thawing reduced the percentage of CD34+ cells positive for the presence of aldehyde dehydrogenase and the number of progenitor colonies. These differences were significant. CONCLUSION: These studies indicate that CD34+ cell counts were maintained when CB samples were stored at 1 to 6 degrees C in test tubes for 3 days. Cryopreservation-thawing resulted in changes in a number of parameters including the percentage of CD34+ cells that were aldehyde dehydrogenase(+) and the number of 7-aminoactinomycin D(+) cells and SYTO16(low) cells. View Publication

Hematopoietic stem cells (HSCs) react to various stress conditions. However,it is unclear whether and how HSCs respond to severe anemia. Here,we demonstrate that upon induction of acute anemia,HSCs rapidly proliferate and enhance their erythroid differentiation potential. In severe anemia,lipoprotein profiles largely change and the concentration of ApoE increases. In HSCs,transcription levels of lipid metabolism-related genes,such as very low-density lipoprotein receptor ( Vldlr ),are upregulated. Stimulation of HSCs with ApoE enhances their erythroid potential,whereas HSCs in Apoe knockout mice do not respond to anemia induction. Vldlr high HSCs show higher erythroid potential,which is enhanced after acute anemia induction. Vldlr high HSCs are epigenetically distinct because of their low chromatin accessibility,and more chromatin regions are closed upon acute anemia induction. Chromatin regions closed upon acute anemia induction are mainly binding sites of Erg. Inhibition of Erg enhanced the erythroid differentiation potential of HSCs. Our findings indicate that lipoprotein metabolism plays an important role in HSC regulation under severe anemic conditions. Subject terms: Haematopoietic stem cells,Fat metabolism,Chromatin,Anaemia View Publication

Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However,mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study,we investigated the effects of two nuclear receptors,liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter,while GCNF strongly suppressed CR-1 expression in these cells. In addition,the CR-1 promoter was unmethylated in NTERA-2 cells,while T47D,ZR75-1,and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells,promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors,suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively,these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer. View Publication