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      Endothelial and perivascular cells maintain haematopoietic stem cells

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      Nature

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          Abstract

          Multiple cell types have been proposed to create niches for haematopoietic stem cells (HSCs). However, the expression patterns of HSC maintenance factors have not been systematically studied and no such factor has been conditionally deleted from any candidate niche cell. Thus, the cellular sources of these factors are undetermined. Stem Cell Factor (SCF) is a key niche component that maintains HSCs. Using Scf gfp knock-in mice we found Scf was primarily expressed by perivascular cells throughout bone marrow. HSC frequency and function were not affected when Scf was conditionally deleted from haematopoietic cells, osteoblasts, Nestin-Cre, or Nestin-CreER-expressing cells. However, HSCs were depleted from bone marrow when Scf was deleted from endothelial cells or Leptin receptor ( Lepr)-expressing perivascular stromal cells. Most HSCs were lost when Scf was deleted from both endothelial and Lepr-expressing perivascular cells. HSCs reside in a perivascular niche in which multiple cell types express factors that promote HSC maintenance.

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          Most cited references 43

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          Exploration, normalization, and summaries of high density oligonucleotide array probe level data.

          In this paper we report exploratory analyses of high-density oligonucleotide array data from the Affymetrix GeneChip system with the objective of improving upon currently used measures of gene expression. Our analyses make use of three data sets: a small experimental study consisting of five MGU74A mouse GeneChip arrays, part of the data from an extensive spike-in study conducted by Gene Logic and Wyeth's Genetics Institute involving 95 HG-U95A human GeneChip arrays; and part of a dilution study conducted by Gene Logic involving 75 HG-U95A GeneChip arrays. We display some familiar features of the perfect match and mismatch probe (PM and MM) values of these data, and examine the variance-mean relationship with probe-level data from probes believed to be defective, and so delivering noise only. We explain why we need to normalize the arrays to one another using probe level intensities. We then examine the behavior of the PM and MM using spike-in data and assess three commonly used summary measures: Affymetrix's (i) average difference (AvDiff) and (ii) MAS 5.0 signal, and (iii) the Li and Wong multiplicative model-based expression index (MBEI). The exploratory data analyses of the probe level data motivate a new summary measure that is a robust multi-array average (RMA) of background-adjusted, normalized, and log-transformed PM values. We evaluate the four expression summary measures using the dilution study data, assessing their behavior in terms of bias, variance and (for MBEI and RMA) model fit. Finally, we evaluate the algorithms in terms of their ability to detect known levels of differential expression using the spike-in data. We conclude that there is no obvious downside to using RMA and attaching a standard error (SE) to this quantity using a linear model which removes probe-specific affinities.
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            Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.

            The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin(+) MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent 'mesenspheres' that can self-renew and expand in serial transplantations. Nestin(+) MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or beta3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin(+) cells and favours their osteoblastic differentiation, in vivo nestin(+) cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin(+) MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin(+) cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.
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              Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus

              Background Several Cre reporter strains of mice have been described, in which a lacZ gene is turned on in cells expressing Cre recombinase, as well as their daughter cells, following Cre-mediated excision of a loxP-flanked transcriptional "stop" sequence. These mice are useful for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes. The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize β-gal in living tissue. Results In view of the general utility of targeting the ubiquitously expressed ROSA26 locus, we constructed a generic ROSA26 targeting vector. We then generated two reporter lines of mice by inserting EYFP or ECFP cDNAs into the ROSA26 locus, preceded by a loxP-flanked stop sequence. These strains were tested by crossing them with transgenic strains expressing Cre in a ubiquitous (β-actin-Cre) or a cell-specific (Isl1-Cre and En1-Cre) pattern. The resulting EYFP or ECFP expression patterns indicated that the reporter strains function as faithful monitors of Cre activity. Conclusions In contrast to existing lacZ reporter lines, where lacZ expression cannot easily be detected in living tissue, the EYFP and ECFP reporter strains are useful for monitoring the expression of Cre and tracing the lineage of these cells and their descendants in cultured embryos or organs. The non-overlapping emission spectra of EYFP and ECFP make them ideal for double labeling studies in living tissues.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                0028-0836
                1476-4687
                21 December 2011
                25 January 2012
                26 July 2012
                : 481
                : 7382
                : 457-462
                Affiliations
                [1 ]Howard Hughes Medical Institute, Children’s Research Institute, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA
                [2 ]Transgenic Animal Model Core, University of Michigan, Ann Arbor, MI, 48109-2216, USA
                [3 ]Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY, 11724
                Author notes
                [4 ]Author for correspondence: Children’s Research Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas, 75390-8502; phone 214-633-1791; fax 214-648-5517; Sean.Morrison@ 123456UTSouthwestern.edu
                nihpa343810
                10.1038/nature10783
                3270376
                22281595
                Funding
                Funded by: Howard Hughes Medical Institute :
                Award ID: || HHMI_
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