159
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Hematopoietic stem cells (HSCs) reside in a perivascular niche but the location remains controversial 1 . HSCs are rare and few can be found in thin tissue sections 2, 3 or upon live imaging 4 , making it difficult to comprehensively localize dividing and non-dividing HSCs. We discovered that α-catulin GFP/+ was expressed by only 0.02% of bone marrow hematopoietic cells, including virtually all HSCs. One in 3.5 α-catulin-GFP +c-kit + cells gave long-term multilineage reconstitution of irradiated mice, indicating that α-catulin-GFP +c-kit + cells contain HSCs with a purity comparable to the best markers available. We were able to optically clear the bone marrow to perform deep confocal imaging, making it possible to image thousands of α-catulin-GFP +c-kit + cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin-GFP +c-kit + cells indicated that HSCs were more common in central marrow than near bone surfaces and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted Leptin Receptor + and Cxcl12 high niche cells. Approximately 85% of HSCs were within 10μm of a sinusoidal blood vessel. Most HSCs were distant from arterioles, transition zone vessels, and bone surfaces. This was true of Ki-67 + dividing HSCs and Ki-67 non-dividing HSCs. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Leptin Receptor +Cxcl12 high cells throughout the bone marrow.

          Related collections

          Most cited references 24

          • Record: found
          • Abstract: found
          • Article: not found

          Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment.

          The identity of cells that establish the hematopoietic microenvironment (HME) in human bone marrow (BM), and of clonogenic skeletal progenitors found in BM stroma, has long remained elusive. We show that MCAM/CD146-expressing, subendothelial cells in human BM stroma are capable of transferring, upon transplantation, the HME to heterotopic sites, coincident with the establishment of identical subendothelial cells within a miniature bone organ. Establishment of subendothelial stromal cells in developing heterotopic BM in vivo occurs via specific, dynamic interactions with developing sinusoids. Subendothelial stromal cells residing on the sinusoidal wall are major producers of Angiopoietin-1 (a pivotal molecule of the HSC "niche" involved in vascular remodeling). Our data reveal the functional relationships between establishment of the HME in vivo, establishment of skeletal progenitors in BM sinusoids, and angiogenesis.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            A highly efficient recombineering-based method for generating conditional knockout mutations.

            Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Single-cell phenotyping within transparent intact tissue through whole-body clearing.

              Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies. Copyright © 2014 Elsevier Inc. All rights reserved.
                Bookmark

                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                13 August 2015
                23 September 2015
                1 October 2015
                29 April 2016
                : 526
                : 7571
                : 126-130
                Affiliations
                [1 ]Howard Hughes Medical Institute
                [2 ]Children’s Research Institute and the Department of Pediatrics
                [3 ]Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
                Author notes
                [*]

                These authors contributed equally

                Article
                NIHMS711524
                10.1038/nature15250
                4850557
                26416744

                Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                Categories
                Article

                Uncategorized

                Comments

                Comment on this article