Analysis of the Hematopoietic Stem Cell Niche

Cristina Lo Celso1, Rachael J. Klein1, David T. Scadden1

1 Harvard University, Cambridge, null
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.5
DOI:  10.1002/9780470151808.sc02a05s3
Online Posting Date:  November, 2007
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Abstract

Hematopoietic stem cells (HSCs) continuously replenish all blood cell lineages not only to maintain the normal rapid turnover of differentiated cells but also to respond to injury and stress. Cell‐extrinsic mechanisms are critical determinants of the fine balance between HSC self‐renewal and differentiation. The bone marrow microenvironment has emerged as a new area of intense study to identify which of its many components constitute the HSC niche and regulate HSC fate. While HSCs have been isolated, characterized and used in clinical practice for many years thanks to the development of very specific assays and technology (i.e., bone marrow transplants and fluorescence activated cell sorting), study of the HSC niche has evolved by combining experimental designs developed in different fields. In this unit we describe a collection of protocols spanning a wide range of techniques that can help every researcher tackling questions regarding the nature of the HSC niche. Curr. Protoc. Stem Cell Biol. 3:2A.5.1‐2A.5.31. © 2007 by John Wiley & Sons, Inc.

Keywords: HSC niche; histology; intravital microscopy; homing; co‐culture

     
 
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Table of Contents

  • Introduction
  • Histological Analysis
  • Basic Protocol 1: Mouse Bone Processing for Histology: Paraffin‐Embedded Blocks and Sections
  • Alternate Protocol 1: Mouse Bone Processing for Histology: Fixed Decalcified Frozen Blocks and Sections
  • Alternate Protocol 2: Mouse Bone Marrow Processing for Histology: Fresh Frozen Blocks and Sections
  • Staining Protocols for Tissue Sections
  • Basic Protocol 2: Tissue Section Staining: Immunofluorescence on Frozen Sections
  • Alternate Protocol 3: Tissue Section Staining: Signal Amplification using Biotin‐Conjugated Secondary Antibody
  • Alternate Protocol 4: Tissue Section Staining: Amplification using Tyramide
  • Alternate Protocol 5: Tissue Section Staining: Immunohistochemistry
  • Alternate Protocol 6: Tissue Section Staining: Immunofluorescence and Immunohistochemistry on Paraffin Sections
  • In Vivo Imaging
  • Basic Protocol 3: Bone Marrow Live Imaging
  • Support Protocol 1: Mouse Anesthesia: Ketamine/Xylazine
  • Support Protocol 2: Mouse Anesthesia: Avertin
  • Support Protocol 3: Mouse Anesthesia: Isoflurane
  • Support Protocol 4: Live Cell Staining
  • Support Protocol 5: Suturing the Scalp
  • Support Protocol 6: Gluing the Scalp
  • Homing and Lodging Assays
  • Basic Protocol 4: Homing Assay
  • Basic Protocol 5: Lodgment Assay
  • Support Protocol 7: Staining of Cells with CFDA‐SE
  • Basic Protocol 6: HSC‐Stroma Co‐Culture: CAFC/LTC‐IC Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Mouse Bone Processing for Histology: Paraffin‐Embedded Blocks and Sections

  Materials
  • Mouse
  • 3% (w/v) paraformaldehyde (PFA, see recipe)
  • Phosphate‐buffered saline (PBS, e.g., Cellgro)
  • PBS/10% (w/v) EDTA, pH 7.5 (EDTA can be purchased from Sigma)
  • 70% ethanol
  • Dissecting tools:
    • Scissors
    • Tweezers
    • Scalpel
  • 24‐well plate
  • Screw‐lid 15‐ml conical tubes (Falcon) or 10‐ml flat bottom tubes

Alternate Protocol 1: Mouse Bone Processing for Histology: Fixed Decalcified Frozen Blocks and Sections

  Materials
  • PBS/20% (w/v) sucrose
  • OCT compound (Tissue‐Tek)
  • Dry ice
  • Cryomolds, 25 × 20 × 5–mm or 10 × 10 × 5–mm (Tissue‐Tek)
  • Cryostat (e.g., Leica, CM 3050S)
  • Disposable high‐profile blades (CL Sturkey, D554DD)
  • Microscope slides (e.g., colorfrost plus from Fisher scientific)
  • Paintbrush (optional)
  • Cryogene tape system (optional, Instrumedics)
  • Additional reagents and equipment to obtain mouse bone for processing ( protocol 1)

Alternate Protocol 2: Mouse Bone Marrow Processing for Histology: Fresh Frozen Blocks and Sections

  Materials
  • Mouse
  • PBS/20% (w/v) sucrose (optional)
  • OCT (Tissue‐Tek)
  • Dry ice
  • Dissecting instruments
  • Cryostat
  • Cryostat disposable blades (e.g., D554XD Extremus from CL Sturkey)
  • Cryogene tape system (Instrumedics) including tape and UV flashlight
  • Cryomolds 25 × 20 × 5–mm or 10 × 10 × 5–mm (Tissue‐Tek)
  • Cryogene‐coated microscope slides (Instrumedics)

Basic Protocol 2: Tissue Section Staining: Immunofluorescence on Frozen Sections

  Materials
  • Cryostat‐cut tissue sections
  • Phosphate‐buffered saline (PBS, e.g., Cellgro)
  • Blocking solution: PBS/10% (v/v) FBS (Invitrogen)
  • Primary antibody of interest, purified or biotin conjugated
  • Washing solution: PBS/0.1% or 0.2% (v/v) Tween (Sigma)
  • Species‐specific fluorophore‐conjugated secondary antibody or fluorophore‐conjugated streptavidin (Invitrogen, Alexa conjugates recommended)
  • Milli‐Q purified water
  • Vectashield: either hard set (which hardens gluing the coverslip in position) or original compound (which remains liquid) with DAPI (Vector Labs) or other mounting medium
  • Pap‐pen (Fisher)
  • Dark incubation chamber, humidified
  • Coplin jars or beakers
  • Thin forceps
  • Coverslips (Fisher)
NOTE: Everything is done on the bench unless otherwise stated. Incubations are in the dark in a humidified chamber, to reduce fluorophore damage and evaporation.

Alternate Protocol 3: Tissue Section Staining: Signal Amplification using Biotin‐Conjugated Secondary Antibody

  • Prepared tissue sections ( protocol 4)
  • Purified unconjugated primary antibody
  • Biotin‐conjugated species‐specific secondary antibody (Dako)
  • Fluorophore‐conjugated streptavidin (Invitrogen, Alexa‐conjugated recommended)

Alternate Protocol 4: Tissue Section Staining: Amplification using Tyramide

  • An HRP‐conjugated reagent, either the secondary antibody or the streptavidin, both available from DAKO
  • 3% (v/v) hydrogen peroxide
  • Additional reagents and equipment for preparing and rehydrating sections using PBS ( protocol 4)

Alternate Protocol 5: Tissue Section Staining: Immunohistochemistry

  Materials
  • Frozen sections
  • Phosphate‐buffered saline (PBS, e.g., Cellgro)
  • 3% (v/v) H 2O 2
  • Blocking solution: PBS/10% (v/v) FBS
  • Primary antibody of interest, purified or biotin conjugated
  • Washing solution: PBS/0.1% or 0.2% (v/v) Tween
  • Secondary antibody, HRP‐ or biotin‐conjugated (Dako)
  • Streptavidin, HRP‐conjugated (DAKO), optional
  • DAB (see recipe)
  • Milli‐Q purified water
  • Hematoxylin
  • Pap‐pen
  • Beakers or containers holding at least 200 ml distilled water for final washes

Alternate Protocol 6: Tissue Section Staining: Immunofluorescence and Immunohistochemistry on Paraffin Sections

  • Xylene (Fisher)
  • 80% and 100% ethanol (Fisher)
  • Distilled water
  • Na citrate or other antigen retrieval buffer (see recipe)
  • 500‐ml to 1‐liter beaker or microwave‐proof container with lid
  • Microwave oven
  • Additional reagents and equipment for tissue section staining ( protocol 7)

Basic Protocol 3: Bone Marrow Live Imaging

  Materials
  • Mice injected with labeled cells ( protocol 13)
  • Anesthetic of choice (Support Protocols protocol 101 through protocol 123)
  • Fluorescent vascular dye (e.g., fluorescently labeled dextran or Angiosense, see recipe)
  • Stained cells of choice, optional
  • Phosphate‐buffered saline (PBS, e.g., Cellgro)
  • Methocell (optional, OmniVision)
  • Buprenorphine (Buprenex)
  • Hair clipper or sharp scissors, optional
  • Surgery thin tweezers and scissors
  • Suturing thread [e.g., Ethilon 6‐0 (0.7 metric) PC‐3, Ethicon, no. 1966G] or glue (Vetbond, 3 M)
  • Suture needle
  • Warmed stage or warmed cylindrical holder
  • Appropriate intravital microscope (e.g., Olympus IV100)

Support Protocol 1: Mouse Anesthesia: Ketamine/Xylazine

  Materials
  • Mouse
  • Ketamine/Xylazine cocktail (see recipe)
  • Syringe
  • Balance
  • Heated pad

Support Protocol 2: Mouse Anesthesia: Avertin

  Materials
  • Mouse
  • Avertin solution (see recipe)
  • Syringe
  • Balance
  • Heated pad

Support Protocol 3: Mouse Anesthesia: Isoflurane

  Materials
  • Mouse
  • Isoflurane (e.g., Nicholas Piramal or Henry Shein)
  • Oxygen (e.g., Nicholas Piramal or Henry Shein)
  • Isoflurane anesthesia machinery (e.g., Henry Shein)

Support Protocol 4: Live Cell Staining

  Materials
  • Cells of choice
  • Phosphate‐buffered saline (PBS)
  • DiD or DiI (Invitrogen)
  • 37°C incubator
  • 1.0‐ml insulin syringe equipped with a 31‐G needle

Support Protocol 5: Suturing the Scalp

  Materials
  • Mouse from imaging experiment
  • Suturing thread [Ethilon 6‐0 (0.7 metric) PC‐3, Ethicon, no.1966G]
  • Suture needle
  • Tweezers

Support Protocol 6: Gluing the Scalp

  Materials
  • Mice
  • Stained cells of choice
  • PBS/2% (v/v) FBS
  • Lineage cocktail antibodies (equal amounts of Mac1, Gr1, Ter119, CD4, CD8, CD3, and B220 biotinylated antibodies, BD Pharmingen), optional
  • Fluorophore‐conjugated streptavidin (BD Pharmingen)
  • Gamma irradiator
  • Needles and syringes
  • Dissecting instruments
  • 40‐µm filters (BD)
  • FACS tubes (BD)
  • FACS machine (Calibur, BD)
  • Additional reagents and equipment for harvesting cells from the donor mice (Lo Celso and Scadden, ), labeling the cells appropriately (Support Protocols protocol 134 and protocol 187) and counting cells using a hemacytometer (Phelan, )

Basic Protocol 4: Homing Assay

  Materials
  • Cells of choice
  • Phosphate‐buffered saline (PBS; e.g., Cellgro)
  • Vectashield
  • Coverslips
  • Thin forceps (to handle coverslips)
  • Additional reagents and solutions for labeling cells (Support Protocols protocol 134 and protocol 187) and dissecting the femur and processing for histological analysis ( protocol 1 or protocol 2)

Basic Protocol 5: Lodgment Assay

  Materials
  • Cells of choice
  • Phosphate‐buffered saline (PBS), 37°C
  • 100 µM CFDA SE stock (see recipe)
  • Medium (e.g., DMEM, Invitrogen) supplemented with 10% (v/v) FBS, 37°C
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS)
  • Additional reagents and equipment for preparing cells of choice ( protocol 17)

Support Protocol 7: Staining of Cells with CFDA‐SE

  Materials
  • Mice
  • Collagenase (Worthington)
  • PBS/2% and 20% FBS
  • Long‐term culture medium (H5100 for human or M5300 for murine cells, see recipe)
  • Trypsin with EDTA (Cellgro # 25‐053‐CI)
  • dH 2O
  • Hematopoietic cells of choice
  • Methylcellulose‐containing medium (see recipe)
  • Dissecting tools
  • Mortar and pestle (Fisher)
  • 70‐µm filters (BD)
  • Scissors
  • Heated water bath with shaker
  • 25‐ or 75‐cm2 flasks (Nunc)
  • Irradiator (see protocol 16 for more details)
  • 96‐well plate
  • 33°C incubator 5% CO 2
  • L‐Calc software (free from Stem Cells Technologies)
  • Additional reagents and equipment for dissecting bones from mice (Lo Celso and Scadden, )
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Figures

  •   FigureFigure 2.A0.1 Schematic representation of the cryogene tape system. Fresh frozen (nonfixed and nondecalcified), OCT‐embedded bones are best sectioned with the cryogene tape system integrated in a cryostat. The embedded bone is positioned on the block holder so that the bone is perpendicular to the cryostat blade and the block is trimmed until the bone marrow cavity is exposed. The cryogene tape is positioned on the block so that the black line marks the bottom end of the block and touches the cryostat blade first.
  •   FigureFigure 2.A0.2 Alternative strategies for amplification of immunofluorescence (IF) staining signal. Different levels of amplification can be used in multilayered IF in order to visualize a certain antigen (Ag) on tissue sections. The primary antibody (I Ab) recognizes the antigen and constitutes the first layer. Species‐specific secondary antibody (II Ab) is the second layer. (A) II Ab used is directly conjugated to a fluorophore (II Ab Fluo, green symbol). (B) II Ab used is biotin‐conjugated (gray symbol) and subsequently bound by fluorophore‐conjugated streptavidin (SA, light gray symbol, Fluo, green symbol). (C) II Ab used is HRP conjugated (red symbol) and subsequently bound by fluorophore‐conjugated tyramide (Tyr, blue symbol). (D) Biotin‐conjugated II Ab is bound by streptavidin‐conjugated HRP and subsequently by fluorophore‐conjugated tyramide. (E) Biotin‐conjugated tyramide is used, followed by fluorophore‐conjugated streptavidin. Spaces between symbols indicate reagents added in subsequent incubations, contact between symbols indicates commercially available covalently bound reagents.
  •   FigureFigure 2.A0.3 Lodging assay. Picture of a section obtained from the hip bone of a non‐irradiated mouse injected with DiD stained lineage‐negative cells 6 hr prior to sacrifice. Green is cytoplasmic bone and bone marrow cells' autofluorescence signal, blue is DAPI nuclear counterstain, purple is DiD membrane dye. Arrow indicates a lineage‐negative cell. Scale bar is 100 µm.
  •   FigureFigure 2.A0.4 Layout of a CAFC/LTC‐IC experiment. Water is distributed in all peripheral wells of a 96‐well plate (blue circles) and serial dilutions of mouse whole bone marrow mononuclear cells (WBM MNC) or human CD34+ cells are seeded on top of stroma in the central wells. Black‐to‐white gradient indicates wells seeded with decreasing number of cells.

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Literature Cited

Literature Cited
   Adams, G.B. and Scadden, D.T. 2006. The hematopoietic stem cell in its place. Nat. Immunol. 7:333‐337.
   Adams, G.B., Chabner, K.T., Alley, I.R., Olson, D.P., Szczepiorkowski, Z.M., Poznansky, M.C., Kos, C.H., Pollak, M.R., Brown, E.M., and Scadden, D.T. 2006. Stem cell engraftment at the endosteal niche is specified by the calcium‐sensing receptor. Nature 439:599‐603.
   Arai, F., Hirao, A., Ohmura, M., Sato, H., Matsuoka, S., Takubo, K., Ito, K., Koh, G.Y., and Suda, T. 2004. Tie2/angiopoietin‐1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118:149‐61.
   Arras, M., Autenried, P., Rettich, A., Spaeni, D., and Rulicke, T. 2001. Optimization of intraperitoneal injection anesthesia in mice: Drugs, dosages, adverse effects, and anesthesia depth. Comp. Med. 51:443‐456.
   Askenasy, N. and Farkas, D.L. 2002. Optical imaging of PKH‐labeled hematopoietic cells in recipient bone marrow in vivo. Stem Cells 20:501‐13.
   Askenasy, N., Zorina, T., Farkas, D.L., and Shalit, I. 2002. Transplanted hematopoietic cells seed in clusters in recipient bone marrow in vivo. Stem Cells 20:301‐310.
   Bouzianas, D.G. 2003. Cobblestone area measuring (CAM) assay: A new way of assessing the potential of human haemopoietic stem cells. Methods Cell Sci. 25:201‐210.
   Breems, D.A., Blokland, E.A., Neben, S., and Ploemacher, R.E. 1994. Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay. Leukemia 8:1095‐1104.
   Bryder, D., Rossi, D., and Weissman, I. 2006. Hematopoietic Stem Cells. The paradigmatic Tissue‐Specific Stem Cell. Am. J. Pathol. 169:338‐346.
   Calvi, L.M., Adams, G.B., Weibrecht, K.W., Weber, J.M., Olson, D.P., Knight, M.C., Martin, R.P., Schipani, E., Divieti, P., Bringhurst, F.R., Milner, L.A., Kronenberg, H.M., and Scadden, D.T. 2003. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425:841‐846.
   Carson, F.L. 1997. Histotechnology: A self‐instructional text. American Society for Clinical Pathology Press, Chicago.
   Cavanagh, L.L., Bonasio, R., Mazo, I.B., Halin, C., Cheng, G., van der Velden, A.W., Cariappa, A., Chase, C., Russell, P., Starnbach, M.N., Koni, P.A., Pillai, S., Weninger, W., and von Andrian, U.H., 2005. Activation of bone marrow‐resident memory T cells by circulating, antigen‐bearing dendritic cells. Nat. Immunol. 6:1029‐1037.
   Chabner, K.T., Adams, G.B., Qiu, J., Moskowitz, M., Marsters, E.S., Topulos, G.P., and Scadden, D.T. 2004. Direct vascular delivery of primitive hematopoietic cells to bone marrow improves localization but not engraftment. Blood 103:4685‐4686.
   Charbord, P. and Moore, K. 2005. Gene expression in stem cell‐supporting stromal cell lines. Ann. N.Y. Acad. Sci. 1044:159‐167.
   Filipe, M. and Lake, B. 1990. Histochemistry in Pathology. Churchill Livingstone, London.
   Iga, A.M., Sarkar, S., Sales, K.M., Winslet, M.C., and Seifalian, A.M. 2006. Quantitating therapeutic disruption of tumor blood flow with intravital video microscopy. Cancer Res. 66:11517‐11519.
   Johnstone, A.P. and Turner, M.W. 1997. Immunochemistry 2: A practical approach. Oxford University Press, Oxford.
   Jung, Y., Wang, J., Song, J., Shiozawa, Y., Wang, J., Havens, A., Wang, Z., Sun, Y.X., Emerson, S.G., Krebsbach, P.H., and Taichman, R.S. 2007. Annexin II expressed by osteoblasts and endothelial cells regulates stem cell adhesion, homing and engraftment following transplantation. Blood 110:82‐90.
   Katayama, Y., Battista, M., Kao, W.M., Hidalgo, A., Peired, A.J., Thomas, S.A., and Frenette, P.S. 2006. Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell 124:407‐421.
   Kiel, M.J., Yilmaz, O.H., Iwashita, T., Yilmaz, O.H., Terhorst, C., and Morrison, S.J. 2005. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121:1109‐1121.
   Kitajima, K., Tanaka, M., Zheng, J., Yen, H., Sato, A., Sugiyama, D., Umehara, H., Sakai, E., and Nakano, T. 2006. Redirecting differentiation of hematopoietic progenitors by a transcription factor, GATA‐2. Blood 107:1857‐1863.
   Kollet, O., Dar, A., Shivtiel, S., Kalinkovich, A., Lapid, K., Sztainberg, Y., Tesio, M., Samstein, R.M., Goichberg, P., Spiegel, A., Elson, A., and Lapidot, T. 2006. Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nat. Med. 12:657‐664.
   Kuebler, W.M., Parthasarathi, K., Lindert, J., and Bhattacharya, J. 2007. Real‐time lung microscopy. J. Appl. Physiol. 102:1255‐1264.
   Lane, D. and Harlow, E. 1999. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Woodbury, NY.
   Lo Celso, C. and Scadden, D.T. 2007. Isolation of Hematopoietic Stem Cells from Bone Marrow. In Journal of Visualized Experiments Issue 2.
   Lo Celso, C., Prowse, D.M., and Watt, F.M. 2004. Transient activation of beta‐catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours. Development 131:1787‐1799.
   Mazo, I.B., Honczarenko, M., Leung, H., Cavanagh, L.L., Bonasio, R., Weninger, W., Engelke, K., Xia, L., McEver, R.P., Koni, P.A., Silberstein, L.E., and von Andrian, U.H. 2005. Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells. Immunity 22:259‐270.
   Montet, X., Figueiredo, J.L., Alencar, H., Ntziachristos, V., Mahmood, U., and Weissleder, R. 2007. Tomographic fluorescence imaging of tumor vascular volume in mice. Radiology 242:751‐758.
   Nilsson, S.K., Johnston, H.M., and Coverdale, J.A. 2001. Spatial localization of transplanted hematopoietic stem cells: Inferences for the localization of stem cell niches. Blood 97:2293‐2299.
   Nilsson, S.K., Johnston, H.M., Whitty, G.A., Williams, B., Webb, R.J., Denhardt, D.T., Bertoncello, I., Bendall, L.J., Simmons, P.J., and Haylock, D.N. 2005. Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells. Blood 106:1232‐1239.
   Pawley, J.B. 1995. Handbook of Biological Confocal Microscopy. Plenum Press, New York.
   Pearce, D.J. and Bonnet, D. 2007. The combined use of Hoechst efflux ability and aldehyde dehydrogenase activity to identify murine and human hematopoietic stem cells. Exp. Hematol. 35:1437‐1446.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Runnels, J.M., Zamiri, P., Spencer, J.A., Veilleux, I., Wei, X., Bogdanov, A., and Lin, C.P. 2006. Imaging molecular expression on vascular endothelial cells by in vivo immunofluorescence microscopy. Mol. Imaging 5:31‐40.
   Scadden, D.T. 2006. The stem‐cell niche as an entity of action. Nature 441:1075‐1079.
   Schofield, R. 1978. The relationship between the spleen colony‐forming cell and the haemopoietic stem cell. Blood Cells 4:7‐25.
   Shizuru, J.A., Negrin, R.S., and Weissman, I.L. 2005. Hematopoietic stem and progenitor cells: Clinical and preclinical regeneration of the hematolymphoid system. Annu. Rev. Med. 56:509‐538.
   Sipkins, D.A., Wei, X., Wu, J.W., Runnels, J.M., Cote, D., Means, T.K., Luster, A.D., Scadden, D.T., and Lin, C.P. 2005. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 435:969‐973.
   Soon, L., Braet, F., and Condeelis, J. 2007. Moving in the right direction‐nanoimaging in cancer cell motility and metastasis. Microsc. Res. Tech. 70:252‐257.
   Stier, S., Ko, Y., Forkert, R., Lutz, C., Neuhaus, T., Grunewald, E., Cheng, T., Dombkowski, D., Calvi, L.M., Rittling, S.R., and Scadden, D.T. 2005. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. J. Exp. Med. 201:1781‐1791.
   Sugiyama, T., Kohara, H., Noda, M., and Nagasawa, T. 2006. Maintenance of the hematopoietic stem cell pool by CXCL12‐CXCR4 chemokine signaling in bone marrow stromal cell niches. Immunity 25:977‐988.
   Sutherland, H.J., Lansdorp, P.M., Henkelman, D.H., Eaves, A.C., and Eaves, C.J. 1990. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc. Natl. Acad. Sci. U.S.A. 87:3584‐3588.
   Sutherland, H.J., Eaves, C.J., Lansdorp, P.M., Thacker, J.D., and Hogge, D.E. 1991. Differential regulation of primitive human hematopoietic cells in long‐term cultures maintained on genetically engineered murine stromal cells. Blood 78:666‐672.
   Taghon, T.N., David, E.S., Zuniga‐Pflucker, J.C., and Rothenberg, E.V. 2005. Delayed, asynchronous, and reversible T‐lineage specification induced by Notch/Delta signaling. Genes Dev. 19:965‐978.
   Taichman, R.S., Reilly, M.J., and Emerson, S.G. 2000. The Hematopoietic microenvironment: Osteoblasts and the hematopoietic microenvironment. Hematology 4:421‐426.
   van Os, R., Kamminga, L.M., and de Haan, G. 2004. Stem cell assays: Something old something new, something borrowed. Stem Cells 22:1181‐1190.
   Wolf, N.S. 1974. Dissecting the hematopoietic microenvironment. I. Stem cell lodgment and commitment, and the proliferation and differentiation of erythropoietic descendants in the S1‐S1d mouse. Cell Tissue Kinet. 7:89‐98.
   Yang, L., Wang, L., Geiger, H., Cancelas, J.A., Mo, J., and Zheng, Y. 2007. Rho GTPase Cdc42 coordinates hematopoietic stem cell quiescence and niche interaction in the bone marrow. Proc. Natl. Acad. Sci. U.S.A. 104:5091‐5096.
   Zhang, J., Niu, C., Ye, L., Huang, H., He, X., Tong, W.G., Ross, J., Haug, J., Johnson, T., Feng, J.Q., Harris, S., Wiedemann, L.M., Mishina, Y., and Li, L. 2003. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 425:836‐841.
   Zhu, J., Garrett, R., Jung, Y., Zhang, Y., Kim, N., Wang, J., Joe, G.J., Hexner, E., Choi, Y., Taichman, R.S., and Emerson, S.G. 2007. Osteoblasts support B lymphocyte commitment and differentiation from hematopoietic stem cells. Blood 109:3706‐3712.
   Zipfel, W.R., Williams, R.M., and Webb, W.W. 2003. Nonlinear magic: Multiphoton microscopy in the biosciences. Nat. Biotechnol. 21:1369‐1377.
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