Isolation and Assessment of Single Long‐Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow

David G. Kent1, Brad J. Dykstra2, Connie J. Eaves3

1 Department of Haematology, University of Cambridge, 2 Harvard Medical School, Boston, Massachusetts, 3 Department of Medical Genetics, University of British Columbia, Vancouver
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.4
DOI:  10.1002/cpsc.10
Online Posting Date:  August, 2016
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Hematopoietic stem cells with long‐term repopulating activity can now be routinely obtained at purities of 40% to 50% from suspensions of adult mouse bone marrow. Here we describe robust protocols for both their isolation as CD45+EPCR+CD150+CD48 (ESLAM) cells using multiparameter cell sorting and for tracking their clonal growth and differentiation activity in irradiated mice transplanted with single ESLAM cells. The simplicity of these procedures makes them attractive for characterizing the molecular and biological properties of individual hematopoietic stem cells with unprecedented power and precision. © 2016 by John Wiley & Sons, Inc.

Keywords: stem cell isolation; flow cytometry; hematopoiesis; single‐cell transplants; multi‐lineage reconstitution

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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of CD45+EPCR+CD150+CD48− (ESLAM) Cells from Adult Mouse Bone Marrow
  • Basic Protocol 2: Transplantation of Single Cells and Tracking Their Regenerated WBC Progeny
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Isolation of CD45+EPCR+CD150+CD48− (ESLAM) Cells from Adult Mouse Bone Marrow

  • C57B1/6J (B6) mice, 8 to 12 weeks old
  • HBSS/2% FBS: Hanks’ balanced salt solution containing 2% (w/v) fetal bovine serum (FBS)
  • 0.8% (w/v) ammonium chloride (NH 4Cl) solution (STEMCELL Technologies, cat no. 07850), ice cold
  • 0.1 mg/ml DNase I (Sigma; optional)
  • HBSS/2% FBS plus 1.2 µl mouse FcR blocking antibody (2.4G2; STEMCELL Technologies, cat. no. 01504)CD45‐FITC (Clone 30‐F11; Biolegend, cat. no. 103108)
  • EPCR‐PE (Clone RMEPCR1560; STEMCELL Technologies, cat. no. 60038PE)
  • CD150‐PECy7 (Clone TC15‐12F12.2; Biolegend, cat. no. 115914)
  • CD48‐APC (Clone HM48‐1; Biolegend, cat. no. 103412)
  • HBSS/2% FBS plus 1 µM 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma, cat no. D9542; note that 7‐AAD, PI, or other viability markers can be used as well)
  • Serum‐free medium (SFM): see recipe or purchase StemSpan serum‐free expansion medium (STEMCELL Technologies, cat. no. 09600)
  • Dissecting instruments including scissors and forceps, sterile
  • Mortar and pestle (optional)
  • 70‐µm or 100‐µm filters for 50‐ml tubes (optional)
  • 50‐ml conical polypropylene centrifuge tubes (e.g., Corning Falcon)
  • 21‐G, 1‐in. general‐use sterile hypodermic needles (BD, cat. no. 305165) and syringes
  • Tabletop centrifuge
  • Flow cytometer sample tubes (e.g., BD, cat. no. 352058 or 352063)
  • 5‐ml filter‐capped tubes (BD, cat. no. 358235)
  • Flow cytometer with sorting capability (e.g., Influx or Aria equipped with a 405, 488, and 640 nm lasers, BD)
  • 96‐well U‐bottom plates (e.g., Nunc, cat. no. 163320; optional, necessary if performing single‐cell isolation as in protocol 2)
  • Additional reagents and equipment for flow cytometry (Robinson et al., )

Basic Protocol 2: Transplantation of Single Cells and Tracking Their Regenerated WBC Progeny

  • Single HSCs sorted into 100 to 200 µl medium in a 96‐well plate ( protocol 1)
  • Irradiated recipient mice (strain W41/W41 or B6, >7 weeks old; see annotation to step 6 of this protocol regarding irradiation)
  • 0.8% (w/v) ammonium chloride (NH 4Cl) solution (STEMCELL Technologies, cat no. 07850) ice cold
  • HBSS/2% FBS: Hanks’ balanced salt solution containing 2% (w/v) fetal bovine serum (FBS)
  • 2× blocking reagent (see recipe)
  • Antibody cocktails for peripheral blood analysis (see recipe)
  • HBSS/2% FBS plus 1 µM 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma, cat no. D9542)
  • Tabletop centrifuge with microtiter plate carrier
  • Inverted microscope (preferably with movable stage)
  • Insulin syringes with 28‐G, 0.5‐in needles (BD, cat no. 329465)
  • 137Cs γ‐ray irradiation machine (or X‐ray irradiator)
  • Infra‐red heat lamp
  • Heparinized capillary tubes (e.g., Fisher, cat no. 22‐362‐56)
  • 12 × 75–mm tubes with caps (BD cat. no. 352057)
  • 96‐well U‐bottom microtiter plates (e.g., Nunc, cat. no. 163320)
  • ScreenMates 1.4‐ml round‐bottom storage tubes in snap rack (Thermo Scientific, cat. no. 4246)
  • Flow cytometer (e.g., BD Fortessa equipped with 405, 488, and 640 nm lasers)
  • Additional reagents and equipment for blood collection (Donovan and Brown, ) and flow cytometry (Robinson et al., )
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Literature Cited

Literature Cited
  Acar, M., Kocherlakota, K.S., Murphy, M.M., Peyer, J.G., Oguro, H., Inra, C.N., Jaiyeola, C., Zhao, Z., Luby‐Phelps, K., and Morrison, S.J. 2015. Deep imaging of bone marrow shows non‐dividing stem cells are mainly perisinusoidal. Nature 526:126‐130. doi: 10.1038/nature15250.
  Adolfsson, J., Borge, O.J., Bryder, D., Theilgaard‐Monch, K., Astrand‐Grundstrom, I., Sitnicka, E., Sasaki, Y., and Jacobsen, S.E. 2001. Upregulation of Flt3 expression within the bone marrow Lin(‐)Sca1(+)c‐kit(+) stem cell compartment is accompanied by loss of self‐renewal capacity. Immunity 15:659‐669. doi: 10.1016/S1074‐7613(01)00220‐5.
  Audet, J., Miller, C.L., Rose‐John, S., Piret, J.M., and Eaves, C.J. 2001. Distinct role of gp130 activation in promoting self‐renewal divisions by mitogenically stimulated murine hematopoietic stem cells. Proc. Natl. Acad. Sci. U.S.A. 98:1757‐1762. doi: 10.1073/pnas.98.4.1757.
  Balazs, A.B., Fabian, A.J., Esmon, C.T., and Mulligan, R.C. 2006. Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow. Blood 107:2317‐2321. doi: 10.1182/blood‐2005‐06‐2249.
  Beerman, I., Bhattacharya, D., Zandi, S., Sigvardsson, M., Weissman, I.L., Bryder, D., and Rossi, D.J. 2010. Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion. Proc. Natl. Acad. Sci. U.S.A. 107:5465‐5470. doi: 10.1073/pnas.1000834107.
  Benveniste, P., Cantin, C., Hyam, D., and Iscove, N.N. 2003. Hematopoietic stem cells engraft in mice with absolute efficiency. Nat. Immunol. 4:708‐713. doi: 10.1038/ni940.
  Benveniste, P., Frelin, C., Janmohamed, S., Barbara, M., Herrington, R., Hyam, D., and Iscove, N.N. 2010. Intermediate‐term hematopoietic stem cells with extended but time‐limited reconstitution potential. Cell Stem Cell 6:48‐58. doi: 10.1016/j.stem.2009.11.014.
  Benz, C., Copley, M.R., Kent, D.G., Wohrer, S., Cortes, A., Aghaeepour, N., Ma, E., Mader, H., Rowe, K., Day, C., Treloar, D., Brinkman, R.R., and Eaves, C.J. 2012. Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs. Cell Stem Cell 10:273‐283. doi: 10.1016/j.stem.2012.02.007.
  Bertoncello, I., Bradley, T.R., Hodgson, G.S., and Dunlop, J.M. 1991. The resolution, enrichment, and organization of normal bone marrow high proliferative potential colony‐forming cell subsets on the basis of rhodamine‐123 fluorescence. Exp. Hematol. 19:174‐178.
  Bradford, G.B., Williams, B., Rossi, R., and Bertoncello, I. 1997. Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment. Exp. Hematol. 25:445‐453.
  Bryder, D., Sasaki, Y., Borge, O.J., and Jacobsen, S.E. 2004. Deceptive multilineage reconstitution analysis of mice transplanted with hemopoietic stem cells, and implications for assessment of stem cell numbers and lineage potentials. Eur. J. Immunol. 172:1548‐1552. doi: 10.4049/jimmunol.172.3.1548.
  Bystrykh, L.V., Verovskaya, E., Zwart, E., Broekhuis, M., and de Haan, G. 2012. Counting stem cells: Methodological constraints. Nat. Methods 9:567‐574. doi: 10.1038/nmeth.2043.
  Challen, G.A., Boles, N.C., Chambers, S.M., and Goodell, M.A. 2010. Distinct hematopoietic stem cell subtypes are differentially regulated by TGF‐beta1. Cell Stem Cell 6:265‐278. doi: 10.1016/j.stem.2010.02.002.
  Chen, C.Z., Li, M., de Graaf, D., Monti, S., Gottgens, B., Sanchez, M.J., Lander, E.S., Golub, T.R., Green, A.R., and Lodish, H.F. 2002. Identification of endoglin as a functional marker that defines long‐term repopulating hematopoietic stem cells. Proc. Natl. Acad. Sci. U.S.A. 99:15468‐15473. doi: 10.1073/pnas.202614899.
  Cheshier, S.H., Morrison, S.J., Liao, X., and Weissman, I.L. 1999. In vivo proliferation and cell cycle kinetics of long‐term self‐renewing hematopoietic stem cells. Proc. Natl. Acad. Sci. U.S.A. 96:3120‐3125. doi: 10.1073/pnas.96.6.3120.
  de Haan, G., Weersing, E., Dontje, B., van Os, R., Bystrykh, L.V., Vellenga, E., and Miller, G. 2003. In vitro generation of long‐term repopulating hematopoietic stem cells by fibroblast growth factor‐1. Dev. Cell 4:241‐251. doi: 10.1016/S1534‐5807(03)00018‐2.
  Dick, J.E., Magli, M.C., Huszar, D., Phillips, R.A., and Bernstein, A. 1985. Introduction of a selectable gene into primitive stem cells capable of long‐term reconstitution of the hemopoietic system of W/Wv mice. Cell 42:71‐79. doi: 10.1016/S0092‐8674(85)80102‐1.
  Donovan, J. and Brown, P. 2006. Blood collection. Curr. Protoc. Immunol. 73:1.7.1‐1.7.9.
  Dykstra, B., Olthof, S., Schreuder, J., Ritsema, M., and de Haan, G. 2011. Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells. J. Exp. Med. 208:2691‐2703. doi: 10.1084/jem.20111490.
  Dykstra, B., Kent, D., Bowie, M., McCaffrey, L., Hamilton, M., Lyons, K., Lee, S.J., Brinkman, R., and Eaves, C. 2007. Long‐term propagation of distinct hematopoietic differentiation programs in vivo. Cell Stem Cell 1:218‐229. doi: 10.1016/j.stem.2007.05.015.
  Dykstra, B., Ramunas, J., Kent, D., McCaffrey, L., Szumsky, E., Kelly, L., Farn, K., Blaylock, A., Eaves, C., and Jervis, E. 2006. High‐resolution video monitoring of hematopoietic stem cells cultured in single‐cell arrays identifies new features of self‐renewal. Proc. Natl. Acad. Sci. U.S.A. 103:8185‐8190. doi: 10.1073/pnas.0602548103.
  Eaves, C.J. 2015. Hematopoietic stem cells: Concepts, definitions, and the new reality. Blood 125:2605‐2613. doi: 10.1182/blood‐2014‐12‐570200.
  Eaves, C., Miller, C., Cashman, J., Conneally, E., Petzer, A., Zandstra, P., and Eaves, A. 1997. Hematopoietic stem cells: Inferences from in vivo assays. Stem Cells 15 Suppl 1:1‐5.
  Ema, H., Morita, Y., and Suda, T. 2014. Heterogeneity and hierarchy of hematopoietic stem cells. Exp. Hematol. 42:74‐82 e72. doi: 10.1016/j.exphem.2013.11.004.
  Ema, H., Takano, H., Sudo, K., and Nakauchi, H. 2000. In vitro self‐renewal division of hematopoietic stem cells. J. Exp. Med. 192:1281‐1288. doi: 10.1084/jem.192.9.1281.
  Ema, H., Sudo, K., Seita, J., Matsubara, A., Morita, Y., Osawa, M., Takatsu, K., Takaki, S., and Nakauchi, H. 2005. Quantification of self‐renewal capacity in single hematopoietic stem cells from normal and Lnk‐deficient mice. Dev. Cell 8:907‐914. doi: 10.1016/j.devcel.2005.03.019.
  Fleming, T.J., Fleming, M.L., and Malek, T.R. 1993. Selective expression of Ly‐6G on myeloid lineage cells in mouse bone marrow. RB6‐8C5 mAb to granulocyte‐differentiation antigen (Gr‐1) detects members of the Ly‐6 family. Eur. J. Immunol. 151:2399‐2408.
  Ford, C.E., Hamerton, J.L., Barnes, D.W., and Loutit, J.F. 1956. Cytological identification of radiation‐chimaeras. Nature 177:452‐454. doi: 10.1038/177452a0.
  Gazit, R., Mandal, P.K., Ebina, W., Ben‐Zvi, A., Nombela‐Arrieta, C., Silberstein, L.E., and Rossi, D.J. 2014. Fgd5 identifies hematopoietic stem cells in the murine bone marrow. J. Exp. Med. 211:1315‐1331. doi: 10.1084/jem.20130428.
  Gerrits, A., Dykstra, B., Kalmykowa, O.J., Klauke, K., Verovskaya, E., Broekhuis, M.J., de Haan, G., and Bystrykh, L.V. 2010. Cellular barcoding tool for clonal analysis in the hematopoietic system. Blood 115:2610‐2618. doi: 10.1182/blood‐2009‐06‐229757.
  Gonzalez‐Murillo, A., Lozano, M.L., Montini, E., Bueren, J.A., and Guenechea, G. 2008. Unaltered repopulation properties of mouse hematopoietic stem cells transduced with lentiviral vectors. Blood 112:3138‐3147. doi: 10.1182/blood‐2008‐03‐142661.
  Goodell, M.A., Brose, K., Paradis, G., Conner, A.S., and Mulligan, R.C. 1996. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183:1797‐1806. doi: 10.1084/jem.183.4.1797.
  Grinenko, T., Arndt, K., Portz, M., Mende, N., Gunther, M., Cosgun, K.N., Alexopoulou, D., Lakshmanaperumal, N., Henry, I., Dahl, A., and Waskow, C. 2014. Clonal expansion capacity defines two consecutive developmental stages of long‐term hematopoietic stem cells. J. Exp. Med. 211:209‐215. doi: 10.1084/jem.20131115.
  Harrison, D.E. 1980. Competitive repopulation: A new assay for long‐term stem cell functional capacity. Blood 55:77‐81.
  Harrison, D.E. and Lerner, C.P. 1991. Most primitive hematopoietic stem cells are stimulated to cycle rapidly after treatment with 5‐fluorouracil. Blood 78:1237‐1240.
  Hodgson, G.S. and Bradley, T.R. 1979. Properties of haematopoietic stem cells surviving 5‐fluorouracil treatment: Evidence for a pre‐CFU‐S cell? Nature 281:381‐382. doi: 10.1038/281381a0.
  Jones, R.J., Wagner, J.E., Celano, P., Zicha, M.S., and Sharkis, S.J. 1990. Separation of pluripotent haematopoietic stem cells from spleen colony‐forming cells. Nature 347:188‐189. doi: 10.1038/347188a0.
  Kent, D.G., Copley, M.R., Benz, C., Wohrer, S., Dykstra, B.J., Ma, E., Cheyne, J., Zhao, Y., Bowie, M.B., Gasparetto, M., Delaney, A., Smith, C., Marra, M., and Eaves, C.J. 2009. Prospective isolation and molecular characterization of hematopoietic stem cells with durable self‐renewal potential. Blood 113:6342‐6350. doi: 10.1182/blood‐2008‐12‐192054.
  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. doi: 10.1016/j.cell.2005.05.026.
  Lemischka, I.R., Raulet, D.H., and Mulligan, R.C. 1986. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 45:917‐927. doi: 10.1016/0092‐8674(86)90566‐0.
  Miller, C.L. and Eaves, C.J. 1997. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho‐myeloid reconstituting ability. Proc. Natl. Acad. Sci. U.S.A. 94:13648‐13653. doi: 10.1073/pnas.94.25.13648.
  Miller, C.L., Rebel, V.I., Lemieux, M.E., Helgason, C.D., Lansdorp, P.M., and Eaves, C.J. 1996. Studies of W mutant mice provide evidence for alternate mechanisms capable of activating hematopoietic stem cells. Exp. Hematol. 24:185‐194.
  Morita, Y., Ema, H., and Nakauchi, H. 2010. Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment. J. Exp. Med. 207:1173‐1182. doi: 10.1084/jem.20091318.
  Morrison, S.J. and Weissman, I.L. 1994. The long‐term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1:661‐673. doi: 10.1016/1074‐7613(94)90037‐X.
  Morrison, S.J., Uchida, N., and Weissman, I.L. 1995. The biology of hematopoietic stem cells. Rev. Cell Dev. Biol. 11:35‐71. doi: 10.1146/annurev.cb.11.110195.000343.
  Naik, S.H., Perie, L., Swart, E., Gerlach, C., van Rooij, N., de Boer, R.J., and Schumacher, T.N. 2013. Diverse and heritable lineage imprinting of early haematopoietic progenitors. Nature 496:229‐232. doi: 10.1038/nature12013.
  Okada, S., Nakauchi, H., Ngayoshi, K., Nishikawa, S., Nishikawa, S.‐I., Miura, Y., and Suda, T. 1991. Enrichment and characterisation of murine hematopoietic stem cells that express c‐kit molecule. Blood 78:1706‐1717.
  Osawa, M., Hanada, K., Hamada, H., and Nakauchi, H. 1996. Long‐term lymphohematopoietic reconstitution by a single CD34‐low/negative hematopoietic stem cell. Science 273:242‐245. doi: 10.1126/science.273.5272.242.
  Ploemacher, R.E. and Brons, R.H. 1989. Separation of CFU‐S from primitive cells responsible for reconstitution of the bone marrow hemopoietic stem cell compartment following irradiation: Evidence for a pre‐CFU‐S cell. Exp. Hematol. 17:263‐266.
  Ramshaw, H.S., Rao, S.S., Crittenden, R.B., Peters, S.O., Weier, H.U., and Quesenberry, P.J. 1995. Engraftment of bone marrow cells into normal unprepared hosts: Effects of 5‐fluorouracil and cell cycle status. Blood 86:924‐929.
  Rebel, V.I., Dragowska, W., Eaves, C.J., Humphries, R.K., and Lansdorp, P.M. 1994. Amplification of Sca‐1+ Lin‐ WGA+ cells in serum‐free cultures containing steel factor, interleukin‐6, and erythropoietin with maintenance of cells with long‐term in vivo reconstituting potential. Blood 83:128‐136.
  Rebel, V.I., Miller, C.L., Thornbury, G.R., Dragowska, W.H., Eaves, C.J., and Lansdorp, P.M. 1996. A comparison of long‐term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse. Exp. Hematol. 24:638‐648.
  Robinson, J.P., Darzynkiewicz, Z., Nolan, J.P., Shankey, T.V., Telford, W., and Watkins, S. 2016. Current Protocols in Cytometry. John Wiley & Sons, Hoboken, N.J.
  Roederer, M. 2001. Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats. Cytometry 45:194–205.
  Rossi, D.J., Bryder, D., Zahn, J.M., Ahlenius, H., Sonu, R., Wagers, A.J., and Weissman, I.L. 2005. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc. Natl. Acad. Sci. U.S.A. 102:9194‐9199. doi: 10.1073/pnas.0503280102.
  Sanjuan‐Pla, A., Macaulay, I.C., Jensen, C.T., Woll, P.S., Luis, T.C., Mead, A., Moore, S., Carella, C., Matsuoka, S., Bouriez Jones, T., Chowdhury, O., Stenson, L., Lutteropp, M., Green, J.C.A., Facchini, R., Boukarabila, H., Grover, A., Gambardella, A., Thongjuea, S., Carrelha, J., Tarrant, P., Atkinson, D., Clark, S.‐A., Nerlov, C., and Jacobsen, S.E.W. 2013. Platelet‐biased stem cells reside at the apex of the haematopoietic stem‐cell hierarchy. Nature 502:232‐236. doi: 10.1038/nature12495.
  Sato, T., Laver, J.H., and Ogawa, M. 1999. Reversible expression of CD34 by murine hematopoietic stem cells. Blood 94:2548‐2554.
  Schofield, R. and Dexter, T.M. 1985. Studies on the self‐renewal ability of CFU‐S which have been serially transferred in long‐term culture or in vivo. Leuk. Res. 9:305‐313. doi: 10.1016/0145‐2126(85)90093‐1.
  Spangrude, G.J., Heimfeld, S., and Weissman, I.L. 1988. Purification and characterisation of mouse hematopoietic stem cells. Science 241:58‐62. doi: 10.1126/science.2898810.
  Sudo, K., Ema, H., Morita, Y., and Nakauchi, H. 2000. Age‐associated characteristics of murine hematopoietic stem cells. J. Exp. Med. 192:1273‐1280. doi: 10.1084/jem.192.9.1273.
  Szilvassy, S.J. 2003. The biology of hematopoietic stem cells. Arch. Med. Res. 34:446‐460. doi: 10.1016/j.arcmed.2003.06.004.
  Szilvassy, S.J., Lansdorp, P.M., Humphries, R.K., Eaves, A.C., and Eaves, C.J. 1989b. Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long‐term repopulating ability. Blood 74:930‐939.
  Szilvassy, S.J., Humphries, R.K., Lansdorp, P.M., Eaves, A.C., and Eaves, C.J. 1990. Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc. Natl. Acad. Sci. U.S.A. 87:8736‐8740. doi: 10.1073/pnas.87.22.8736.
  Szilvassy, S.J., Fraser, C.C., Eaves, C.J., Lansdorp, P.M., Eaves, A.C., and Humphries, R.K. 1989a. Retrovirus‐mediated gene transfer to purified hemopoietic stem cells with long‐term lympho‐myelopoietic repopulating ability. Proc. Natl. Acad. Sci. U.S.A. 86:8798‐8802. doi: 10.1073/pnas.86.22.8798.
  Tajima, F., Deguchi, T., Laver, J.H., Zeng, H., and Ogawa, M. 2001. Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells. Blood 97:2618‐2624. doi: 10.1182/blood.V97.9.2618.
  Till, J.E. and Mc, C.E. 1961. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14:213‐222. doi: 10.2307/3570892.
  Till, J.E. and McCulloch, E.A. 1980. Hemopoietic stem cell differentiation. Biochim. Biophys. Acta 605:431‐459.
  Trevisan, M. and Iscove, N.N. 1995. Phenotypic analysis of murine long‐term hemopoietic reconstituting cells quantitated competitively in vivo and comparison with more advanced colony‐forming progeny. J. Exp. Med. 181:93‐103. doi: 10.1084/jem.181.1.93.
  Trevisan, M., Yan, X.Q., and Iscove, N.N. 1996. Cycle initiation and colony formation in culture by murine marrow cells with long‐term reconstituting potential in vivo. Blood 88:4149‐4158.
  Uchida, N., Dykstra, B., Lyons, K.J., Leung, F.Y., and Eaves, C.J. 2003. Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics. Exp. Hematol. 31:1338‐1347. doi: 10.1016/j.exphem.2003.09.001.
  Uchida, N., Dykstra, B., Lyons, K., Leung, F., Kristiansen, M., and Eaves, C. 2004. ABC transporter activities of murine hematopoietic stem cells vary according to their developmental and activation status. Blood 103:4487‐4495. doi: 10.1182/blood‐2003‐11‐3989.
  Wagers, A.J. and Weissman, I.L. 2006. Differential expression of alpha2 integrin separates long‐term and short‐term reconstituting Lin‐/loThy1.1(lo)c‐kit+ Sca‐1+ hematopoietic stem cells. Stem Cells 24:1087‐1094. doi: 10.1634/stemcells.2005‐0396.
  Wagers, A.J., Sherwood, R.I., Christensen, J.L., and Weissman, I.L. 2002. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 297:2256‐2259. doi: 10.1126/science.1074807.
  Wiesmann, A., Phillips, R.L., Mojica, M., Pierce, L.J., Searles, A.E., Spangrude, G.J., and Lemischka, I. 2000. Expression of CD27 on murine hematopoietic stem and progenitor cells. Immunity 12:193‐199. doi: 10.1016/S1074‐7613(00)80172‐7.
  Wilson, A., Laurenti, E., Oser, G., van der Wath, R.C., Blanco‐Bose, W., Jaworski, M., Offner, S., Dunant, C.F., Eshkind, L., Bockamp, E., Lió P., Macdonald, H.R., and Trumpp, A. 2008. Hematopoietic stem cells reversibly switch from dormancy to self‐renewal during homeostasis and repair. Cell 135:1118‐1129. doi: 10.1016/j.cell.2008.10.048.
  Wilson, N.K., Kent, D.G., Buettner, F., Shehata, M., Macaulay, I.C., Calero‐Nieto, F.J., Sanchez Castillo, M., Oedekoven, C.A., Diamanti, E., Schulte, R., Ponting, C.P., Voet, T., Caldas, C., Stingl, J., Green, A.R., Theis, F.J., and Göttgens, B. 2015. Combined single‐cell functional and gene expression analysis resolves heterogeneity within stem cell populations. Cell Stem Cell. 16:712‐724. doi: 10.1016/j.stem.2015.04.004.
  Wolf, N.S., Kone, A., Priestley, G.V., and Bartelmez, S.H. 1993. In vivo and in vitro characterization of long‐term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342‐rhodamine 123 FACS selection. Exp. Hematol. 21:614‐622.
  Yamamoto, R., Morita, Y., Ooehara, J., Hamanaka, S., Onodera, M., Rudolph, K.L., Ema, H., and Nakauchi, H. 2013. Clonal analysis unveils self‐renewing lineage‐restricted progenitors generated directly from hematopoietic stem cells. Cell 154:1112‐1126. doi: 10.1016/j.cell.2013.08.007.
  Yilmaz, O.H., Kiel, M.J., and Morrison, S.J. 2006. SLAM family markers are conserved among hematopoietic stem cells from old and reconstituted mice and markedly increase their purity. Blood 107:924‐930. doi: 10.1182/blood‐2005‐05‐2140.
  Zhang, C.C. and Lodish, H.F. 2005. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood 105:4314‐4320. doi: 10.1182/blood‐2004‐11‐4418.
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