TLX1 (HOX11) Immortalization of Embryonic Stem Cell–Derived and Primary Murine Hematopoietic Progenitors

Robert G. Hawley1, Teresa S. Hawley2, Alan B. Cantor3

1 The George Washington University Medical Center, Washington, D.C., 2 Flow Cytometry Core Facility, The George Washington University Medical Center, Washington, D.C., 3 Dana‐Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.7
DOI:  10.1002/9780470151808.sc01f07s7
Online Posting Date:  December, 2008
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Abstract

The ability to generate genetically engineered cell lines is of great experimental value. They provide a renewable source of material that may be suitable for biochemical analyses, chromatin immunoprecipitation assays, structure‐function studies, gene function assignment, and transcription factor target gene identification. This unit describes protocols for TLX1 (HOX11)‐mediated immortalization of murine hematopoietic progenitors derived from in vitro differentiated murine embryonic stem cells, or from primary mouse fetal liver or bone marrow. A wide variety of hematopoietic cell types have been immortalized using these procedures including erythroid, megakaryocytic, monocytic, myelocytic, and multipotential cell types. These lines are typically cytokine dependent for their survival and growth. Curr. Protoc. Stem Cell Biol. 7:1F.7.1‐1F.7.19. © 2008 by John Wiley & Sons, Inc.

Keywords: murine cell immortalization; TLX1; HOX11; hematopoietic in vitro ES cell differentiation; hematopoietic progenitors

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: TLX1 Immortalization of ES Cell In Vitro Differentiated Hematopoietic Progenitors
  • Alternate Protocol 1: TLX1 Immortalization of Primary Murine Hematopoietic Cells
  • Support Protocol 1: Maintenance of TLX1 (HOX11) Retroviral Producer Cell Lines
  • Support Protocol 2: Cloning of Immortalized Cells in Methylcellulose
  • Support Protocol 3: Cryopreservation of Immortalized Cells
  • Support Protocol 4: Immunophenotypic Analysis of Immortalized Cell Lines by FACS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: TLX1 Immortalization of ES Cell In Vitro Differentiated Hematopoietic Progenitors

  Materials
  • Murine ES cells, gel‐adapted (grown on gelatinized plates, not on feeder cells) and of low passage number (see unit 1.4)
  • IMDM‐ES‐15 with MTG, LIF, and pen/strep (see recipe)
  • 0.05% (w/v) and 0.25% (w/v) trypsin/EDTA (Invitrogen, cat. no. 25200 and 25300, respectively)
  • IMDM‐ES‐5 with MTG and pen/strep (see recipe)
  • Primary ES cell differentiation medium (see recipe)
  • TLX1‐retroviral producer cell line (see Support Protocol):
    • GP+E‐86/MSCV‐HOX11 for cotransduction of the neomycin phosphotransferase (neo) gene conferring resistance to the neomycin analog Geneticin in mammalian cells or
    • GP+E‐86/MSCVhyg‐HOX11 for cotransduction of the hygromycin B phosphotransferase (hyg) drug resistance gene
  • Isocove's modified Dulbecco's medium (IMDM; Invitrogen, cat. no. 12440)
  • IMDM‐ES‐15: IMDM supplemented with 15% (v/v) ES‐FBS
  • Coculture medium (see recipe)
  • IMDM‐ES‐15 with glutamine and pen/strep (see recipe)
  • Immortalized cell medium (see recipe)
  • 50 mg/ml Geneticin (Invitrogen cat. no. 10131‐035) or 50 mg/ml hygromycin B (Mediatech) stock
  • Gelatinized 25‐cm2 tissue culture flasks (see recipe)
  • 15‐ml and 50‐ml conical centrifuge tubes (sterile)
  • Tissue culture centrifuge (refrigerated, benchtop centrifuge with swinging bucket rotor)
  • 100‐mm petri dishes
  • 100‐mm and 60‐mm tissue culture dishes
  • 20‐G needle and 3‐ml syringe
  • Hemacytometer
  • Cell irradiator (optional)
  • Additional reagents and equipment for counting viable cells (unit 1.3)

Alternate Protocol 1: TLX1 Immortalization of Primary Murine Hematopoietic Cells

  • Adult mouse
  • Mouse dissection tools
  • 70% ethanol spray solution
  • 1.5‐ml microcentrifuge tubes, sterile
  • 1× PBS, sterile: diluted from 10× phosphate‐buffered saline without calcium and magnesium (Sigma, cat. no. P7059) to 1× with water, and sterilized by passing through a 0.22‐µm filter
  • 70‐µm cell strainer (BD Falcon; optional)
  • 22‐G needle and 1‐ml syringe (for bone marrow progenitors)
  • 23‐G needle and 1‐ml syringe (for fetal liver progenitors)
  • 25‐G needle and 1‐ml syringe (for fetal liver progenitors)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Support Protocol 1: Maintenance of TLX1 (HOX11) Retroviral Producer Cell Lines

  Materials
  • Suspension of immortalized cells ( protocol 1 or protocol 2)
  • Methylcellulose medium (see recipe)
  • Immortalized cell medium (see recipe), containing the appropriate selection antibiotic
  • Tissue culture centrifuge (refrigerated, benchtop centrifuge with swinging bucket rotor)
  • 15‐ml centrifuge tubes (sterile)
  • 5‐ml syringe fitted with a 16‐G needle
  • 35‐mm tissue culture dish (Falcon, cat. no. 35‐1008)
  • 150‐mm bacterial dishes (Falcon, cat. no. 35‐1058)
  • Inverted microscope
  • 2‐ to 20‐µl micropipettor and sterile, disposable tips
  • 96‐well flat bottom tissue culture plate (Costar, cat. no. 3596)
  • 24‐well tissue culture plate (Falcon, cat. no. 35‐3047)
  • Additional reagents and equipment for counting cells (unit 1.3)

Support Protocol 2: Cloning of Immortalized Cells in Methylcellulose

  Materials
  • Suspension of immortalized cells ( protocol 1, protocol 2, or protocol 4)
  • Immortalized cell medium (see recipe), ice cold
  • 2× freezing medium (see recipe), ice cold
  • Tissue culture centrifuge (refrigerated, benchtop centrifuge with swinging bucket rotor)
  • 15‐ml centrifuge tubes (sterile)
  • 1.8‐ml cryovials (Nunc, cat. no. 377267)
  • −80°C freezer
  • Liquid nitrogen storage tank

Support Protocol 3: Cryopreservation of Immortalized Cells

  Materials
  • Cell line suspensions ( protocol 1 or protocol 2)
  • FACS wash buffer (see recipe)
  • Fluorochrome‐conjugated antibodies and isotype matched control antibodies (see Table 1.7.1); choice of fluorochromes depends on the flow cytometer being used and the combination of markers being analyzed
  • Tissue culture centrifuge (refrigerated, benchtop centrifuge with swinging bucket rotor)
  • 15‐ml conical centrifuge tube
  • 12 × 75–mm FACS tubes (BD Falcon, cat. no. 35‐2054)
  • Flow cytometer (FACS analyzer)
  • Additional reagents and equipment for counting cells (unit 1.3)
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Figures

Videos

Literature Cited

Literature Cited
   Akashi, K., Traver, D., Miyamoto, T., and Weissman, I.L. 2000. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404:193‐197.
   Baron, M.H. 2005. Developmental Hematopoiesis: Methods and Protocols. Humana Press, Totowa, N.J.
   Calvo, K.R., Sykes, D.B., Pasillas, M., and Kamps, M.P. 2000. Hoxa9 immortalizes a granulocyte‐macrophage colony‐stimulating factor‐dependent promyelocyte capable of biphenotypic differentiation to neutrophils or macrophages, independent of enforced Meis expression. Mol. Cell. Biol. 20:3274‐3285.
   Cantor, A.B., Katz, S.G., and Orkin, S.H. 2002. Distinct domains of the GATA‐1 cofactor FOG‐1 differentially influence erythroid versus megakaryocytic maturation. Mol. Cell. Biol. 22:4268‐4279.
   Coghill, E., Eccleston, S., Fox, V., Cerruti, L., Brown, C., Cunningham, J., Jane, S., and Perkins, A. 2001. Erythroid Kruppel‐like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice. Blood 97:1861‐1868.
   Deguchi, K., Ayton, P.M., Carapeti, M., Kutok, J.L., Snyder, C.S., Williams, I.R., Cross, N.C., Glass, C.K., Cleary, M.L., and Gilliland, D.G. 2003. MOZ‐TIF2‐induced acute myeloid leukemia requires the MOZ nucleosome binding motif and TIF2‐mediated recruitment of CBP. Cancer Cell 3:259‐271.
   Dixon, D.N., Izon, D.J., Dagger, S., Callow, M.J., Taplin, R.H., Kees, U.R., and Greene, W.K. 2007. TLX1/HOX11 transcription factor inhibits differentiation and promotes a non‐haemopoietic phenotype in murine bone marrow cells. Br. J. Haematol. 138:54‐67.
   Du, C., Redner, R.L., Cooke, M.P., and Lavau, C. 1999. Overexpression of wild‐type retinoic acid receptor alpha (RARalpha) recapitulates retinoic acid‐sensitive transformation of primary myeloid progenitors by acute promyelocytic leukemia RARalpha‐fusion genes. Blood 94:793‐802.
   Du, Y., Jenkins, N.A., and Copeland, N.G. 2005. Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells. Blood 106:3932‐3939.
   Dube, I.D., Kamel‐Reid, S., Yuan, C.C., Lu, M., Wu, X., Corpus, G., Raimondi, S.C., Crist, W.M., Carroll, A.J., and Minowada, J. 1991. A novel human homeobox gene lies at the chromosome 10 breakpoint in lymphoid neoplasias with chromosomal translocation t(10;14). Blood 78:2996‐3003.
   Fischbach, N.A., Rozenfeld, S., Shen, W., Fong, S., Chrobak, D., Ginzinger, D., Kogan, S.C., Radhakrishnan, A., Le Beau, M.M., Largman, C., and Lawrence, H.J. 2005. HOXB6 overexpression in murine bone marrow immortalizes a myelomonocytic precursor in vitro and causes hematopoietic stem cell expansion and acute myeloid leukemia in vivo. Blood 105:1456‐1466.
   Fuller, S.A., Takahashi, M., and Hurrell, J.G.R. 2001. Cloning of hybridoma cell lines by limiting dilution. Curr. Protoc. Mol. Biol. 1:11.8.1‐11.8.2.
   Gonda, T.J., Ramsay, R.G., and Johnson, G.R. 1989. Murine myeloid cell lines derived by in vitro infection with recombinant c‐myb retroviruses express myb from rearranged vector proviruses. EMBO J. 8:1767‐1775.
   Green, S.M., Lowe, A.D., Parrington, J., and Karn, J. 1989. Transformation of growth factor‐dependent myeloid stem cells with retroviral vectors carrying c‐myc. Oncogene 4:737‐751.
   Hatano, M., Roberts, C.W., Minden, M., Crist, W.M., and Korsmeyer, S.J. 1991. Deregulation of a homeobox gene, HOX11, by the t(10;14) in T cell leukemia. Science 253:79‐82.
   Hawley, R.G., Fong, A.Z., Lu, M., and Hawley, T.S. 1994a. The HOX11 homeobox‐containing gene of human leukemia immortalizes murine hematopoietic precursors. Oncogene 9:1‐12.
   Hawley, R.G., Lieu, F.H., Fong, A.Z., and Hawley, T.S. 1994b. Versatile retroviral vectors for potential use in gene therapy. Gene Ther. 1:136‐138.
   Kamps, M.P. and Wright, D.D. 1994. Oncoprotein E2A‐Pbx1 immortalizes a myeloid progenitor in primary marrow cultures without abrogating its factor‐dependence. Oncogene 9:3159‐3166.
   Karasuyama, H. and Melchers, F. 1988. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18:97‐104.
   Kawabe, T., Muslin, A.J., and Korsmeyer, S.J. 1997. HOX11 interacts with protein phosphatases PP2A and PP1 and disrupts a G2/M cell‐cycle checkpoint. Nature 385:454‐458.
   Keller, G., Wall, C., Fong, A.Z., Hawley, T.S., and Hawley, R.G. 1998. Overexpression of HOX11 leads to the immortalization of embryonic precursors with both primitive and definitive hematopoietic potential. Blood 92:877‐887.
   Kennedy, M.A., Gonzalez‐Sarmiento, R., Kees, U.R., Lampert, F., Dear, N., Boehm, T., and Rabbitts, T.H. 1991. HOX11, a homeobox‐containing T‐cell oncogene on human chromosome 10q24. Proc. Natl. Acad. Sci. U.S.A. 88:8900‐8904.
   Lavau, C., Szilvassy, S.J., Slany, R., and Cleary, M.L. 1997. Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX‐ENL. EMBO J. 16:4226‐4237.
   Lee, J.C., Hapel, A.J., and Ihle, J.N. 1982. Constitutive production of a unique lymphokine (IL 3) by the WEHI‐3 cell line. J. Immunol. 128:2393‐2398.
   Lu, M., Gong, Z.Y., Shen, W.F., and Ho, A. 1991. The tcl‐3 proto‐oncogene altered by chromosomal translocation in T‐cell leukemia codes for a homeobox protein. EMBO J. 10:2905‐2910.
   Owens, B.M., Zhu, Y.X., Suen, T.C., Wang, P.X., Greenblatt, J.F., Goss, P.E., and Hawley, R.G. 2003. Specific homeodomain‐DNA interactions are required for HOX11‐mediated transformation. Blood 101:4966‐4974.
   Owens, B.M., Hawley, T.S., Spain, L.M., Kerkel, K.A., and Hawley, R.G. 2006. TLX1/HOX11‐mediated disruption of primary thymocyte differentiation prior to the CD4+CD8+ double‐positive stage. Br. J. Haematol. 132:216‐229.
   Pal, S., Cantor, A.B., Johnson, K.D., Moran, T.B., Boyer, M.E., Orkin, S.H., and Bresnick, E.H. 2004. Coregulator‐dependent facilitation of chromatin occupancy by GATA‐1. Proc. Natl. Acad. Sci. U.S.A. 101:980‐985.
   Perkins, A.C. and Cory, S. 1993. Conditional immortalization of mouse myelomonocytic, megakaryocytic and mast cell progenitors by the Hox‐2.4 homeobox gene. EMBO J. 12:3835‐3846.
   Pinto do, O.P., Richter, K., and Carlsson, L. 2002. Hematopoietic progenitor/stem cells immortalized by Lhx2 generate functional hematopoietic cells in vivo. Blood 99:3939‐3946.
   Pronk, C.J., Rossi, D.J., Mansson, R., Attema, J.L., Norddahl, G.L., Chan, C.K., Sigvardsson, M., Weissman, I.L., and Bryder, D. 2007. Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy. Cell Stem Cell 1:428‐442.
   Riz, I. and Hawley, R.G. 2005. G1/S transcriptional networks modulated by the HOX11/TLX1 oncogene of T‐cell acute lymphoblastic leukemia. Oncogene 24:5561‐5575.
   Riz, I., Akimov, S.S., Eaker, S.S., Baxter, K.K., Lee, H.J., Marino‐Ramirez, L., Landsman, D., Hawley, T.S., and Hawley, R.G. 2007. TLX1/HOX11‐induced hematopoietic differentiation blockade. Oncogene 26:4115‐4123.
   Shaw, G.C., Cope, J.J., Li, L., Corson, K., Hersey, C., Ackermann, G.E., Gwynn, B., Lambert, A.J., Wingert, R.A., Traver, D., Trede, N.S., Barut, B.A., Zhou, Y., Minet, E., Donovan, A., Brownlie, A., Balzan, R., Weiss, M.J., Peters, L.L., Kaplan, J., Zon, L.I., and Paw, B.H. 2006. Mitoferrin is essential for erythroid iron assimilation. Nature 440:96‐100.
   Schnabel, C.A., Jacobs, Y., and Cleary, M.L. 2000. HoxA9‐mediated immortalization of myeloid progenitors requires functional interactions with TALE cofactors Pbx and Meis. Oncogene 19:608‐616.
   Su, X., Drabkin, H., Clappier, E., Morgado, E., Busson, M., Romana, S., Soulier, J., Berger, R., Bernard, O.A., and Lavau, C. 2006. Transforming potential of the T‐cell acute lymphoblastic leukemia‐associated homeobox genes HOXA13, TLX1, and TLX3. Genes Chromosomes Cancer 45:846‐855.
   Suh, HC., Leeanansaksiri, W., Ji, M., Klarmann, K.D., Renn, K., Gooya, J., Smith, D., McNiece, I., Lugthart, S., Valk, P.J., Delwel, R., and Keller, J.R. 2008. Id1 immortalizes hematopoietic progenitors in vitro and promotes a myeloproliferative disease in vivo. Oncogene 27:5612‐5623.
   Templin, C., Kotlarz, D., Rathinam, C., Rudolph, C., Schatzlein, S., Ramireddy, K., Rudolph, K. L., Schlegelberger, B., Klein, C., and Drexler, H. 2008. Establishment of immortalized multipotent hematopoietic progenitor cell lines by retroviral‐mediated gene transfer of beta‐catenin. Exp. Hematol. 36:204‐215.
   Varnum‐Finney, B., Xu, L., Brashem‐Stein, C., Nourigat, C., Flowers, D., Bakkour, S., Pear, W.S., and Bernstein, I.D. 2000. Pluripotent, cytokine‐dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat. Med. 6:1278‐1281.
   Yu, W.M., Hawley, T.S., Hawley, R.G., and Qu, C.K. 2002. Immortalization of yolk sac‐derived precursor cells. Blood 100:3828‐3831.
   Yu, W.M., Hawley, T.S., Hawley, R.G., and Qu, C.K. 2003. Catalytic‐dependent and ‐independent roles of SHP‐2 tyrosine phosphatase in interleukin‐3 signaling. Oncogene 22:5995‐6004.
   Zhang, P., Nelson, E., Radomska, H.S., Iwasaki‐Arai, J., Akashi, K., Friedman, A.D., and Tenen, D.G. 2002. Induction of granulocytic differentiation by 2 pathways. Blood 99:4406‐4412.
   Zhang, X.B., Schwartz, J.L., Humphries, R.K., and Kiem, H.P. 2007. Effects of HOXB4 overexpression on ex vivo expansion and immortalization of hematopoietic cells from different species. Stem Cells 25:2074‐2081.
   Zinkel, S.S., Hurov, K.E., Ong, C., Abtahi, F.M., Gross, A., and Korsmeyer, S.J. 2005. A role for proapoptotic BID in the DNA‐damage response. Cell 122:579‐591.
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