Isolation of Hematopoietic Stem Cells from Mouse Embryonic Stem Cells

Shannon L. McKinney‐Freeman1, Olaia Naveiras1, George Q. Daley2

1 Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, 2 Harvard Stem Cell Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.3
DOI:  10.1002/9780470151808.sc01f03s4
Online Posting Date:  February, 2008
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Abstract

This unit describes a protocol for the isolation of cells from murine embryonic stem cells with hematopoietic stem cell activity, defined by the ability to reconstitute, long term, multiple lineages of the hematopoietic system of lethally irradiated mice. The protocol subjects hematopoietic progenitors specified in differentiating embryoid bodies to ectopic HoxB4 expression (delivered via retroviral infection), followed by coculture and expansion on OP9 stromal cells in the presence of hematopoietic cytokines for 10 days. The protocol results in the generation of hundreds of millions of cells that can rescue mice from lethal irradiation. Although little is known about the phenotype and frequency of the actual hematopoietic stem cell–like cell within the population of cells generated by this protocol, the protocol establishes a system in which these cells can be further studied and the results ultimately translated to the human system. Curr. Protoc. Stem Cell Biol. 4:1F.3.1‐1F.3.10. © 2008 by John Wiley & Sons, Inc.

Keywords: embryonic stem cell; mESC; hematopoietic stem cell; HSC; blood; bone marrow transplantation; HoxB4

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Differentiation of Embryonic Stem Cells as Embryoid Bodies
  • Basic Protocol 2: Infection of Day 6 Embryoid Body Cells with MSCV‐HoxB4‐IRES‐GFP
  • Support Protocol 1: OP9 Stromal Cell Culture
  • Basic Protocol 3: Expansion of Infected EB‐Derived Cells on OP9 Stroma
  • Basic Protocol 4: Reconstitution of Recipient Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Differentiation of Embryonic Stem Cells as Embryoid Bodies

  Materials
  • mESC growing as a confluent culture on an MEF feeder layer (see unit 1.3) in a 25‐cm2 flask
  • Phosphate‐buffered saline (PBS, Invitrogen, cat. no. 20012‐027)
  • 0.25% (w/v) trypsin (Invitrogen, cat. no. 15090‐046)
  • Differentiation medium (see recipe)
  • 0.4% (w/v) trypan blue (Sigma cat. no. T8154)
  • 25‐cm2 tissue culture flask
  • 15‐ and 50‐ml conical tubes
  • Hemacytometer
  • Inverted tissue culture microscope
  • Multichannel micropipettor (e.g., ePet by BioHit, VWR), capable of accurately dispensing 15 µl
  • 10‐ and 15‐cm nonadherent petri dishes
  • Plate shaker (e.g., orbital shaker by Ikaworks, VWR)

Basic Protocol 2: Infection of Day 6 Embryoid Body Cells with MSCV‐HoxB4‐IRES‐GFP

  Materials
  • Day 6 EBs ( protocol 1)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 20012‐027)
  • Dissociation enzymes (see recipe)
  • Enzyme‐free dissociation buffer (Invitrogen, cat. no. 13151‐014)
  • 10% IMDM (see recipe)
  • 0.4% (w/v) trypan blue (Sigma cat. no. T8154)
  • OP9 stromal cells: plated in 6‐well tissue‐culture plates 24 hr prior to use ( protocol 3)
  • MSCV‐HoxB4‐IRES‐GFP viral supernatant: ecotrophic pseudotype prepared according to standard techniques (Pear, ) and titered (Cepko, )
  • Protamine sulfate (Sigma, cat. no. P3369)
  • 10% IMDM + cytokine cocktail (see recipe)
  • 15‐ml conical tubes
  • Water bath, 37°C
  • Inverted tissue culture microscope
  • Hemacytometer
  • Centrifuge with rotor adapted for 6‐well plates

Support Protocol 1: OP9 Stromal Cell Culture

  Materials
  • OP9 stromal cells (ATCC #CRL‐2749)
  • 20% α‐MEM (see recipe)
  • 6‐well tissue‐culture plates

Basic Protocol 3: Expansion of Infected EB‐Derived Cells on OP9 Stroma

  Materials
  • Infected mESC cultures grown on OP9 stromal cells in 6‐well plates ( protocol 2)
  • 0.05% (w/v) trypsin/EDTA (Invitrogen, cat. no. 25300‐062)
  • 10% IMDM + cytokine cocktail (see recipe)
  • 50‐ml conical tubes
  • 75‐cm2 tissue culture flask

Basic Protocol 4: Reconstitution of Recipient Mice

  Materials
  • OP9‐expanded cells ( protocol 4)
  • Serum‐free IMDM (Invitrogen) or PBS (Invitrogen)
  • Recipient mice: 6‐ to 8‐week‐old Rag‐2−/−γc−/− mice weighing 15 to 22 grams (Taconic Farms)
  • Sterile H 2O
  • Cesium‐source γ‐irradiator
  • 1‐ml syringe
  • 30½‐G needles
  • Autoclaved mouse cages
  • Additional reagents and equipment for evaluating cells and organs by fluorescence microscopy or flow cytometry (e.g., see Robinson et al., ; http://www.cyto.purdue.edu)
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Figures

Videos

Literature Cited

Literature Cited
   Brent, L., Sherwood, R.A., Linch, D.C., and Gale, R.E. 1990. Failure of embryonic mouse cells to engraft in immunocompetent allogeneic recipients. Br. J. Haematol. 74: 549‐551.
   Burt, R.K., Verda, L., Kim, D.A., Oyama, Y., Luo, K., and Link, C. 2004. Embryonic stem cells as an alternate marrow donor source: Engraftment without graft‐versus‐host disease. J. Exp. Med. 199: 895‐904.
   Cepko, C. 1996. Preparation of a specific retrovirus producer cell line. Curr. Protoc. Mol. Biol. 63: 9.10.1‐9.10.13.
   Chen, U., Kosco, M., and Staerz, U. 1992. Establishment and characterization of lymphoid and myeloid mixed‐cell populations from mouse late embryoid bodies, “embryonic‐stem‐cell fetuses”. Proc. Natl. Acad. Sci. U.S.A. 89: 2541‐2545.
   Keller, G. 2005. Embryonic stem cell differentiation: Emergence of a new era in biology and medicine. Genes Dev. 19: 1129‐1155.
   Kyba, M., Perlingeiro, R.C., and Daley, G.Q. 2002. HoxB4 confers definitive lymphoid‐myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 109: 29‐37.
   Lengerke, C., Schmitt, S., Bowman, T.V., Jang, I.H., Maouche‐Chretien, L., McKinney‐Freeman, S., Davidson, A.J., Hammerschmidt, M., Rentzsch, F., Green, J.B.A., Zon, L.I., and Daley, G.Q. 2008. BMP and Wnt specify hematopoietic fate by activation of the Cdx‐Hox pathway. Cell Stem Cell 2: 72‐82.
   Muller, A.M. and Dzierzak, E.A. 1993. ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients. Development 118: 1343‐1351.
   Nakano, T., Kodama, H., and Honjo, T. 1994. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265: 1098‐1101.
   Palacios, R., Golunski, E., and Samaridis, J. 1995. In vitro generation of hematopoietic stem cells from an embryonic stem cell line. Proc. Natl. Acad. Sci. U.S.A. 92: 7530‐7534.
   Pear, W. 1996. Transient transfection methods for preparation of high‐titer retroviral supernatants. Curr. Protoc. Mol. Biol. 68: 9.11.1‐9.11.18.
   Robinson, J.P., Darzynkiewicz, Z., Hoffman, R., Nolan, J.P., Orfao, A., Rabinovitch, P.S., and Watkins, S. (eds.) 2008. Current Protocols in Cytometry. John Wiley & Sons, Hoboken, N.J.
   Sauvageau, G., Thorsteinsdottir, U., Eaves, C.J., Lawrence, H.J., Largman, C., Lansdorp, P.M., and Humphries, R.K. 1995. Overexpression of HoxB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev. 9: 1753‐1765.
   Verda, L., An Kim, D., Ikehara, S., Statkute, L., Bronesky, D., Petrenko, Y., Oyama, Y., He, X., Link, C., Vahanian, N.N., and Burt, R.K. 2007. Hematopoietic mixed chimerism derived from allogeneic embryonic stem cells prevents autoimmune diabetes mellitus in NOD mice. Stem Cells [Epub. Nov. 1, 2007].
   Wang, Y., Yates, F., Naveiras, O., Ernst, P., and Daley, G.Q. 2005. Embryonic stem cell‐derived hematopoietic stem cells. Proc. Natl. Acad. Sci. U.S.A. 102: 19081‐19086.
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