Hematoendothelial Differentiation of Human Embryonic Stem Cells

Maxim A. Vodyanik1, Igor I. Slukvin2

1 University of Wisconsin, Madison, Wisconsin, 2 WiCell Research Institute, Madison, Wisconsin
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 23.6
DOI:  10.1002/0471143030.cb2306s36
Online Posting Date:  September, 2007
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Abstract

Human embryonic stem cells (hESCs) represent a unique population of cells capable of self‐renewal and differentiation into all types of somatic cells, including hematopoietic and endothelial cells. Since the pattern of hematopoietic and endothelial development observed in the embryo can be reproduced using ESCs differentiated in culture, hESCs can be used as a model for studies of specification and diversification of hematoendothelial progenitors. In addition, hESCs can be seen as a scalable source of hematopoietic and endothelial cells for in vitro studies. This unit describes a method for efficient differentiation of hESCs into hematopoietic progenitors and endothelial cells through coculture with mouse OP9 bone marrow stromal cells, as well as an approach for their analysis and isolation. Support protocols are provided for culture of mouse embryonic fibroblasts, evaluation of hematopoietic and endothelial differentiation by flow cytometry and colony‐forming assay, removal of OP9 cells, and propagation of hESC‐derived endothelial cells. Curr. Protoc. Cell Biol. 36:23.6.1‐23.6.28. © 2007 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; hematopoietic development; in vitro differentiation; hematopoietic progenitors; endothelial cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Hematoendothelial Differentiation of hESCs in OP9 Coculture
  • Support Protocol 1: Assessment of Hematoendothelial Differentiation by Flow Cytometry
  • Support Protocol 2: Hematopoietic Precursor Colony‐Forming Assay
  • Support Protocol 3: Removal of OP9 Cells from hESC/OP9 Coculture
  • Basic Protocol 2: Isolation of hESC‐Derived Hematopoietic Progenitors and Endothelial Cells
  • Support Protocol 4: Propagation of hESCs on Mouse Embryonic Fibroblasts
  • Support Protocol 5: Preparation of MEF Feeder Plates
  • Support Protocol 6: Culture of OP9 Cells
  • Support Protocol 7: Propagation of Endothelial Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Hematoendothelial Differentiation of hESCs in OP9 Coculture

  Materials
  • 6‐well plates with undifferentiated hESCs on day 5 to 6 of culture ( protocol 6)
  • 1× phosphate‐buffered saline, without calcium and magnesium (PBS; GIBCO)
  • Differentiation medium (see recipe), warmed to 37°C
  • 10‐cm dishes with OP9 cells on day 8 to 12 after plating ( protocol 8)
  • Collagenase IV solution (see recipe)
  • 0.05% (w/v) trypsin/0.5 mM EDTA solution (1×; GIBCO)
  • Cell washing/MACS buffer (see recipe)
  • 15‐ and 50‐ml polypropylene centrifuge tubes
  • Temperature controlled centrifuge
  • 70‐µm nylon filters (cell strainers; Falcon)
  • Additional reagents and equipment for counting cells (unit 1.1)

Support Protocol 1: Assessment of Hematoendothelial Differentiation by Flow Cytometry

  Materials
  • Cells collected from hESC/OP9 coculture ( protocol 1)
  • FACS buffer with and without fetal bovine serum (FBS; see recipe)
  • Mouse anti‐human CD31‐PE mAb (clone WM59; BD Pharmingen)
  • Mouse anti‐human CD43‐FITC mAb (clone 1G10; BD Pharmingen)
  • Mouse anti‐human TRA‐1‐85‐APC mAb (clone TRA‐1‐85; R&D Systems)
  • Mouse anti‐human CD235a‐PE mAb (clone CLB‐ery‐1; Caltag)
  • Mouse anti‐human CD45‐APC mAb (clone HI30; BD Pharmingen)
  • Mouse anti‐human CD29‐FITC/PE mAb (clone MEM‐101A; Caltag)
  • Mouse anti‐human CD34‐APC mAb (clone 581; BD Pharmingen), optional
  • Mouse IgG1 FITC control mAb (clone MOPC‐21; BD Pharmingen)
  • Mouse IgG1 PE control mAb (clone MOPC‐21; BD Pharmingen)
  • Mouse IgG1 APC control mAb (clone MOPC‐21; BD Pharmingen)
  • 7‐aminoactinomycin D (7AAD) staining solution (Via‐Probe; BD Pharmingen)
  • 5‐ml polystyrene test tubes (Falcon)
  • Temperature‐controlled centrifuge
  • Flow cytometer (FACSCalibur; BD Immunocytometry Systems)
  • FlowJo flow cytometry analysis software (Tree Star)

Support Protocol 2: Hematopoietic Precursor Colony‐Forming Assay

  Materials
  • MethoCult H4435 GF+ complete medium (Stem Cell Technologies): thaw, divide into 3‐ml single‐test aliquots (using a 10‐ml syringe and blunt‐end needle), and refreeze; store up to 6 months at −20°C
  • 6 × 104 cells ( protocol 1, step 17)/0.3 ml differentiation medium
  • Differentiation medium (see recipe)
  • Sterile distilled water
  • Blunt‐end needles (Stem Cell Technologies) and 5‐ and 10‐ml syringes, optional
  • 37°C water bath
  • Disposable 5‐ml serological pipet
  • 3.5‐cm low‐adherence CFU assay dishes (Stem Cell Technologies)
  • 3.5‐cm regular plastic tissue culture dishes
  • 10‐cm regular plastic tissue culture dishes
  • Inverted microscope
  • Gridded scoring dishes (Stem Cell Technologies)

Support Protocol 3: Removal of OP9 Cells from hESC/OP9 Coculture

  Materials
  • Cell suspension ( protocol 1), hold on ice
  • Cell washing/MACS buffer (see recipe), degassed under vacuum and ice cold
  • Hamster anti‐mouse CD29‐PE mAb (clone HM beta 1‐1; Serotec)
  • Anti‐PE magnetic microbeads (Miltenyi Biotec)
  • Mouse anti‐human TRA‐1‐85‐APC mAb (clone TRA‐1‐85; R&D Systems)
  • 7‐aminoactinomycin D (7AAD) staining solution (Via‐Probe; BD Pharmingen)
  • 15‐ml graduated polypropylene tubes
  • Temperature controlled centrifuge, 4°C
  • MACS rotation mixer (Miltenyi Biotec)
  • 35‐µm preseparation filters (Miltenyi Biotec)
  • Midi‐MACS magnet/stand (Miltenyi Biotec)
  • LD (depletion) columns (Miltenyi Biotec)
  • Additional reagents and equipment for counting cells (unit 1.1) and analyzing cells by flow cytometry ( protocol 2)
NOTE: Cells and cell washing/MACS buffer must be kept on ice during the separation procedure.

Basic Protocol 2: Isolation of hESC‐Derived Hematopoietic Progenitors and Endothelial Cells

  Materials
  • Cell suspension ( protocol 1), hold on ice
  • Cell washing/MACS buffer (see recipe), degassed under vacuum and ice cold
  • Mouse anti‐human CD43‐FITC mAb (clone 1G10; BD Pharmingen)
  • Anti‐FITC magnetic microbeads, (Miltenyi Biotec)
  • Basic (nonconjugated) microbeads (Miltenyi Biotec), optional
  • Mouse anti‐human CD31‐PE (clone WM59; BD Pharmingen)
  • Anti‐PE magnetic microbeads, (Miltenyi Biotec)
  • FACS buffer without FBS (see recipe)
  • 7‐aminoactinomycin D (7AAD) solution (Via‐Probe; BD Pharmingen)
  • Mouse IgG1‐PE isotype control mAb (clone MOPC‐21; BD Pharmingen)
  • Mouse IgG1‐APC isotype control mAb (clone MOPC‐21; BD Pharmingen)
  • Mouse anti‐human CD235a‐PE (clone CLB‐ery‐1; Caltag)
  • Mouse anti‐human CD45‐APC (clone HI30; BD Pharmingen)
  • FBS (GIBCO), heat inactivated
  • LD (depletion) columns, (Miltenyi Biotec)
  • 15‐ml graduated polypropylene tubes
  • Temperature‐controlled centrifuge, 4°C
  • MACS rotation mixer, (Miltenyi Biotec)
  • 35‐µm preseparation filters (Miltenyi Biotec)
  • LS (positive selection) columns (Miltenyi Biotec)
  • Midi‐MACS magnet/stand (Miltenyi Biotec)
  • Cell sorter (FACS Vantage SE; BD Immunocytometry Systems)
  • 5‐ml polypropylene tubes (Falcon)
  • Additional reagents and equipment for counting cells (unit 1.1) and performing FACS ( protocol 2)
NOTE: Cells and cell washing/MACS buffer must be kept on ice during the separation procedure.

Support Protocol 4: Propagation of hESCs on Mouse Embryonic Fibroblasts

  Materials
  • hESC cultures (H1 or H9 hESC lines; National Stem Cell Bank; see )
  • 6‐well plates with pre‐plated irradiated MEFs ( protocol 7)
  • hESC growth medium (see recipe)
  • 1× phosphate‐buffered saline, without calcium and magnesium (PBS; GIBCO), room temperature
  • Collagenase IV solution (see recipe)
  • 37°C water bath
  • 5‐ml glass pipet, sterile

Support Protocol 5: Preparation of MEF Feeder Plates

  Materials
  • Gelatin‐coated 10‐cm dishes and 6‐well plates (see recipe)
  • CF‐1 strain MEFs (e.g., National Stem Cell Bank or ATCC: frozen stock at passage 3; 2–3 × 106 cells/vial)
  • MEF growth medium (see recipe)
  • 1× phosphate‐buffered saline, without calcium and magnesium (PBS; GIBCO)
  • 0.05% (w/v) trypsin/0.5 mM EDTA solution (1×; GIBCO)
  • 15‐ml polypropylene tubes
  • Gamma irradiator
  • Additional reagents and equipment for counting cells (unit 1.1)

Support Protocol 6: Culture of OP9 Cells

  Materials
  • OP9 growth medium (see recipe).
  • 0.05% (w/v) trypsin/0.5 mM EDTA solution (1×; GIBCO)
  • OP9 cells (ATCC #CRL‐2749)
  • 1× phosphate‐buffered saline, without calcium and magnesium (PBS; GIBCO)
  • Gelatin‐coated 10‐cm dishes (see recipe)
  • 15‐ml polypropylene tubes
  • Inverted microscope

Support Protocol 7: Propagation of Endothelial Cells

  Materials
  • MACS‐isolated endothelial (CD31+CD43) cells on day 7 to 8 of hESC/OP9 coculture ( protocol 5, step 22)
  • Fibronectin‐coated 10‐cm tissue culture dishes (see recipe)
  • Endothelial cell growth medium (ECGM; see recipe)
  • 1× phosphate‐buffered saline, without calcium and magnesium (PBS; GIBCO)
  • HyQ‐Tase cell detachment solution (HyClone)
  • Endothelial serum‐free medium (ESFM; GIBCO)
  • Temperature controlled centrifuge, 4°C
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Figures

Videos

Literature Cited

   Amit, M., Carpenter, M.K., Inokuma, M.S., Chiu, C.P., Harris, C.P., Waknitz, M.A., Itskovitz‐Eldor, J., and Thomson, J.A. 2000. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227:271‐278.
   Bazil, V., Brandt, J., Tsukamoto, A., and Hoffman, R. 1995. Apoptosis of human hematopoietic progenitor cells induced by crosslinking of surface CD43, the major sialoglycoprotein of leukocytes. Blood 86:502‐511.
   Burkert, U., von Ruden, T., and Wagner, E.F. 1991. Early fetal hematopoietic development from in vitro differentiated embryonic stem cells. New Biol. 3:698‐708.
   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.
   Cho, S.K., Webber, T.D., Carlyle, J.R., Nakano, T., Lewis, S.M., and Zuniga‐Pflucker, J.C. 1999. Functional characterization of B lymphocytes generated in vitro from embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 96:9797‐9802.
   Coulombel, L. 2004. Identification of hematopoietic stem/progenitor cells: Strength and drawbacks of functional assays. Oncogene 23:7210‐7222.
   Daley, G.Q. 2003. From embryos to embryoid bodies: Generating blood from embryonic stem cells. Ann. N.Y. Acad. Sci. 996:122‐131.
   Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. 1985. The in vitro development of blastocyst‐derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands, and myocardium. J. Embryol. Exp. Morphol. 87:27‐45.
   Draper, J.S., Smith, K., Gokhale, P., Moore, H.D., Maltby, E., Johnson, J., Meisner, L., Zwaka, T.P., Thomson, J.A., and Andrews, P.W. 2004. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat. Biotechnol. 22:53‐54.
   Eaves, C.J. and Lambie, K. 1995. Atlas of Human Hematopoietic Colonies. StemCell Technologies, Vancouver, Canada.
   Eto, K., Murphy, R., Kerrigan, S.W., Bertoni, A., Stuhlmann, H., Nakano, T., Leavitt, A.D., and Shattil, S.J. 2002. Megakaryocytes derived from embryonic stem cells implicate CalDAG‐GEFI in integrin signaling. Proc. Nat. Acad. Sci. U.S.A. 99:12819‐12824.
   Fairchild, P.J., Brook, F.A., Gardner, R.L., Graca, L., Strong, V., Tone, Y., Tone, M., Nolan, K.F., and Waldmann, H. 2000. Directed differentiation of dendritic cells from mouse embryonic stem cells. Curr. Biol. 10:1515‐1518.
   Galic, Z., Kitchen, S.G., Kacena, A., Subramanian, A., Burke, B., Cortado, R., and Zack, J.A. 2006. T lineage differentiation from human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 103:11742‐11747.
   Gaur, M., Kamata, T., Wang, S., Moran, B., Shattil, S.J., and Leavitt, A.D. 2006. Megakaryocytes derived from human embryonic stem cells: A genetically tractable system to study megakaryocytopoiesis and integrin function. J. Thromb. Haemost. 4:436‐442.
   Gutierrez‐Ramos, J.C. and Palacios, R. 1992. In vitro differentiation of embryonic stem cells into lymphocyte precursors able to generate T and B lymphocytes in vivo. Proc. Natl. Acad. Sci. U.S.A. 89:9171‐9175.
   Hole, N., Graham, G.J., Menzel, U., and Ansell, J.D. 1996. A limited temporal window for the derivation of multilineage repopulating hematopoietic progenitors during embryonal stem cell differentiation in vitro. Blood 88:1266‐1276.
   Kaufman, D.S., Hanson, E.T., Lewis, R.L., Auerbach, R., and Thomson, J.A. 2001. Hematopoietic colony‐forming cells derived from human embryonic stem cells. Proc. Nat. Acad. Sci. U.S.A. 98:10716‐10721.
   Keller, G. 2005. Embryonic stem cell differentiation: Emergence of a new era in biology and medicine. Genes. Dev. 19:1129‐1155.
   Keller, G., Kennedy, M., Papayannopoulou, T., and Wiles, M.V. 1993. Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol. Cell Biol. 13:473‐486.
   Kodama, H., Nose, M., Niida, S., and Nishikawa, S. 1994. Involvement of the c‐kit receptor in the adhesion of hematopoietic stem cells to stromal cells. Exp. Hem. 22:979‐984.
   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.
   Lensch, M.W. and Daley, G.Q. 2004. Origins of mammalian hematopoiesis: In vivo paradigms and in vitro models. Curr. Top. Dev. Biol. 60:127‐196.
   Lieber, J.G., Webb, S., Suratt, B.T., Young, S.K., Johnson, G.L., Keller, G.M., and Worthen, G.S. 2004. The in vitro production and characterization of neutrophils from embryonic stem cells. Blood 103:852‐859.
   Nakano, T. 2003. Hematopoietic stem cells: Generation and manipulation. Trends Immunol. 24:589‐594.
   Nakano, T., Kodama, H., and Honjo, T. 1994. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265:1098‐1101.
   Nakano, T., Kodama, H., and Honjo, T. 1996. In vitro development of primitive and definitive erythrocytes from different precursors. Science 272:722‐724.
   Ng, E.S., Davis, R.P., Azzola, L., Stanley, E.G., and Elefanty, A.G. 2005. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood 106:1601‐1603.
   Nishikawa, S.I., Nishikawa, S., Hirashima, M., Matsuyoshi, N., and Kodama, H. 1998. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE‐cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development 125:1747‐1757.
   Potocnik, A.J., Nielsen, P.J., and Eichmann, K. 1994. In vitro generation of lymphoid precursors from embryonic stem cells. Embo. J. 13:5274‐5283.
   Potocnik, A.J., Kohler, H., and Eichmann, K. 1997. Hemato‐lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells. Proc. Nat. Acad. Sci. U.S.A. 94:10295‐300.
   Qiu, C., Hanson, E., Olivier, E., Inada, M., Kaufman, D.S., Gupta, S., and Bouhassira, E.E. 2005. Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development. Exp. Hem. 33:1450‐1458.
   Robertson, S., Kennedy, M., and Keller, G. 1999. Hematopoietic commitment during embryogenesis. Ann. N.Y. Acad. Sci. 872:9‐16.
   Schmitt, R.M., Bruyns, E., and Snodgrass, H.R. 1991. Hematopoietic development of embryonic stem cells in vitro: Cytokine and receptor gene expression. Genes Dev. 5:728‐740.
   Schmitt, T.M., de Pooter, R.F., Gronski, M.A., Cho, S.K., Ohashi, P.S., and Zuniga‐Pflucker, J.C. 2004. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat. Immunol. 5:410‐417.
   Schuldiner, M., Yanuka, O., Itskovitz‐Eldor, J., Melton, D.A., and Benvenisty, N. 2000. From the cover: Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Nat. Acad. Sci. U.S.A. 97:11307‐11312.
   Senju, S., Hirata, S., Matsuyoshi, H., Masuda, M., Uemura, Y., Araki, K., Yamamura, K., and Nishimura, Y. 2003. Generation and genetic modification of dendritic cells derived from mouse embryonic stem cells. Blood 101:3501‐3508.
   Slukvin, I.I., Vodyanik, M.A., Thomson, J.A., Gumenyuk, M.E., and Choi, K.D. 2006. Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway. J. Immunol. 176:2924‐2932.
   Thomson, J.A., Itskovitz‐Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145‐1147.
   Vodyanik, M.A., Bork, J.A., Thomson, J.A., and Slukvin, I.I. 2005. Human embryonic stem cell‐derived CD34+ cells: Efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood 105:617‐626.
   Vodyanik, M.A., Thomson, J.A., and Slukvin, I.I. 2006. Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures. Blood 108:2095‐2105.
   Wang, L., Menendez, P., Shojaei, F., Li, L., Mazurier, F., Dick, J.E., Cerdan, C., Levac, K., and Bhatia, M. 2005a. Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression. J. Exp. Med. 201:1603‐1614.
   Wang, Y., Yates, F., Naveiras, O., Ernst, P., and Daley, G.Q. 2005b. Embryonic stem cell‐derived hematopoietic stem cells. Proc. Natl. Acad. Sci. U.S.A. 102:19081‐19086.
   Williams, B.P., Daniels, G.L., Pym, B., Sheer, D., Povey, S., Okubo, Y., Andrews, P.W., and Goodfellow, P.N. 1988. Biochemical and genetic analysis of the OKa blood group antigen. Immunogenetics 27:322‐329.
   Woll, P.S., Martin, C.H., Miller, J.S., and Kaufman, D.S. 2005. Human embryonic stem cell‐derived NK cells acquire functional receptors and cytolytic activity. J. Immunol. 175:5095‐5103.
   Yoshida, H., Hayashi, S., Kunisada, T., Ogawa, M., Nishikawa, S., Okamura, H., Sudo, T., and Shultz, L.D. 1990. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345:442‐444.
   Zambidis, E.T., Peault, B., Park, T.S., Bunz, F., and Civin, C.I. 2005. Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development. Blood 106:860‐870.
   Zhang, H., Saeki, K., Kimura, A., Nakahara, M., Doshi, M., Kondo, Y., Nakano, T., and Yuo, A. 2006. Efficient and repetitive production of hematopoietic and endothelial cells from feeder‐free monolayer culture system of primate embryonic stem cells. Biol. Reprod. 74:295‐306.
   Zhang, W.J., Park, C., Arentson, E., and Choi, K. 2005. Modulation of hematopoietic and endothelial cell differentiation from mouse embryonic stem cells by different culture conditions. Blood 105:111‐114.
Key References
   Coulombel, L. 2004. See above.
  Provides a critical overview of in vivo and in vitro functional assays used for analysis hematopoietic stem cells and progenitors.
   Vodyanik, M.A., Thomson, J.A., and Slukvin, I.I. 2006. See above.
  Describes methods for generation and pathways of hematopoietic and endothelial differentiation from hESCs cocultured on OP9.
   Zambidis, E.T., Peault, B., Park, T.S., Bunz, F., and Civin, C.I. 2005. See above.
  Describes procedures for and pathways of hematopoietic differentiation from hESCs using the embryoid body method.
Internet Resources
  http://www.nationalstemcellbank.org
  National Stem Cell Bank. Distributes hESC lines and provides technical support to the hESC research community and technical training in the culture of hESCs. In addition, information regarding karyotype and HLA genotype of distributed cells is provided. Protocols for culture, freezing, and thawing of hESCs as well as for derivation, culture, and propagation of mouse embryonic fibroblasts are also available.
  http://www.stemcell.com
  Stem Cell Technologies. Protocols for hematopoietic stem cell/progenitor research.
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