Differentiation of Human Erythroid Cells in Culture

E. Fibach1, E. Prus1

1 Hadassah University Hospital, Ein‐Kerem, Jerusalem
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.7
DOI:  10.1002/0471142735.im22f07s69
Online Posting Date:  November, 2005
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Abstract

A culture procedure for growing erythroid progenitors in liquid medium is described. The procedure is divided into two phases. The first is an erythropoietin (EPO)–independent phase in which peripheral blood mononuclear cells are isolated and cultured in the presence of a combination of growth factors, but in the absence of EPO. During this phase, early erythroid‐committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an EPO‐supplemented medium, continue to proliferate and mature into hemoglobin‐containing nucleated erythroid cells. The culture procedure yields populations that are large, relatively pure, and synchronized (in terms of differentiation), and which recapitulate in vivo erythropoiesis. Since the cells are grown in suspension, samples of cells can be withdrawn at any time, without disturbing the cultures, and assayed for various parameters, e.g., morphology, size, number, viability, apoptosis, cell cycle, surface antigens, or gene expression. Several procedures for analyzing the cultured cells with respect to their hemoglobin content during their differentiation are included.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Peripheral Blood Mononuclear Cells for Erythroid Cultures
  • Basic Protocol 2: Erythroid Cell Culture Procedure
  • Benzidine Staining of Hemoglobins
  • Support Protocol 1: Immunofluorescence Staining of Hemoglobins
  • Support Protocol 2: Preparation of Samples for HPLC Analysis of Hemoglobins
  • Support Protocol 3: Cation‐Exchange HPLC for Separation of Hemoglobins
  • Support Protocol 4: Reversed‐Phase HPLC for Analysis of Globin Chains
  • Support Protocol 5: Lysing RBC by Osmotic Shock
  • Support Protocol 6: Removing RBC by Percoll Separation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparation of Peripheral Blood Mononuclear Cells for Erythroid Cultures

  Materials
  • Peripheral blood or buffy coat fraction
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • Ficoll‐Hypaque (density = 1.077 g/ml)
  • 15‐ or 50‐ml centrifuge tubes
  • Centrifuge

Basic Protocol 2: Erythroid Cell Culture Procedure

  Materials
  • Complete MEM medium (see recipe)
  • Preselected FBS (see recipe); do not heat‐inactivate
  • 50 mg/ml cyclosporin A (Sandoz)
  • Conditioned medium from 5637 human bladder carcinoma cell line (see recipe)
  • Mononuclear cells, washed (see protocol 1)
  • 10% (w/v) deionized BSA (see recipe)
  • 2‐mercaptoethanol (Sigma)
  • 4 mg/ml dexamethasone sodium phosphate (Elkin‐Sinn Pharmaceuticals, Cherry Hill N.J.)
  • Human holo‐transferrin (Sigma)
  • Human recombinant stem cell factor (CytoLab Ltd.; http://www.cytolab.com/)
  • Human recombinant erythropoietin (EPO; Ortho Pharmaceutical)
  • 25‐ or 75‐cm2 tissue culture flasks

Benzidine Staining of Hemoglobins

  Materials
  • Benzidine dihydrochloride (Sigma)
  • Acetic acid, glacial
  • 33% H 2O 2
  • 3,3′‐dimethoxybenzidine (Sigma)
  • Methanol, absolute
  • Ethanol, absolute
  • Giemsa stain
  • Brown bottles to accommodate volumes of stock solution prepared
  • Shandon Cytospin cytocentrifuge (Thermo Electron Corp.)
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A) and preparing cytospin slides (unit 21.4)
CAUTION: Benzidine is carcinogenic. Use caution when handling; prepare the stock solutions in the chemical hood, wearing gloves and a mask. When staining cells, wear gloves. Dispose of waste properly.

Support Protocol 1: Immunofluorescence Staining of Hemoglobins

  Materials
  • Cultured cells (see protocol 2)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 4% paraformaldehyde in PBS, pH 7.4 (see appendix 2A for PBS)
  • 1:4 (v/v) methanol:acetone
  • Phycoerythrin (PE)–conjugated monoclonal antibody against human HbF (IQProducts; http://www.iqproducts.nl/)
  • Additional reagents and equipment for flow cytometry (Chapter 5)

Support Protocol 2: Preparation of Samples for HPLC Analysis of Hemoglobins

  Materials
  • Erythroid cells growing in culture (see protocol 2)
  • PBS ( appendix 2A)
  • Additional reagents and equipment for cation‐exchange HPLC (see protocol 5) and/or reversed‐phase HPLC (see protocol 6)

Support Protocol 3: Cation‐Exchange HPLC for Separation of Hemoglobins

  Materials
  • Potassium cyanide (KCN; Aldrich)
  • Bis‐Tris (Sigma)
  • Anhydrous sodium acetate (e.g., Sigma) or sodium acetate trihydrate (e.g., Mallinckrodt)
  • Glacial acetic acid
  • Synchropak CM300 column (Eichrom Technologies, Inc.)
  • HPLC apparatus (see, e.g., unit 9.2)

Support Protocol 4: Reversed‐Phase HPLC for Analysis of Globin Chains

  Materials
  • Acetonitrile (Sigma)
  • Trifluoroacetic acid (TFA; Aldrich)
  • 250 mm × 4.0–mm large‐pore Vydac C4 column
  • HPLC apparatus (see, e.g., unit 9.2)

Support Protocol 5: Lysing RBC by Osmotic Shock

  Materials
  • Interphase layer from Ficoll separation (see protocol 1)
  • PBS ( appendix 2A)
  • ACK lysing solution (unit 3.1), freshly prepared
  • 50‐ml centrifuge tubes
  • Tabletop centrifuge

Support Protocol 6: Removing RBC by Percoll Separation

  Materials
  • 10× PBS (see appendix 2A for 1×)
  • Percoll (Sigma)
  • Phase II erythroid cell culture (see protocol 2)
  • 50‐ml centrifuge tubes
  • Tabletop centrifuge
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Figures

Videos

Literature Cited

Literature Cited
   Amoyal, I., Goldfarb, A., and Fibach, E. 2003. Flow cytometric analysis of hydroxyurea effects on fetal hemoglobin production in cultures of beta‐thalassemia erythroid precursors. Hemoglobin 27:77‐87.
   Clarke, B.J. and Housman, D. 1977. Characterization of an erythroid precursor cell of high proliferative capacity in normal human peripheral blood. Proc. Natl. Acad. Sci. U.S.A 74:1105‐1109.
   Fibach, E. 2001. Cell culture and animal models to screen for promising fetal hemoglobin–stimulating compounds. Semin. Hematol. 38:374‐381.
   Fibach, E., Manor, D., Oppenheim, A., and Rachmilewitz, E.A. 1989. Proliferation and maturation of human erythroid progenitors in liquid culture. Blood 73:100‐103.
   Fibach, E., Bianchi, N., Borgatti, M., Prus, E., and Gambari, R. 2003. Mithramycin induces fetal hemoglobin production in normal and thalassemic human erythroid precursor cells. Blood 102:1276‐1281.
   Huisman, T.H. 1987. Separation of hemoglobins and hemoglobin chains by high‐performance liquid chromatography. J. Chromatogr. 418:277‐304.
   Myers, C.D., Katz, F.E., Joshi, G., and Millar, J.L. 1984. A cell line secreting stimulating factors for CFU‐GEMM culture. Blood 64:152‐155.
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