Differentiation and Characterization of Myeloid Cells

Dipti Gupta1, Hetavi Parag Shah1, Krishnakumar Malu1, Nancy Berliner2, Peter Gaines1

1 Department of Biological Sciences, University of Massachusetts Lowell, Lowell, Massachusetts, 2 Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.5
DOI:  10.1002/0471142735.im22f05s104
Online Posting Date:  February, 2014
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Abstract

Ex vivo differentiation of myeloid cells begins with an enriched population of bone marrow–derived hematopoietic stem cells generated by lineage depletion and/or positive selection for CD34+ antigen (human) or Sca‐1+ (mouse) cells, which are then expanded and subsequently induced in vitro in a process that recapitulates normal myeloid development. Myeloid cell lines include two human leukemic cell lines, NB‐4 and HL‐60, which have been demonstrated to undergo retinoic acid–induced myeloid development; however, both cell lines exhibit defects in the up‐regulation of late‐expressed neutrophil‐specific genes. Multiple murine factor–dependent cell models of myelopoiesis are also available that express the full range of neutrophil maturation markers, including: 32Dcl3 cells, which undergo G‐CSF‐induced myeloid maturation; EML/EPRO cells, which develop into mature neutrophils in response to cytokines and retinoic acid; and ER‐Hoxb8 cells, which undergo myeloid maturation upon removal of estradiol in the maintenance medium. In this unit, the induction of myeloid maturation in each of these model systems is described, including their differentiation to either neutrophils or macrophages, if applicable. Commonly used techniques to test for myeloid characteristics of developing cells are also described, including flow cytometry and real time RT‐PCR. Curr. Protoc. Immunol. 104:22F.5.1‐22F.5.28. © 2014 by John Wiley & Sons, Inc.

Keywords: stem cell progenitors; promyelocytes; myeloid induction; neutrophil characteristics; macrophages; cell‐surface markers; granule protein gene expression

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Neutrophil Differentiation of Human CD34+ Progenitors
  • Basic Protocol 2: Neutrophil Differentiation of Lineage‐Depleted Mouse Progenitors
  • Alternate Protocol 1: Monocyte/Macrophage Differentiation of Lineage‐Depleted Mouse Progenitors
  • Alternate Protocol 2: CFU‐GM Colony Formation
  • Basic Protocol 3: Neutrophil Differentiation of Human Leukemic Cell Lines
  • Alternate Protocol 3: Monocyte/Macrophage Differentiation of Human Leukemic Cell Lines
  • Basic Protocol 4: Differentiation of 32Dcl3 Cells to Myeloid Cells
  • Basic Protocol 5: Myeloid Differentiation of Multipotent EML Cells
  • Basic Protocol 6: Myeloid Differentiation of ER‐Hoxb8 Progenitors
  • Support Protocol 1: Preparation of WEHI‐3B Conditioned Medium
  • Support Protocol 2: Analysis of Cell Surface Myeloid Markers
  • Support Protocol 3: Analysis of Neutrophil‐Specific Granule Protein Gene Expression
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Neutrophil Differentiation of Human CD34+ Progenitors

  Materials
  • Purified hematopoietic stem cell progenitors (for human cells, CD34+ progenitors isolated from bone marrow or peripheral blood; unit 7.4)
  • Complete IMDM medium: IMDM (e.g., Life Technologies) supplemented with 10% heat‐denatured fetal bovine serum (FBS; appendix 2A), 2 mM L‐glutamine, 5 U/ml penicillin G, and 5 µg/ml streptomycin sulfate
  • Recombinant cytokines: SCF, IL‐3, and G‐CSF (all available from Peprotech)
  • 15‐ml conical tubes (e.g., BD Falcon)
  • Tabletop centrifuge
  • 6‐well tissue culture plates
  • Additional reagents and equipment for counting cells ( appendix 3A), trypan blue exclusion test for cell viability ( appendix 3B), preparation of cytospin smears (unit 21.4), and Wright‐Giemsa staining ( appendix 3C)

Basic Protocol 2: Neutrophil Differentiation of Lineage‐Depleted Mouse Progenitors

  Materials
  • IMDM supplemented with 2% heat‐inactivated fetal bovine serum (FBS; appendix 2A) or 10% heat‐inactivated horse serum (HS; appendix 2A), 2 mM L‐glutamine, 5 U/ml penicillin G, and 5 µg/ml streptomycin sulfate
  • RPMI 1640 medium (e.g., Invitrogen), ice cold
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Cell staining buffer: PBS ( appendix 2A) containing 3% FBS and 0.1% sodium azide
  • Anti‐mouse CD16/CD32 monoclonal antibody (e.g. Mouse Fc Block, BD Pharmingen, cat. no. 553141)
  • Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set (BD Biosciences, cat. no. 558451)
  • Recombinant cytokines: SCF, IL‐3, and G‐CSF (Peprotech, resuspended from lyophilized material in PBS plus 0.1% FBS to a final concentration of 50 µg/ml)
  • Mouse BD Fc Block purified anti‐mouse CD16/CD32 (BD Biosciences, cat. no. 553141)
  • Dissection supplies for cleaning bones from hind legs (sterile scalpels and forceps)
  • Sterile gauze pads
  • 23‐G needles and 3‐ or 5‐ml syringes (3 or 5 ml)
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • 70‐µm nylon cell strainer (BD Falcon)
  • Tabletop centrifuge
  • Sterile “snap‐cap” 12 × 75–mm round‐bottom polypropylene tubes (BD Falcon, cat. no. 35263, or equivalent tubes)
  • Cell separation magnet (BD IMagnet, Becton Dickinson, cat. no. 552311)
  • 6‐well tissue culture plates
  • Additional reagents and equipment for euthanasia (unit 1.8), counting cells using a hemacytometer ( appendix 3A), and trypan blue exclusion test for cell viability ( appendix 3B)

Alternate Protocol 1: Monocyte/Macrophage Differentiation of Lineage‐Depleted Mouse Progenitors

  Additional Materials (also see protocol 2)
  • M‐CSF (Peprotech), resuspended from lyophilized material in PBS ( appendix 2A) containing 0.1% (v/v) FBS to a final concentration of 50 µg/ml.

Alternate Protocol 2: CFU‐GM Colony Formation

  Additional Materials (also see protocol 1)
  • Cytokine‐free base methylcellulose medium (with fetal bovine serum; available from Stem Cell Technologies; MethoCult H4230 for human or M3231 for mouse)
  • IMDM (serum free)
  • Purified CD34+ cells (unit 7.4)
  • Complete IMDM: IMDM supplemented with heat‐denatured 2% FBS ( appendix 2A), 2 mM L‐glutamine, 5 U/ml penicillin G, and 5 µg/ml streptomycin sulfate
  • Cytokines (depending on the conditions required; human or mouse recombinant SCF, IL‐3, G‐CSF, and GM‐CSF can be purchased from Peprotech; human or mouse EPO can be purchased from BioLegend)
  • Sterile round‐bottom snap‐cap polypropylene tubes (12 × 75 mm; BD Falcon, cat. no. 352063, or equivalent tubes)
  • 3‐, 5‐ and 10‐ml luer‐lock sterile syringes
  • Blunt‐end 16‐G needles (Stem Cell Technologies, cat. no. 28110/28120)
  • 6‐well tissue culture plates or 35‐mm tissue culture dishes
  • 16‐G needles
  • Additional reagents and equipment for NBT staining assay (unit 7.23)

Basic Protocol 3: Neutrophil Differentiation of Human Leukemic Cell Lines

  Materials
  • HL‐60 (ATCC) or NB‐4 cells (can be obtained by contacting Dr. Michel Lanotte, mlanotte@chu‐stlouis.fr)
  • Complete RPMI medium: RPMI‐1640 medium supplemented with 10% heat‐inactivated FBS, 2 mM L‐glutamine, 5 U/ml penicillin G, and 5 µg/ml streptomycin sulfate
  • Dimethylsulfoxide (DMSO)
  • 10 mM ATRA stock solution (see recipe)
  • Additional reagents and equipment for preparation of cytospin smears (unit 21.4) and Wright‐Giemsa staining ( appendix 3C)

Alternate Protocol 3: Monocyte/Macrophage Differentiation of Human Leukemic Cell Lines

  Additional Materials (also see protocol 5)
  • Phorbol 12‐myristate 13‐acetate (PMA; Sigma‐Aldrich, cat. no. P1585, resuspended in dimethylsulfoxide at 1 mg/ml stock concentration)
  • 1α,25(OH) 2D 3 (Sigma‐Aldrich, cat. no. D1530; resuspend in ethanol at 10 µm stock concentration)

Basic Protocol 4: Differentiation of 32Dcl3 Cells to Myeloid Cells

  Materials
  • Myeloblastic 32Dcl3 cells
  • IMDM supplemented with 10% FBS, 10% WEHI‐conditioned medium (see protocol 10), 2 mM L‐glutamine, 5 U/ml penicillin G, and 5 µg/ml streptomycin sulfate
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • G‐CSF (Peprotech)
  • Additional reagents and equipment for preparation of cytospin smears (unit 21.4) and Wright‐Giemsa staining ( appendix 3C)

Basic Protocol 5: Myeloid Differentiation of Multipotent EML Cells

  Materials
  • EML cells (ATCC no. CRL‐11691)
  • IMDM supplemented with 20% heat‐inactivated horse serum, 15% BHK/MKL‐conditioned medium (see protocol 10), 2 mM L‐glutamine, 5 U/ml penicillin G, and 5 µg/ml streptomycin sulfate
  • IMDM supplemented with 20% heat‐inactivated horse serum, 10% BHK/HM5‐conditioned medium (see protocol 10) or 10 ng/ml mouse GM‐CSF (Peprotech), 2 mM L‐glutamine, 5 U/ml penicillin G, and 5 µg/ml streptomycin sulfate
  • WEHI‐conditioned medium (see protocol 10) or mouse IL‐3 (Peprotech, prepared with DPBS (without Ca or Mg) plus 0.1% FBS at 50 µg/ml for stock solution)
  • 10 mM ATRA stock solution (see recipe)
  • Dulbecco's phosphate buffered saline (DPBS) without Ca or Mg (Thermo Fisher Scientific, cat. no. SH30028)
  • 6‐well tissue culture plates
  • 75‐cm2 tissue culture flasks
  • Additional reagents and equipment for preparation of cytospin smears (unit 21.4) and Wright‐Giemsa staining ( appendix 3C)

Basic Protocol 6: Myeloid Differentiation of ER‐Hoxb8 Progenitors

  Materials
  • SCF ER‐Hoxb8 and GM‐CSF ER‐Hoxb8 cell lines (obtained directly from M. Kamps, UCSD School of Medicine, La Jolla, California)
  • Opti‐MEM medium (Invitrogen) supplemented with 10% heat‐inactivated certified FBS, 2 mM L‐glutamine, 5 U/ml penicillin G, and 5 µg/ml streptomycin sulfate
  • 50 µg/ml stem cell factor stock solution (SCF; Peprotech; prepared in PBS containing 0.1% FBS)
  • 10 mM β‐estradiol stock solution (Sigma, cat no. E‐2758; dissolve in 100% ethanol with shaking for 30 min at room temperature, stored at −20°C)
  • RPMI‐1640 medium supplemented with 10% heat‐inactivated certified FBS, 2 mM L‐glutamine, 5 U/ml penicillin G, and 5 µg/ml streptomycin sulfate
  • 50 µg/ml Granulocyte Macrophage Colony Stimulating Factor stock solution (GM‐CSF; Peprotech; prepared in PBS containing 0.1% FBS)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 6‐well tissue culture dishes
  • 15‐ml conical tubes (e.g., BD Falcon)
  • Additional reagents and equipment for preparation of cytospin smears (unit 21.4) and Wright‐Giemsa staining ( appendix 3C)

Support Protocol 1: Preparation of WEHI‐3B Conditioned Medium

  Materials
  • Cells of interest (e.g., WEHI‐3B, BHK/MKL, BHK/HM‐5) and corresponding growth medium supplemented with 2 mM L‐glutamine, 5 U/ml penicillin G, and 5 µg/ml streptomycin sulfate
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1× Trypsin‐EDTA
  • 15‐ml conical centrifuge tubes
  • 75‐ and 150‐cm2 tissue culture flasks
  • 0.45‐µm filters

Support Protocol 2: Analysis of Cell Surface Myeloid Markers

  Materials
  • Cell samples: undifferentiated versus differentiated cells, 1 × 106 cells/sample
  • Cell labeling buffer (see recipe), ice cold
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • Fc receptor blocking antibody (anti‐CD16/CD32; for mouse cells bearing FcγII and FcγIII receptors; e.g., Fc block, Pharmingen)
  • Fluorochrome‐labeled antibody to Gr‐1, Mac‐1 or F4/80 (Pharmingen or Santa Cruz)
  • 12 × 75–mm flow cytometry tubes (e.g., Falcon)
  • Microcentrifuge
  • Additional reagents and equipment for trypan blue exclusion ( appendix 3B)

Support Protocol 3: Analysis of Neutrophil‐Specific Granule Protein Gene Expression

  Materials
  • 1 × 107 cells/ml cell samples
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • TRI reagent (Molecular Research Center, http://www.mrcgene.com/; also available from Invitrogen as TRIzol)
  • Chloroform
  • Isopropyl alcohol
  • 80% ethanol (prepared with DEPC‐treated water)
  • DEPC‐treated water ( appendix 2A)
  • SuperScript III First‐Strand Synthesis System (Invitrogen, cat. no. 18080‐051)
  • RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen, cat. no. 10777‐019, also supplied in above mentioned kit)
  • 2 U/µl E. coli RNase H (Invitrogen, cat. no. 18021‐014, also supplied in abovementioned kit)
  • SsoAdvanced SYBR Green Supermix (BioRad, cat. no. 172‐5261)
  • 2.5 pmol/µl Forward Primer
  • 2.5 pmol/µl Reverse Primer
  • 1.5‐ml RNase‐free microcentrifuge tubes
  • 70°C heating block
  • Spectrophotometer (e.g., Nanodrop from Thermo Scientific)
  • 0.5‐ml microcentrifuge tubes (e.g., VWR, cat. no. 89000‐010), autoclaved
  • 65°C, 50°C, 70°C and 37°C water baths
  • iCycler with MyiQ single color real‐Time PCR detection system (BioRad)
  • 96‐well PCR plates (e.g. Fisherbrand, cat no. 14‐230‐232)
  • Domed 8‐strip caps (e.g., BioRad, cat. no. TCS‐0801)
  • Additional reagents and equipment for spectrophotometeric determination of nucleic acid concentration ( appendix 3L)
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Figures

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Literature Cited

Literature Cited
  Berliner, N., Hsing, A., Graubert, T., Sigurdsson, F., Zain, M., Bruno, E., and Hoffman, R. 1995. Granulocyte colony‐stimulating factor induction of normal human bone marrow progenitors results in neutrophil‐specific gene expression. Blood 85:799‐803.
  Choi, K., Vodyanik, M., and Slukvin, I.I. 2012. The hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells. Nat. Protoc. 6:296‐313.
  Cowland, J.B. and Borregaard, N. 1999. The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules. J. Leukoc. Biol. 66:989‐995.
  Dalton, W.T. Jr, Ahearn, M.J., McCredie, K.B., Freireich, E.J., Stass, S.A., and Trujillo, J.M. 1988. HL‐60 cell line was derived from a patient with FAB‐M2 and not FAB‐M3. Blood 71:242‐247.
  Douer, D., Ramezani, L., Parker, J., and Levine, A.M. 2000. All‐trans‐retinoic acid effects the growth, differentiation and apoptosis of normal human myeloid progenitors derived from purified CD34+ bone marrow cells. Leukemia 14:874‐881.
  Graubert, T., Johnston, J., and Berliner, N. 1993. Cloning and expression of the cDNA encoding mouse neutrophil gelatinase: Demonstration of coordinate secondary granule protein gene expression during terminal neutrophil maturation. Blood 82:3192‐3197.
  Khanna‐Gupta, A., Kolibaba, K., Zibello, T.A., and Berliner, N. 1994. NB4 cells show bilineage potential and an aberrant pattern of neutrophil secondary granule protein gene expression. Blood 84:294‐302.
  Lanotte, M., Martin‐Thouvenin, V., Najman, S., Balerini, P., Valensi, F., and Berger, R. 1991. NB4, a maturation inducible cell line with the t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood 77:1080‐1086.
  Lawson, N.D., Khanna‐Gupta, A., and Berliner, N. 1998a. Isolation and characterization of the cDNA for mouse neutrophil collagenase: Demonstration of shared negative regulatory pathways for neutrophil secondary granule protein gene expression. Blood 91:2517‐2524.
  Lawson, N.D., Krause, D.S., and Berliner, N. 1998b. Normal neutrophil differentiation and secondary granule gene expression in the EML and MPRO cell lines. Exp. Hematol. 26:1178‐1185.
  Le Cabec, V., Calafat, J., and Borregaard, N. 1997. Sorting of the specific granule protein, NGAL, during granulocytic maturation of HL‐60 cells. Blood 89:2113‐2121.
  Lee, K., Chang, M., Ahn, J., Yu, D., Jung, S., Choi, J., Noh, Y., Lee, Y., and Ahn, M. 2002. Differential gene expression in retinoic acid‐induced differentiation of acute promyelocytic leukemia cells, NB4 and HL‐60 cells. Biochem. Biophys. Res. Commun. 296: 1125‐1133.
  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.
  Olins, A.L., Hoang, T.V., Zwerger, M., Herrmann, H., Zentgraf, H., Noegel, A.A., Karakesisoglou, I., Hodzic, D., and Olins, D.E. 2009. The LINC‐less granulocyte nucleus. Eur. J. Cell. Biol. 88:203‐214.
  Song, X. and Norman, A.W. 1998. 1,25‐dihydrozyvitamin D3 and phorbol ester mediate the expression of alkaline phosphatase in NB4 acute promyelocytic leukemia cells. Leuk. Res. 22:69‐76.
  Subramanian, G., Cahudhury P., Malu, K., Fowler, S., Manmode, R., Gotur, D., Zwerger, M., Ryan, D., Roberti, R., and Gaines, P. 2012. Lamin B receptor regulates the growth and maturation of myeloid progenitors via its sterol reductase domain: Implications for cholesterol biosynthesis in regulating myelopoeisis. J. Immunol. 188:85‐102.
  Tocci, A., Parolini, I., Gabbianelli, M., Testa, U., Luchetti, L., Samoggia, P., Masella, B., Russo, G., Valtieri, M., and Peschle, C. 1996. Dual action of retinoic acid on human embryonic/fetal hematopoiesis: Blockade of primitive progenitor proliferation and shift from multipotent/erythroid/monocytic to granulocytic differentiation program. Blood 88:2878‐2888.
  Tsai, S. and Collins, S.J. 1993. A dominant negative retinoic acid receptor blocks neutrophil differentiation at the promyelocyte stage. Proc. Natl. Acad. Sci. U.S.A. 90:7153‐7157.
  Tsai, S., Bartelmez, S., Sitnicka, E., and Collins, S. 1994. Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant‐negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development. Genes Dev. 8:2831‐2841.
  Wang, G.G., Calvo, K.R, Pasillas, M.P., Sykes, D.B., Häcker, H., and Kamps, M.P. 2006. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat. Methods. 3:287‐293.
  Xu, Y.Z., Thuraisingam, T., de Lima Morais, D.A., Rola‐Pleszczynski, M., and Radzioch, D. 2010. Nuclear translocation of β‐actin is involved in transcriptional regulation during macrophage differentiation of HL‐60 cells. Mol. Biol. Cell 21:811‐820.
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