In Vitro Differentiation and Measurement of B Cell Progenitor Activity in Culture

Taku Kouro1, Takafumi Yokota1, Robert Welner1, Paul W. Kincade1

1 Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.2
DOI:  10.1002/0471142735.im22f02s66
Online Posting Date:  May, 2005
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Abstract

As well as other blood cells, B lymphocytes originate from hematopoietic stem cells. However, it is not fully understood how their production is controlled. In the serum‐free, stromal‐cell‐free B cell differentiation culture described here, early steps of the B lineage differentiation process are reproduced under defined conditions. This assay is useful for examining the direct effects of various soluble factors on B cell progenitors because it does not contain stromal cells or unknown factors. Additionally, this assay yields sufficient cloning to measure B cell progenitors from single cell cultures. Stromal cell coculture assays are also described that cover a wider category of precursors such as human B cell progenitors.

Keywords: B lymphocyte; progenitors; differentiation; culture; stromal cell; cytokine

     
 
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Table of Contents

  • Basic Protocol 1: Serum‐Free, Stromal Cell‐Free B Cell Differentiation Culture
  • Support Protocol 1: Titration of Cytokine Concentration used in the B Cell Differentiation Cultures
  • Basic Protocol 2: Stromal Cell Cocultures for Murine B Cell Differentiation
  • Basic Protocol 3: Single‐Cell Differentiation Assay of B Cell Progenitors
  • Basic Protocol 4: Stromal Cell Coculture for Human Lymphocyte Differentiation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Serum‐Free, Stromal Cell‐Free B Cell Differentiation Culture

  Materials
  • Recombinant mouse cytokines: stem cell factor, Flt3 ligand, and IL‐7 (R & D Systems)
  • Complete X‐VIVO15 serum‐free culture medium (see recipe)
  • Aseptically prepared lineage‐negative lymphoid progenitors (see unit 22.1)
  • Staining/wash buffer (see recipe)
  • 2.4G2 hybridoma culture supernatant (ATCC no. HB‐197) or purified anti‐mouse CD16/CD32 (FcγIII/II receptor monoclonal antibody (BD Pharmingen)
  • Labeled antibodies (BD Pharmingen): FITC‐conjugated anti‐CD11b/Mac‐1 MAb, PE‐conjugated anti‐CD19 MAb
  • 24‐well culture plate
  • 5‐ml round‐bottom tubes
  • Refrigerated centrifuge with microtiter plate carrier
  • 96‐well round‐bottom microtiter plates
  • Flow cytometer: e.g., FACScan or FACSCalibur (Becton Dickinson)
  • Additional reagents and equipment for cell counting ( appendix 3B) and flow cytometry (Chapter 5)

Support Protocol 1: Titration of Cytokine Concentration used in the B Cell Differentiation Cultures

  • Mouse bone marrow cells depleted of lineage marker–positive cells by MACS (Lin cells; see unit 22.1, Basic Protocol, steps 1 to 14)

Basic Protocol 2: Stromal Cell Cocultures for Murine B Cell Differentiation

  Materials
  • MS‐5 cells (available from Dr. Kazuhiro J. Mori, ) or OP‐9 cells (available from Riken Cell Bank, http://www.brc.riken.jp/lab/cell/english; e‐mail, )
  • Lineage‐negative lymphohematopoietic progenitors from adult mouse bone marrow (unit 22.1, Basic Protocol 1) or from fetal mouse liver (unit 22.1, Basic Protocol 2)
  • MS‐5 growth medium (see recipe) or OP‐9 growth medium (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Trypsin/EDTA solution (e.g., Cellgro cat. no. 25‐052‐CV, available from Mediatech)
  • Recombinant mouse cytokines: stem cell factor, Flt3 ligand, and IL‐7 (R & D Systems)
  • Hanks' buffered salt solution (HBSS; appendix 2A) containing 0.53 mM EDTA
  • Staining/wash buffer (see recipe)
  • Labeled antibodies: FITC‐conjugated anti‐CD11b(Mac‐1) MAb (BD Pharmingen), PE‐conjugated anti‐CD19 MAb (BD Pharmingen), biotinylated anti‐VCAM‐1 antibody (clone 429 MVCAM.A; BD PharMingen cat no. 01812D)
  • Streptavidin‐conjugated PE‐Texas Red (Caltag cat. no. SA1017) or APC (BD PharMingen cat. no. 13049A)
  • 10‐cm culture dishes (e.g., Corning Inc. cat. no. 430167)
  • 24‐well (e.g., Becton Dickinson cat. no. 353047) or 6‐well culture plate (e.g., Becton Dickinson cat. no. 353046)
  • 15‐ml tubes
  • 96‐well round‐bottom microtiter plates
  • Refrigerated centrifuge with microtiter plate carrier
  • Flow cytometer: e.g., FACScan or FACSCalibur (Becton Dickinson)
  • Additional materials and equipment for cell counting ( appendix 3B) and flow cytometry (Chapter 5)

Basic Protocol 3: Single‐Cell Differentiation Assay of B Cell Progenitors

  Materials
  • Complete X‐VIVO15 serum‐free culture medium (see recipe)
  • Recombinant mouse cytokines: stem cell factor, Flt3 ligand, and IL‐7 (R & D Systems)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fluorescent MAb–labeled cell preparation containing lymphoid progenitors (see unit 22.1)
  • Staining/wash buffer (see recipe)
  • 96‐well round‐bottom culture plates (sterile culture‐treated plates, e.g., Nunc cat. no. 163320)
  • Fluorescence‐activated cell sorter with single‐cell deposition ability (e.g., MoFlo from Dako Cytomation)
  • Inverted microscope
  • 96‐well round‐bottom microtiter plates (for cell staining; nonsterile ELISA plates, e.g., Greiner, cat. no. 650061)
  • Refrigerated centrifuge with microtiter plate carrier
  • Additional reagents and equipment for preparing lymphoid progenitors (unit 22.1), flow cytometry and cell sorting (Chapter 5), and staining and flow cytometric analysis of progenitor cells (see protocol 1, steps to )

Basic Protocol 4: Stromal Cell Coculture for Human Lymphocyte Differentiation

  Materials
  • MS‐5 cells (see protocol 3)
  • MS‐5 growth medium (see recipereecipe)
  • Aseptically prepared human lymphoid progenitors (see unit 22.3, Basic Protocol 3)
  • Recombinant human cytokines (R & D Systems): stem cell factor, G‐CSF, Flt3 ligand (optional), and IL‐15 (optional)
  • Dup‐697 (Cayman Chemical; optional)
  • Labeled antibodies (BD Pharmingen and Caltag): FITC‐labeled anti–human CD64 or CD33; PE‐ (or APC)–labeled anti–human CD19; APC‐ (or PE)–labeled anti–human CD56; biotinylated anti‐VCAM‐1 antibody (clone 429 MVCAM.A, BD PharMingen cat. no. 01812D)
  • 24‐well tissue culture plates or 25‐cm2 cell culture flasks
  • Additional reagents and equipment for stromal cell coculture (see protocol 3)
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Figures

Videos

Literature Cited

Literature Cited
   Ball, T.C., Hirayama, F., and Ogawa, M. 1995. Lymphohematopoietic progenitors of normal mice. Blood 85:3086–3092.
   Borge, O.J., Adolfsson, J., and Jacobsen, A.M. 1999. Lymphoid‐restricted development from multipotent candidate murine stem cells: Distinct and complimentary functions of the c‐kit and flt3‐ ligands. Blood 94:3781–3790.
   Hayashi, S., Kunisada, T., Ogawa, M., Sudo, T., Kodama, H., Suda, T., and Nishikawa, S. 1990. Stepwise progression of B lineage differentiation supported by interleukin 7 and other stromal cell molecules. J. Exp. Med. 171:1683–1695.
   Igarashi, H., Kouro, T., Yokota, T., Comp, P.C., and Kincade, P.W. 2001. Age and stage dependency of estrogen receptor expression by lymphocyte precursors. Proc. Natl. Acad. Sci. U.S.A. 98:15131–15136.
   Medina, K.L. and Kincade, P.W. 1994. Pregnancy‐related steroids are potential negative regulators of B lymphopoiesis. Proc. Natl. Acad. Sci. U.S.A. 91:5382–5386.
   Montecino‐Rodriguez, E., Leathers, H., and Dorshkind, K. 2001. Bipotential B‐macrophage progenitors are present in adult bone marrow. Nat. Immunol. 2:83–88.
   Nishihara, M., Wada, Y., Ogami, K., Ebihara, Y., Ishii, T., Tsuji, K., Ueno, H., Asano, S., Nakahata, T., and Maekawa, T. 1998. A combination of stem cell factor and granulocyte colony‐stimulating factor enhances the growth of human progenitor B cells supported by murine stromal cell line MS‐5. Eur. J. Immunol. 28:855–864.
   Nishikawa, S., Ogawa, M., Kunisada, T., and Kodama, H. 1988. B lymphopoiesis on stromal cell clone: Stromal cell clones acting on different stages of B cell differentiation. Eur. J. Immunol. 18:1767–1771.
   Prieyl, J.A. and LeBien, T.W. 1996. Interleukin 7 independent development of human B cells. Proc. Natl. Acad. Sci. U.S.A. 93:10348–10353.
   Rawlings, D.J., Quan, S.G., Kato, R.M., and Witte, O.N. 1995. Long‐term culture system for selective growth of human B‐cell progenitors. Proc. Natl. Acad. Sci. U.S.A. 92:1570–1574.
   Rossi, M.I., Yokota, T., Medina, K.L., Garrett, K.P., Comp, P.C., Schipul, A.H. Jr., and Kincade, P.W. 2003. B lymphopoiesis is active throughout human life, but there are developmental age‐related changes. Blood 101:576–584.
   Whitlock, C.A. and Witte, O.N. 1982. Long‐term culture of B lymphocytes and their precursors from murine bone marrow. Proc. Natl. Acad. Sci. U.S.A. 79:3608–3612.
   Yokota, T., Meka, C.S., Medina, K.L., Igarashi, H., Comp, P.C., Takahashi, M., Nishida, M., Oritani, K., Miyagawa, J., Funahashi, T., Tomiyama, Y., Matsuzawa, Y., and Kincade, P.W. 2002. Paracrine regulation of fat cell formation in bone marrow cultures via adiponectin and prostaglandins. J. Clin. Invest. 109:1303–1310.
   Yokota, T., Meka, C.S., Kouro, T., Medina, K.L., Igarashi, H., Takahashi, M., Oritani, K., Funahashi, T., Tomiyama, Y., Matsuzawa, Y., and Kincade, P.W. 2003. Adiponectin, a fat cell product, influences the earliest lymphocyte precursors in bone marrow cultures by activation of the cyclooxygenase‐prostaglandin pathway in stromal cells. J. Immunol. 171:5091–5099.
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