Generation of Cultured Oligodendrocyte Progenitor Cells from Rat Neonatal Brains

Eric C. Larsen1, Yoichi Kondo1, Cale D. Fahrenholtz1, Ian D. Duncan1

1 Department of Medical Sciences, University of Wisconsin‐Madison, Madison, Wisconsin
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.1
DOI:  10.1002/9780470151808.sc02d01s6
Online Posting Date:  August, 2008
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Abstract

The oligodendrocyte progenitor cell (OPC) is one of the most studied progenitor cells of the body. It has been extensively researched in tissue culture and more recently in vivo using a wide range of markers that recognize transcription factors and cell surface markers and identify its earliest development from neural stem cells onward. Isolation of OPCs in large numbers and in purified preparations has been sought after as a source of cells for the repair of human myelin disorders. It has been proposed that such cells could be used as an exogenous source of cells for the treatment of lesions in multiple sclerosis and the less common genetic myelin disorders such as Pelizaeus‐Merzbacher disease. Prior to clinical trials, such approaches can be tested in animal models. Here, we describe the isolation of rat OPCs in culture conditions that provide large numbers of purified populations of cells. Curr. Protoc. Stem Cell Biol. 6:2D.1.1‐2D.1.13. © 2008 by John Wiley & Sons, Inc.

Keywords: oligodendrocytes; oligodendrocyte progenitor cells; oligospheres; neurospheres; differentiation; transplantation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of OPC Cultures from Rat Neonates
  • Alternate Protocol 1: Generation of Oligospheres from Neural Precursor Cells
  • Support Protocol 1: Production of B104‐Conditioned Medium
  • Support Protocol 2: In Vitro Differentiation of Cultured OPCs
  • Support Protocol 3: Transplantation of Cultured OPCs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of OPC Cultures from Rat Neonates

  Materials
  • Rat neonates (post‐natal day 0 to 6)
  • Isoflurane (Abbot, cat. no. 5260‐04‐05)
  • 70% ethanol
  • Hank's Balanced Salt Solution (HBSS), without Ca2+ and Mg2+ (Invitrogen, cat. no. 14175)
  • Oligosphere medium (see recipe)
  • Sphere freezing medium: 80% DMEM/10% FBS/10% DMSO
  • Liquid nitrogen
  • Small, airtight container
  • Towel
  • Class II biological hazard flow hood or laminar‐flow hood
  • Surgical scissors, sterile (Fine Science Tools, cat. no. 14090‐11)
  • Forceps, sterile (no. 5 and no. 55; Fine Science Tools, cat. nos. 11252‐20 and 11255‐20)
  • Rat tooth forceps, sterile (Fine Science Tools, cat. no. 11022‐14)
  • 60‐mm and 100‐mm tissue culture dishes, sterile (Nunc, cat. nos. 150288 and 172958)
  • Dissecting microscope
  • Curved spring scissors, sterile (Fine Science Tools, cat. no. 15023‐10)
  • 15‐ml tube, sterile (Fisher, cat. no. 05‐539‐5)
  • Fire‐polished Pasteur pipets (Fisher, cat. no. 13‐678‐20D)
  • 25‐cm2 flasks, sterile (Nunc, cat. no. 136196)
  • Humidified incubator set at 37°C and 5% CO 2
  • Cryovial (Nunc, cat. no. 375418)
  • Freezing container (Nunc, cat. no. 5100‐0001)
  • −80°C freezer
  • 37°C water bath

Alternate Protocol 1: Generation of Oligospheres from Neural Precursor Cells

  • Neurosphere medium (see recipe)

Support Protocol 1: Production of B104‐Conditioned Medium

  Materials
  • B104 neuroblastoma cells (generously provided by Dr. M. Dubois‐Dalcq)
  • B104 feeding medium (see recipe)
  • Trypsin/EDTA (Invitrogen, cat. no. 25200)
  • Trypan blue (Invitrogen, cat. no. 15250‐061)
  • B104 collection medium (see recipe)
  • 37°C water bath
  • Sterile 15‐ml and 50‐ml tubes (Fisher, cat. nos. 05‐539‐5 and 05‐539‐8, respectively)
  • Sterile 75‐cm2 (TPP, cat. no. 90076) and 175‐cm2 (Corning, cat. no. 431080) culture flasks
  • Hemacytometer (Hausser Scientific, cat. no. 1490)
  • 0.22‐µm filter (Millipore, cat. no. SCGPU05RE)
  • Additional reagents and equipment for performing a viable cell count using a hemacytometer and trypan blue (unit 1.3)

Support Protocol 2: In Vitro Differentiation of Cultured OPCs

  Materials
  • Oligospheres (from the protocol 1 or protocol 2)
  • Hank's Balanced Salt Solution (HBSS), without Ca2+ and Mg2+
  • 2 mg/ml bovine serum albumin (BSA; Sigma, cat. no. A‐7906) in HBSS (Invitrogen, cat. no. 14175)
  • Oligosphere differentiation medium (see recipe)
  • Trypan blue (Invitrogen, cat. no. 15250‐061)
  • 15‐ml tubes, sterile
  • Fire‐polished Pasteur pipets
  • Hemacytometer (Hausser Scientific, cat. cat. no. 1490)
  • Additional reagents and equipment for performing a viable cell count using a hemacytometer and trypan blue (unit 1.3)

Support Protocol 3: Transplantation of Cultured OPCs

  • Crushed ice
  • 0.5‐ml microcentrifuge tube
  • Gauze
  • Pulled glass micropipets (see recipe)
  • Programmable syringe pump (Kent Scientific, cat. no. GENIE)
  • Heating pad
  • Isoflurane anesthesia system (including vaporizer and O 2 cylinders)
  • Stereotaxic frame (Stoelting, cat. no. 51600)
  • Spring scissors (Fine Scientific Tools, cat. no. 15023‐10)
  • Bone‐cutting spring scissors (Fine Scientific Tools, cat. no. 16144‐13)
  • High‐speed microdrill (Fine Scientific Tools, cat. no. 18000‐17)
  • 0.5‐mm diameter steel burr (Fine Scientific Tools, cat. no. 19007‐05)
  • Surgical spade
  • 31‐G insulin syringe (BD, cat. no. 328468; bend the needle tip with a needle holder such that the needle has an angle of ∼90‐120°)
  • Micromanipulator
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Figures

Videos

Literature Cited

Literature Cited
   Asakura, K., Hunter, S.F., and Rodriguez, M. 1997. Effects of transforming growth factor β and platelet‐derived growth factor on oligodendrocyte precursors: Insights gained from a neuronal cell line. J. Neurochem. 68:2281‐2290.
   Avellana‐Adalid., V., Nait‐Oumesmar, B., Lachapelle, F., and Baron‐Van Evercooren, A. 1996. Expansion of rat oligodendrocyte progenitors into proliferative “oligospheres” that retain differentiation potential. J. Neurosci. Res. 45:558‐570.
   Bradl, M., Bauer, J., Inomata, T., Zielasek, J., Nave, K.‐A., Toyka, K., Lassmann, H., and Wekerle, H. 1999. Transgenic Lewis rats overexpressing the proteolipid protein gene: Myelin degeneration and its effect on T cell‐mediated experimental autoimmune encephalomyelitis. Acta Neuropathol. 97:595‐606.
   Csiza, C.K. and de Lahunta, A. 1979. Myelin deficiency (md): A neurologic mutant in the Wistar rat. Am. J. Pathol. 95:215‐223.
   Delaney, K.H., Kwiecien, J.M., Wegiel, J., Wisniewski, H.M., Percy, D.H., and Fletch, A.L. 1995. Familial dysmyelination in a Long Evans rat mutant. Lab. Anim. Sci. 45:547‐553.
   Duncan, I.D. 2005. Oligodendrocytes and stem cell transplantation: Their potential in the treatment of leukoencephalopathies. J. Inherit. Metab. Dis. 28:357‐368.
   Duncan, I.D. 2008. Replacing cells in multiple sclerosis. J. Neurol. Sci. 265:89‐92.
   Gold, R., Linington, C., and Lassmann, H. 2006. Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research. Brain 129:1953‐1971.
   Learish, R.D., Brustle, O., Zhang, S.‐C., and Duncan, I.D. 1999. Intraventricular transplantation of oligodendrocyte progenitors into a fetal myelin mutant results in widespread formation of myelin. Ann. Neurol. 46:716‐722.
   Tontsch, U., Archer, D.R., Dubois‐Dalcq, M., and Duncan, I.D. 1994. Transplantation of an oligodendrocyte cell line leading to extensive myelination. Proc. Natl. Acad. Sci. U.S.A. 91:11616‐11620.
   Utzschneider, D.A., Archer, D.R., Kocsis, J.D., Waxman, S.G., and Duncan, I.D. 1994. Transplantation of glial cells enhances action potential conduction of amyelinated spinal cord axons in the myelin‐deficient rat. Proc. Natl. Acad. Sci. U.S.A. 91:53‐57.
   Zhang, S.‐C., Lundberg, C., Lipsitz, D., O'Connor, L.T., and Duncan, I.D. 1998a. Generation of oligodendroglial progenitors from neural stem cells. J. Neurocytol. 27:475‐489.
   Zhang, S.‐C., Lipsitx, D., and Duncan, I.D. 1998b. Self‐renewing canine oligodendroglial progenitors expanded as oligospheres. J. Neurosci. Res. 54:181‐190.
   Zhang, S.‐C., Goetz, B.D., and Duncan, I.D. 2003. Suppression of activated microglia promotes survival and function of transplanted oligodendroglial progenitors. Glia 41:191‐198.
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