Isolation and Culture of Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem Cells, and Retinal Progenitor Cells

Sara Gualdoni1, Michael Baron1, Jörn Lakowski1, Sarah Decembrini1, Rachel A. Pearson2, Robin R. Ali2, Jane C. Sowden1

1 Developmental Biology Unit, UCL Institute of Child Health, University College London, London, United Kingdom, 2 Department of Genetics, UCL Institute of Ophthalmology, University College London, London, United Kingdom
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1H.4
DOI:  10.1002/9780470151808.sc01h04s19
Online Posting Date:  December, 2011
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Abstract

The protocols described in this unit provide detailed information on how to isolate and expand, in culture, ciliary epithelial cells (CECs), previously identified as retinal stem cells, from the adult mouse eye, and embryonic retinal progenitor cells (RPCs) from the developing retina. CECs are initially cultured in floating conditions as neurospheres and then expanded in monolayer cultures. RPCs are cultured in floating conditions. Detailed protocols for retinal differentiation, as well as exogenous gene expression using lentivirus are also described. Curr. Protoc. Stem Cell Biol. 19:1H.4.1‐1H.4.15. © 2011 by John Wiley & Sons, Inc.

Keywords: ciliary epithelial cells (CECs); retinal stem cells; retinal progenitor cells (RPCs); monolayer cultures; neurospheres; retinal differentiation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Ciliary Epithelial Cells from Adult Mouse Eye and Neurosphere Culture
  • Basic Protocol 2: Expansion and Maintenance of Ciliary Epithelial Cells as Monolayer Culture
  • Support Protocol 1: Isolation and Culture of Embryonic Retinal Progenitor Cells
  • Basic Protocol 3: Retinal Differentiation
  • Basic Protocol 4: Lentiviral Infection of Mouse Ciliary Epithelial Cells for Transgene Overexpression
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Ciliary Epithelial Cells from Adult Mouse Eye and Neurosphere Culture

  Materials
  • Adult mouse eyes
  • Papain dissociation system kit (Worthington, cat. no. LK003150) composed of:
    • Earle's balanced salt solution (EBSS) with bicarbonate and phenol red
    • Papain containing L‐cysteine and EDTA to reconstitute to 20 U/ml in 1 mM L‐cysteine with 0.5 mM EDTA (see recipe)
    • DNase I to reconstitute to 2000 U/ml (see recipe)
    • Ovomucoid protease inhibitor with BSA to reconstitute to 10 mg/ml (see recipe)
  • Papain/DNase solution (see recipe)
  • DNase/ovomucoid inhibitor solution (see recipe)
  • Neurosphere‐forming medium (see recipe)
  • 70% ethanol
  • Dissecting tools including:
    • 7‐mm curved micro‐scissors
    • 23‐G × 1/4 in. (0.6 × 30 mm) needle
    • Sharp Watchmaker forceps (5 point)
  • 10‐cm culture dishes
  • Laminar‐flow hood with stereomicroscope
  • 1.5‐ml microcentrifuge tubes
  • Glass pipets
  • 1000‐µl pipet tips
  • Microcentrifuge
  • 12‐well culture plate
NOTE: For the components provided in the papain dissociation kit, after reconstitution, aliquots of each component are kept at −20°C and can be thawed once. Before each use, equilibrate each component with O 2:CO 2 (inside cell incubator), until pink in color. The EBSS contains a pH sensitive indicator dye.

Basic Protocol 2: Expansion and Maintenance of Ciliary Epithelial Cells as Monolayer Culture

  Materials
  • Neurospheres (see protocol 1)
  • Proliferation medium (see recipe)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen, cat. no. 14190‐094)
  • Accutase solution (Sigma‐Aldrich, cat. no. A 6964)
  • 15‐ml tubes (e.g., Falcon)
  • 1000‐µl pipets
  • Centrifuge
  • Poly‐L‐ornithine/fibronectin‐coated culture plates (six, 12‐well plates; see recipe)
  • Hemacytometer

Support Protocol 1: Isolation and Culture of Embryonic Retinal Progenitor Cells

  Materials
  • Mouse embryos at the appropriate age [embryonic day (E), 11.5‐12.5]
  • Earle's balanced salt solution (EBSS) with bicarbonate and phenol red
  • 0.1% Type I collagenase solution (see recipe)
  • Neurosphere‐forming medium (see recipe)
  • 70% ethanol
  • Dissecting tools:
    • Sharp forceps (5 point)
    • Blades, sterile
  • 10‐cm culture dishes
  • Laminar‐flow hood
  • 15‐ml tubes (e.g., Falcon)
  • 1000‐µl pipet tips
  • 70‐µm mesh strainer
  • 24‐well culture plate

Basic Protocol 3: Retinal Differentiation

  Materials
  • 3 to 5 weeks in vitro monolayer CECs ( protocol 2)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen, cat. no. 14190‐094)
  • Accutase solution (Sigma‐Aldrich, cat. no. A6964)
  • CECs neurospheres ( protocol 1)
  • RPCs neurospheres ( protocol 3)
  • Proliferation medium (see recipe)
  • Retinal differentiation‐step1 medium (see recipe)
  • Retinal differentiation‐step2 medium (see recipe)
  • Standard differentiation medium (see recipe)
  • 15‐ml tubes (e.g., Falcon)
  • Centrifuge
  • Hemacytometer
  • Poly‐L‐ornithine/laminin‐coated culture plates (24‐well plates; see recipe)

Basic Protocol 4: Lentiviral Infection of Mouse Ciliary Epithelial Cells for Transgene Overexpression

  Materials
  • Lentivirus (sufficient volume to obtain 15 MOI of virus/well)
  • Standard differentiation medium (see recipe)
  • Proliferation medium (see recipe)
  • Retinal differentiation medium‐step1 (see recipe)
  • Retinal differentiation medium‐step2 (see recipe)
  • Virkon solution
  • Additional reagents and equipment for preparing the monolayer CECs ( protocol 4)
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Figures

Videos

Literature Cited

Literature Cited
   Ahmad, I., Tang, L., and Pham, H. 2000. Identification of neural progenitors in the adult mammalian eye. Biochem. Biophys. Res. Commun. 270:517‐521.
   Akagi, T., Mandai, M., Ooto, S., Hirami, Y., Osakada, F., Kageyama, R., Yoshimura, N., and Takahashi, M. 2004. Otx2 homeobox gene induces photoreceptor‐specific phenotypes in cells derived from adult iris and ciliary tissue. Invest. Ophthalmol. Vis. Sci. 45:4570‐4575.
   Cicero, S.A., Johnson, D., Reyntjens, S., Frase, S., Connell, S., Chow, L.M., Baker, S.J., Sorrentino, B.P., and Dyer, M.A. 2009. Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 106:6685‐6690.
   Coles, B.L., Angenieux, B., Inoue, T., Del Rio‐Tsonis, K., Spence, J.R., McInnes, R.R., Arsenijevic, Y., and van der Kooy, D. 2004. Facile isolation and the characterization of human retinal stem cells. Proc. Natl. Acad. Sci. U.S.A. 101:15772‐15777.
   Das, A.V., James, J., Rahnenführer, J., Thoreson, W.B., Bhattacharya, S., Zhao, X., and Ahmad, I. 2005. Retinal properties and potential of the adult mammalian ciliary epithelium stem cells. Vision Res. 45:1653‐1666.
   Djojosubroto, M., Bollotte, F., Wirapati, P., Radtke, F., Stamenkovic, I., and Arsenijevic, Y. 2009. Chromosomal number aberrations and transformation in adult mouse retinal stem cells in vitro. Invest. Ophthalmol. Vis. Sci. 50:5975‐5987.
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   Gualdoni, S., Baron, M., Lakowski, J., Decembrini, S., Smith, A.J., Pearson, R.A., Ali, R.R., and Sowden, J.C. 2010. Adult ciliary epithelial cells, previously identified as retinal stem cells with potential for retinal repair, fail to differentiate into new rod photoreceptors. Stem Cells 28:1048‐1059.
   Hollyfield, J.G. 1968. Differential addition of cells to the retina in Rana pipiens tadpoles. Dev. Biol. 18:163‐179.
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   Jomary, C., Jones, S.E., and Lotery, A.J. 2009. Generation of light‐sensitive photoreceptor phenotypes by genetic modification of human adult ocular stem cells with Crx. Invest. Ophthalmol. Vis. Sci. 51:181‐189.
   Kokkinopoulos, I., Pearson, R.A., Macneil, A., Dhomen, N.S., MacLaren, R.E., Ali, R.R., and Sowden, J.C. 2008. Isolation and characterisation of neural progenitor cells from the adult Chx10(orJ/orJ) central neural retina. Mol. Cell. Neurosci. 38:359‐373.
   MacNeil, A., Pearson, R.A., MacLaren, R.E., Smith, A.J., Sowden, J.C., and Ali, R.R. 2007. Comparative analysis of progenitor cells isolated from the iris, pars plana, and ciliary body of the adult porcine eye. Stem Cells 25:2430‐2438.
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   Osakada, F., Ikeda, H., Mandai, M., Wataya, T., Watanabe, K., Yoshimura, N., Akaike, A., Sasai, Y., and Takahashi, M. 2008. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat. Biotechnol. 26:215‐224.
   Straznicky, K. and Gaze, R.M. 1971. The growth of the retina in Xenopus laevis: An autoradiographic study. J. Embryol. Exp. Morphol. 26:67‐79.
   Tropepe, V., Coles, B.L., Chiasson, B.J., Horsford, D.J., Elia, A.J., McInnes, R.R., and van der Kooy, D. 2000. Retinal stem cells in the adult mammalian eye. Science 287:2032‐2036.
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