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

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Abstract
Table of Contents
Materials
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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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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 O₂ :CO₂ (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

Figure 1.H0.1 Development of CEC monolayer culture. (A ) Diagram of the adult eye, showing the anatomical position of the CE and associated structures. (B ) DIC image of a transverse section of an adult mouse eye showing the region of the ciliary epithelium (CE) and neural retina (NR). (C ) CECs were grown as neurospheres for 7 days in vitro, before expansion as monolayers in proliferating medium. Scale bar 50 µm. Reproduced from Gualdoni et al. ()

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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.
	Giordano, F., De Marzo, A., Vetrini, F., and Marigo, V. 2007. Fibroblast growth factor and epidermal growth factor differently affect differentiation of murine retinal stem cells in vitro. Mol. Vis. 13:1842‐1850.
	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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	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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Isolation and Culture of Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem Cells, and Retinal Progenitor Cells

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited