Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Summary

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Abstract

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO₃-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Introduction

This protocol describes the generation of a large-eyed preclinical model of geographic atrophy (GA) that allows the evaluation of integration of transplanted hESC-RPE in the subretinal space. The methods described in detail here have been used in 3 recent publications that demonstrate the production of an enriched, pure, and functional population of RPE cells from hESC¹, as well as the creation of outer retinal damage and a GA-like phenotype induced by the subretinal injection of physiologic salt solutions (i.e., BSS and PBS) or NaIO₃ in the rabbit eye^2,3. We further....

Protocol

The following protocol follows the animal care guidelines of Karolinska Instituet. All animal experiments using New Zealand albino rabbits (Table of Materials) have been approved by the regional animal ethics committee (Stockholms Norra Djurförsöksetiska Nämnd) (permit: dnr 56/15). The use of hESC (dnr 2011/745-31/3) and the transfer and manipulation of hESC-RPE (dnr 2013/813-31/2) is also in accordance with the Swedish legislation and Karolinska Institutet regulations, and has been approved by the regional human ethics committee (Regionala Etikprövningsnämnden i Stockholm).

1. Subretinal Injection of....

Results

Representative in vivo images of BAF, IR-cSLO, and SD-OCT of a normal albino rabbit retina are shown in Figure 2. Note the different retinal layers with their distinctive levels of light reflection captured by the SD-OCT instrument.

In Figures 1A and Figure 1B, the setup to create sub-retinal blebs is illustrated: a lid retractor i.......

Discussion

In this protocol, the generation of a large-eyed model of GA and its preclinical use for evaluating hESC-RPE integration in vivo is described.

For translation of regenerative therapies for GA and related diseases into the clinic⁷, it is important to develop and optimize methods that faithfully capture the clinical methods for transplantation and imaging. The rabbit is in this aspect attractive: it has a relatively large eye that permits intraocular surgery and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NutriStem hESC XF differentiation medium –bFGF and –TGFb	Biological Industries	06-5100-01-1A
TrypLE Select 1x	Gibco, ThermoFisher Scientific Corp	12563-011
PBS without Ca2+ and Mg2+	Gibco, ThermoFisher Scientific Corp	14190-094
Cell strainer 40 μm Nylon	VWR	732-2757
Needle 30 G 0.5’’; 0.3 mm x 13 mm	BD Microlance	304827
Acrodisc 25 mm Syringe Filter	Acrodisc	PN4612
0.4% trypan blue	ThermoFisher Scientific Corp	15250061	Use at 0.2%
NaIO3	Sigma-Aldrich Corp	S4007
BSS	Alcon Nordic A/S	65079550
70% Ethanol	Solveco AB	1047
Ketaminol, 100 mg/mL	Intervet, Boxmeer	511519	Use 35 mg/kg ketamine
Rompun vet, 20 mg/mL	Bayer Animal Health	22545	Use 5 mg/kg xylazine
Triescence, 40 mg/mL	Alcon Nordic A/S	412915	2 mg intraviterial
Cyklopentolat-phenylephrine, 0.75% + 2.5%	APL	321968	Use 1 drop in each eye
Viscotears	Laboratoires Théa	597562
Topical saline	Apotea AB	7053249369080
Allfatal vet. 100 mg/mL	Omnidea	77168	Use 100 mg/mL pentobarbital
Extension tube (Hammer)	MedOne Surgical Inc	3223
25 G/38 G polytip subretinal cannula	MedOne Surgical Inc	3219	25 G/38 G
Single Use Flat Lens	Volk	#VWFD10
Barraquer Colibri lid retractor	AgnTho's AB	42-020-030
Non-valved trocars	Alcon Nordic A/S	8065751448
Clawed forceps	Bausch & Lomb Nordic AB	ET1811
Alcon Accurus 400VS Vitrectomy machine	Alcon Nordic A/S	8065740238
Accurus 25+ Gauge Vitrectomy TotalL Plus Pak	Alcon Nordic A/S	8065751493
SD-OCT device	Heidelberg Engineering	Spectralis HRA+OCT	Use Heidelberg Eye Explorer version 1.9.10.0
24 well plates	Sarstedt	83.3922
Neubauer hemocytometer	VWR	631-0925
New Zealand albino rabbits	Lidköpings Rabbit Farm, Sweden
hESC-RPE cells			See reference number 1
Buprenodale Vet, 0.3 mg/ml	Dechra	660500	Use 0.5 mL buprenorphine subcutaneously