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 demonstrated that sub-retinal suspension transplants of hESC-RPE form extensive functional monolayers with photoreceptor rescue capacity².

Several advantages accompany the use of the rabbit eye for the generation of a GA model of disease. Firstly, the size of the rabbit eye, which is 70% the volume of an adult human eye, allows clinically meaningful transplantation using a cell density that is much lower than routinely used in small rodent eyes (1,000 cells/µL vs. 50,000 cells/µL)^4,5. Secondly, surgery in rodents is usually transscleral through the choroid, which compromises the retinal barrier and potentially triggers an inflammatory response and a possible rejection⁶. Both factors together may lead to multilayering and clumping of transplanted cells, and an overall poor integration of the transplanted cells in a disrupted native retinal tissue. However, the large-eyed rabbit model allows performing a surgical technique with instrumentation identical to a clinical setting. Thirdly, a large-eyed model also permits high-resolution in vivo imaging and monitoring of the transplanted cells and the overlying retina through time^1,2,3. Thus, we describe a clinically relevant and cost-efficient preclinical model that should be an attractive alternative to rodents for anyone with an interest in research of the normal and diseased retina and the sub-retinal space.

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 Sodium Iodate (NaIO₃) into a Large-eyed Animal Model

1. Anesthetize animals by intramuscular administration in the thigh with a mixture of 35 mg/kg ketamine and 5 mg/kg xylazine in saline using a 30 G syringe, and dilate pupils with topical eyedrops using a mix of 0.75% cyclopentolate and 2.5% phenylephrine. Proper anesthetization is confirmed if the animal does not react to a hard pinch of its back leg.
2. Place the rabbit under the surgical microscope with the head facing the surgeon (Figure 1A). Use a lid retractor to remove eyelids and nictitans membrane with a sterile cloth to minimize the risk of contamination. Use balanced salt solution (BSS) to prevent dryness while under anesthesia in both eyes.
3. For microsurgery, use a 2-port (or optional 3-port) 25 G transvitreal pars plana technique with non-valved trocars for the insertion of microsurgical instruments (Figure 1B). The multifunction vitrectomy machine has ports to connect an infusion cannula, endoillumination, vitrector, and endolaser. For subretinal injections, only the endoillumination is mandatory, which makes a 2-port set-up sufficient. Insert the endoillumation through the upper left trocar and use the upper right trocar for the subretinal injection cannula.
   1. For a 3-port set-up, use the lower temporal trocar for the BSS infusion cannula. If optional instruments (such as a vitrector) are used, insert them through the upper right trocar.
   2. Insert the upper 2 trocars 1-2 mm from the limbus using clawed forceps to grab and displace the conjunctiva overlying the insertion site.
   3. Make sure the trocars are inserted transsclerally at a 30-45° limbus parallel angle, and proceed to the middle of the tip of the trocar. Then turn the trocar 90° and advance into the eye, aiming at the posterior pole of the eye. This procedure will avoid post-surgical leakage from the sclerotomies, and also decrease the risk of endophthalmitis (i.e., bacterial infection in the eye). See also Figure 2B for trocar positions.
   4. Put a single use flat contact lens on the cornea to visualize the retina, with synthetic tears as contact gel between the eye and the contact lens (Figure 1A).
   5. Draw 500 µL of NaIO₃ into a 1 mL syringe connected to an extension tube (operated by the assistant) and a 38 G polytip cannula (operated by the surgeon).
   6. Insert the endoillumination probe through the upper left trocar and the injection cannula through the upper right trocar, and advance the cannula through the vitreous space towards the retina, aiming for the area just below the optic nerve head.
   7. Allow the tip of the cannula to slowly touch the retina until a focal whitening is visible. The injection itself will penetrate the retina, allowing for subretinal delivery. Do not let the cannula penetrate the retina, since this may cause hemorrhage.
   8. Inject 50 µL of NaIO₃ subretinally over a 5 s period. Since there is a natural cleavage plane between the retina and the underlying choroid, a clearly visible semitransparent bleb should gradually form during the injection.
   9. During the injection, slowly retract the needle, but make sure the tip is maintained within the bleb to minimize reflux.
   10. After removal of the endoillumation and injection cannula, remove the trocars using clawed forceps, and apply light pressure for 30 s to the self-sealing suture-less sclerotomies using the tip or the blunt end of the forceps.
4. Post-surgically give 10 mL of saline subcutaneously to prevent dehydration. Do not give post-surgical topical steroids or antibiotics. For analgesics give 0.5 mL of buprenorphine 0.3 mg/ml subcutaneously after surgery, as well as the day after surgery.
5. After usage, wash all instruments by immersing them for a couple of seconds firstly in 70% ethanol, secondly in 45% ethanol, and lastly in distilled water. Dry them properly with a paper towel.
6. Attend animals until they regain sufficient consciousness and place them all single encaged. If required (e.g., for immunohistochemistry purposes), euthanize animals by intravenous injection of 100 mg/kg pentobarbital (see Table of Materials).
7. Wait 7 days to proceed with the transplantation of hESC-RPE cells.

2. Subretinal Transplantation of hESC-RPE Cells in Treated Animals

1. Administer 2 mg (100 µL) of intravitreal triamcinolone in anesthetized animals using a 30 g injection needle inserted 1-2 mm from the limbus in the lower temporal quadrant 1 week before transplantation of hESC-RPE, and re-administer it every 3 months. Make sure to point the tip towards the posterior pole of the eye to avoid a lens touch.
2. Culture a hESC-RPE monolayer as described previously¹.
3. Remove cell differentiation media (see Table of Materials) of a 24-well confluent hESC-RPE monolayer and wash each well with 500 µL of PBS without Ca²⁺ and Mg²⁺. Repeat this action once more, for a total of 2 washes.
4. Discard supernatant, add 500 µL of trypsin per well, and incubate for 12 min at 37 °C.
5. Tilt the plate and carefully remove trypsin (cells should remain attached to the plate). Collect cells in 800 µL of fresh 37 °C prewarmed differentiation media by gentle pipetting, or even scraping if needed, to obtain a single cell suspension.
   NOTE: Use a 40 µm cell strainer if cell clumps are observed.
6. Count cells in a hemocytometer chamber using 0.2% Trypan Blue, according to the manufacturer's instructions.
7. Add 5 mL of differentiation media and centrifuge cells at room temperature, 300 x g for 5 min.
8. Discard supernatant and resuspend pellet in freshly filter-sterilized PBS (passed through a 25 mm syringe filter) to a final concentration of 1,000 cells/µL.
9. Aliquot the previous cell suspension into 600 µL aliquots and keep on ice until and during surgery.
10. Anesthetize animals by intramuscular administration in the thigh with a mixture of 35 mg/kg ketamine and 5 mg/kg xylazine in saline using a 30 G syringe. Dilate pupils with topical eyedrops using amix of 0.75% cyclopentolate and 2.5% phenylephrine. Proper anesthetization is confirmed if the animal does not react to a hard pinch in its back leg.
11. Place the rabbit with the head facing the surgeon. Use a lid retractor to remove eyelids and nictitans membrane with a sterile cloth to minimize the risk of contamination. Use BSS to prevent dryness while under anesthesia in both eyes.
12. For microsurgery, use a 2-port (or optional 3-port) 25 G transvitreal pars plana technique with trocars placed in the same positions as described in step 1.3 and Figure 2. If the previous sclerotomies are visible, perform trocar insertion just adjacent, but not through these, to minimize the risk of postoperative leakage.
    1. Put a single use flat contact lens on the cornea to visualize the retina, with synthetic tears as the contact gel between the eye and the contact lens (Figure 1A).
    2. After proper tip positioning (see steps 1.3.5 and 1.3.6), inject 50 µL of a gently-mixed hESC-RPE suspension (50,000 cells) subretinally. Aim for the center of the NaIO₃ pretreated area distinguished by a characteristic "metallic" endo-illumination reflex. The neurosensory retina should separate easily creating a clearly visible bleb. Flush the needle with sterile H₂O in between rabbits/after use to avoid needle clogging due to cell clumps. Change the needle when clogging is noticed.
    3. During the injection, slowly retract the needle but make sure the tip is maintained within the bleb to minimize reflux.
    4. After removal of the endoillumation and injection cannula, remove the trocars using clawed forceps, and apply light pressure for 30 s to the self-sealing suture-less sclerotomies using the tip or the blunt end of the forceps.
13. Post-surgically give 10 mL of saline subcutaneously to prevent dehydration. Do not give post-surgical topical steroids or antibiotics. For analgesics give 0.5 mL of buprenorphine 0.3 mg/ml subcutaneously after surgery, as well as the day after surgery.
14. After usage, wash all instruments by immersing them for a couple of seconds, firstly in 70% ethanol, secondly in 45% ethanol, and lastly in distilled water. Dry them properly with a paper towel.
15. Attend animals until they regain sufficient consciousness and place them all single encaged. If required (e.g. for immunohistochemistry purposes), euthanize animals by intravenous injection of 100 mg/kg pentobarbital.

3. In Vivo Retinal and Subretinal Imaging

1. Use a spectral domain optical coherence tomography (SD-OCT) device with the accompanying software (see Table of Materials) to obtain cross-sectional b-scans of the treated animals, according to manufacturer's instructions. To avoid image blurring, make sure to keep the cornea moist by flushing with topical saline every 30-60 s.
   1. Place the anesthetized and pupil-dilated animals (see steps 1.1 and 2.10) in an adjustable mount to obtain an unobstructed path from the instrument light source to the rabbit retina.
   2. Obtain at least 3 OCT scans with simultaneous infrared-confocal scanning laser ophthalmoscopy (IR-cSLO) reflectance reference images representing the upper, central, and lower portion of the injected area.
   3. Obtain en-face fundus images with cSLO blue, green, infrared, and multicolor laser reflectance (i.e., multiple simultaneous laser colors), respectively.
   4. Capture blue light autofluorescence (BAF) images using the blue-light laser capability of the SD-OCT device.

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