Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Summary

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

Abstract

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal diseases at advanced stages. We have shown human recombinant retina-specific laminin isoform matrix is able to support the differentiation of human embryonic stem cells (hESCs) to photoreceptor progenitors. In addition, sub-retinal injection of these cells has also shown partial restoration in the rd10 rodent and rabbit models. Sub-retinal injection is known to be an established method that has been used to deliver pharmaceutical compounds to the photoreceptor cells and retinal pigmented epithelial (RPE) layer of the eye due to its proximity to the target space. It has also been used to deliver adeno-associated viral vectors into the sub-retinal space to treat retinal diseases. The sub-retinal delivery of pharmaceutical compounds and cells in the murine model is challenging due to the constraint in the size of the murine eyeball. This protocol describes the detailed procedure for the preparation of hESC-derived photoreceptor progenitor cells for injection and the sub-retinal delivery technique of these cells in genetic retinitis pigmentosa mutant, rd10 mice. This approach allows cell therapy to the targeted area, in particular the outer nuclear layer of the retina, where diseases leading to photoreceptor degeneration occur.

Introduction

Inherited retinal diseases and age-related macular degeneration lead to photoreceptor cell loss and eventual blindness. The retinal photoreceptor is the outer segment layer of the retina comprised of specialized cells responsible for phototransduction (i.e., conversion of light to neuronal signals). The rod and cone photoreceptor cells are adjacent to the retinal pigmented layer (RPE)¹. Photoreceptor cell replacement therapy to compensate the cell loss has been an emerging and developing therapeutic approach. Embryonic stem cells (ESCs)^2,3,4, induced pluripotent stem cells (iPSCs)-derived RPE cells, and retinal progenitor cells (RPCs)^4,5,6,7,8 were used to restore the damaged photoreceptor cells. Sub-retinal space, a confined space between the retina and the RPE, is an attractive location to deposit these cells to replace damaged photoreceptor cells, RPE, and Mueller cells due to its vicinity^9,10,11.

Gene and cell therapies have been utilizing the sub-retinal space for regenerative medicine for various retinal diseases in pre-clinical studies. This includes the delivery of functional copies of the gene or gene editing tools in the form of either anti-sense oligonucleotide therapy^12,13 or CRISPR/Cas9 or base editing via adeno-associated virus (AAV) based strategy^14,15,16, implantation of materials (e.g., RPE sheet, retinal prosthetics^17,18,19) and differentiated stem cell-derived retinal organoids^20,21,22 to treat retinal and RPE-related diseases. Clinical trials using hESC-RPE³¹ in the sub-retinal space to treat RPE65-associated Leber congenital amaurosis (LCA)^23,24, CNGA3-linked achromatopsia²⁵, MERTK-associated retinitis pigmentosa²⁶, choroideremia^27,28,29,30 have been proven to be an effective approach. Direct injection of cells to the vicinity of the damaged area greatly improves the chance of cell settlement at the appropriate region, synaptic integration, and eventual visual improvement.

Even though sub-retinal injection in human and large-eyed models (i.e., pig^32,33,34,35, rabbit^{36,37,38,39,40}, and non-human primate^41,42,43) has been established, such injection in the murine model is still challenging due to the constrain of the eyeball size and enormous lens occupying the mouse eye^44,45,46. However, genetically modified models are only readily available in small animals and not in large animals (i.e., rabbits and non-human primates), therefore sub-retinal injection in mice draws attention to investigate novel therapeutic approaches in retinal genetic disorders. Three major approaches are being used to deliver cells or AAVs into the sub-retinal space, namely the trans-corneal route, trans-scleral route, and the pars plana route (See Figure 2). Trans-corneal and trans-scleral routes are associated with cataract formation, synechiae, choroidal bleeding, and reflux from the injection site^{11,44,45,47,48,49}. We adopted the pars plana approach as a direct visualization of the injection process, and the injection site can be achieved in real-time under the microscope.

We recently described a method that can differentiate human embryonic stem cells (hESCs) into photoreceptor progenitors under xenofree, chemically defined conditions using recombinant human retina-specific laminin isoform LN523. Since LN523 was found to be present in the retina, we hypothesized that the extracellular matrix niche of the human retina could be recapitulated in vitro and thereby support photoreceptor differentiation from the hESCs³⁶. Single-cell transcriptomic analysis showed that photoreceptor progenitors co-expressing cone-rod homeobox and recoverin were generated after 32 days. A retinal degeneration 10 (rd10) mutant mouse model that mimics autosomal human retinitis pigmentosa was used to evaluate the efficacy of the day 32 hESC-derived photoreceptor progenitors in-vivo. The hESC-derived photoreceptor progenitor cells were injected into the sub-retinal space of rd10 mice at P20, where photoceptor dysfunction and degeneration are ongoing³⁶. Here, we describe a detailed protocol for the preparation of the post-cryopreserved hESC-derived photoreceptor progenitors and delivery into the sub-retinal space of rd10 mice. This method can also be used to administer AAVs, cell suspensions, peptides, or chemicals into the sub-retinal space in mice.

Protocol

The in vivo experiments were done in accordance with the guidelines and protocol approved by the Institutional Animal Care and Use Committee of SingHealth (IACUC) and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in Ophthalmic and Vision Research. The pups were immunosuppressed from P17 (pre-transplantation) to P30 (post-transplantation) by feeding them drinking water containing cyclosporine (260 g/L).

1. Preparation of Day 32 hESC-derived photoreceptor progenitors after cryopreservation

1. Pre-warm photoreceptor differentiation medium (PRDM) in a 37 °C water bath.
2. Retrieve a cryovial containing day 32 hESC-derived photoreceptor progenitor cells from liquid nitrogen. Keep on dry ice.
3. Thaw the cryovial at 37 °C in a water bath for 3-5 min. Resuspend the day 32 cells in 1 mL of PRDM and centrifuge at 130 x g for 4 min.
4. Remove the supernatant and resuspend cells in 1 mL of PRDM.
5. Remove 10 µL of the mixture for cell counting. Mix cells using 0.2% trypan blue according to the manufacturer's instructions. Pipette cell mixture into the cell counting chamber slide. Determine cell number and viability by automated cell counter.
6. Proceed to the next step when the cell viability is above 70%. Centrifuge the remaining cell suspension at 130 x g for 4 min. Remove the supernatant and resuspend the cell pellet in PRDM at the concentration of 3 x 10⁵ cells/µL for transplantation.
7. Observe for visible cell clumps. Resuspend cell clumps repeatedly using a 10 µL pipette tip in the microfuge tube until no visible cell clump is observed. Load into the 33G injection syringe to observe for extrusion of cell solution through the needle.

2. Sub-retinal delivery of the hESCs in rd10 mice

1. Preparation of the animals
   1. Anaesthetize the mouse (P20, male/female, 3-6 g) using a combination of ketamine (20 mg/kg body weight) and xylazine (2 mg/kg body weight) in a 1 mL tuberculin syringe attached to a 27G needle by intraperitoneal approach. Administer a subcutaneous injection of buprenorphine (0.05 mg/kg) to the mouse as a pre-emptive analgesic.
   2. After administering the anesthesia, instill a drop each of 1% tropicamide and 2.5% phenylephrine for pupil dilation. Apply an ophthalmic gel to the cornea to prevent the eye from dryness and anesthesia-related cataract.
   3. Place the mouse in an empty cage until fully anesthetized. Assess the proper anesthetic level by pinching the paw pad and confirm if the animal does not react to the hard pinch.
   4. When the mouse is fully anesthetized, place the animal on a warm pad set at 38 °C.
2. Sub-retinal delivery of the cells
   1. To use a 1-port trans-vitreal pars plana approach, as done here, perform sub-retinal injection in a sterile environment. Use an upright operating microscope with a direct light path to perform the injection.
   2. Prepare the 10 µL glass syringe by removing the needle hub. Mount the 33G blunt needle onto the glass syringe. Take the metal hub cover and carefully secure the needle on the syringe.
   3. Flush with distilled water to check for any signs of leakage and patency of the needle. Empty the syringe and put it at the side carefully.
   4. Place the anesthetized mouse on a pillow, with the treatment eye looking up straight to the objective of the microscope. Apply the 0.5% proparacaine hydrochloride and wait for 30 s. Apply 150 µL of ophthalmic gel on the eye and place a round cover slip on it.
   5. Perform a rough examination of the eye by observing the cornea, iris, pupil, lens, and conjunctiva. Through the pupil, visualize the fundus of the mouse eye by adjusting the focal plane. Adjust the head until the optic head is positioned at the center of the pupil and minimize the movement of the head by proper positioning on the pillow.
   6. Gently tap the base of the tube with the hESCs multiple times to get a uniform cell suspension. By using the 10 µL glass microliter syringe with a 33G blunt needle, withdraw 2 µL of cells/media. Withdraw the cells right before the injection to avoid cell settlement/clumping in the syringe.
   7. Using a 30G disposable needle, make a sclerectomy wound 2 mm behind the limbus. Keep the angle of the needle at ~45° to avoid touching the lens. Once the tip of the needle is visualized in the eye, gently withdraw the needle. Discard the needle into the sharp bin after use to avoid needle prick injury.
   8. Take the glass syringe and insert the blunt needle into the sclerectomy wound. Without touching the lens, advance the blunt needle until it reaches the opposite retina of the entry wound. Ensure the injection area is clear of major retinal blood vessels to avoid bleeding.
   9. Gently penetrate the retina until a pressure branch on the sclera is seen. Keep the blunt end of the needle parallel to the sclera to avoid leaking of the cells into the vitreous space.
   10. Slowly inject 2 µL of cell suspension or PRDM media (control) into the sub-retinal space while gentle pressure is maintained on the syringe. With a successful injection, a visible bleb (i.e., raised retina with the cell suspension/media in it) should be formed at the injection site.
       NOTE: Only gentle pressure should be used during injection to avoid retina tearing and blockage of the cells at the needle tip.
   11. After confirming the bleb, wait for 10 s to let the cells settle down. Gently retract the needle from the eye.
3. Intraoperative optical coherence tomography (OCT) scanning of the bleb (optional)
   1. Position the mouse eye to visualize the bleb under the microscope by slowly moving the head. Secure the position by gently holding the head. No additional pupil dilation is necessary. Do not remove the cover slip and the gel on the eye. It gives a clear optical media to visualize the bleb.
   2. Perform the intra-operative OCT using the built-in iOCT function of the operating microscope.
   3. Press the Cube option on the OCT screen and position the scanning area on the bleb by pressing the Arrow buttons. Adjust the OCT by sliding Centering and Focus for the best OCT quality. Press Capture/Scan to acquire the OCT scan of the bleb area. Review the images to check the quality of the scans.
4. Recovery
   1. Remove the cover slip and clean the gel from the eye with gauze. Apply antibiotic ointment one time after the injection to prevent infection.
   2. Allow the animal to recover from anesthesia under a warm light until they regain sufficient consciousness to maintain sternal recumbency and return to the home cage. Monitor the animal for at least 3 days post-injection for signs of inflammation, infection, and distress.
      NOTE: The 150 W warm light should be at least 30 cm away from the animal cage, and caution should be taken to avoid a burn injury.
   3. Administer a subcutaneous injection of buprenorphine (0.05 mg/kg) 8 hourly for 1 day or per veterinarian’s recommendation. If inflammation or infection of the eye is observed, consult a veterinary professional for appropriate treatment.
5. Cleaning and sterilization of the instruments
   1. Flush the 10 µL glass microliter syringe and the 33G blunt needle with 100% ethanol 10x. Wash away the ethanol by flashing the syringe with distilled water.
   2. Dissemble the glass syringe and the needle. Dry the syringe for storage.

Results

The 10 µL glass syringe was assembled according to the manufacturer's instructions (Figure 1), and the blunt needle used to deliver the cell suspension/media is shown in Figure 1B. Different approaches for sub-retinal injection are illustrated in Figure 2. We describe the pars plana approach in this protocol (Figure 2C). The blunt needle mounted on a glass syringe was inserted through a sclerot...

Discussion

The sub-retinal injection has been used for cell suspension transplantation to treat RPE and retinal diseases^{23,25,26,27,28,31,40}. This approach is highly essential in rodent studies not only for cell transplantation and gene therapy approaches but also to evaluate novel therapeutic compound...

Materials

Name	Company	Catalog Number	Comments
0.3% Tobramycin	Novartis	NDC  0078-0813-01	Tobrex (3.5 g)
0.3% Tobramycin and 0.1% Dexamethasone	Novartis	NDC 0078-0876-01	Tobradex (3.5 g)
0.5% Proparacaine hydrochloride	Alcon	NDC 0998-0016-15	0.5% Alcaine (15 mL)
1 mL Tuberculin syringe	Turemo	SS01T2713
1% Tropicamide	Alcon	NDC 0998-0355-15	1% Mydriacyl (15 mL)
2.5% Phenylephrine hydrochloride	Alcon	NDC 0998-0342-05	2.5% Mydfrin (5 mL)
24-well tissue culture plate	Costar	3526
30 G Disposable needle	Becton Dickinson (BD)	305128
33 G, 20 mm length blunt needles	Hamilton	7803-05
Automated Cell Counter	NanoEnTek	Model: Eve
B27 without Vitamin A	Life Technologies	12587001	2%36
Buprenorphine	Ceva		Vetergesic vet (0.3 mg/mL)
CKI-7	Sigma	C0742	5 µM36
Cyclosporine	Novartis		260 g/L in drinking water
Day 32 hESC-derived photoreceptor progenitor cells	DUKE-NUS Medical School		Human embryonic stem cells are differentiated for 32 days. See protocol in Ref 36.
Gauze	Winner Industries Co. Ltd.	1SNW475-4
Glasgow Minimum Essential Medium	Gibco	11710–035
hESC cell line H1	WiCell Research Institute	WA01
Human brain-derived neurotrophic factor (BDNF)	Peprotech	450-02-50	10 ng/mL36
Human ciliary neurotrophic factor (CNTF)	Prospec-Tany Technogene	CYT-272	10 ng/mL36
Ketamine hydrochloride (100 mg/mL)	Ceva Santé Animale	KETALAB03
LN-521	Biolamina	LN521-02	1 µg36
mFreSR	STEMCELL Technologies	5854
Microlitre glass syringe (10 mL)	Hamilton	7653-01
N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT)	Selleckchem	S2215	10 µM36
N-2 supplement	Life Technologies	A13707-01	1%36
Non-essential amino acids (NEAA)	Gibco	11140–050	1x36
NutriStem XF Media	Satorius	05-100-1A
Operating microscope	Zeiss	OPMI LUMERA 700	With Built-in iOCT function
PRDM (Photoreceptor differentiation medium, 50ml)	DUKE-NUS Medical School		See media composition36. Basal Medium, 10 µM DAPT, 10 ng/mL BDNF, 10 ng/mL CNTF, 0.5 µM Retinoic acid, 2% B27 and 1% N2. Basal Medium: 1x GMEM, 1 mM sodium pyruvate, 0.1 mM B-mercaptoethanol, 1x Non-essential amino acids (NEAA).
Pyruvate	Gibco	11360–070	1 mM36
Rd10 mice	Jackson Laboratory	B6.CXB1-Pde6brd10/J mice	Gender: male/female, Age: P20 (injection), Weight: 3-6 g
Retinoic acid	Tocris Bioscience	0695/50	0.5 µM36
Round Cover Slip (12 mm)	Fisher Scientific	12-545-80
SB431542	Sigma	S4317	0.5 µM36
Vidisic Gel (10 g)	Dr. Gerhard Mann
Xylazine hydrochloride (20 mg/mL)	Troy Laboratories	LI0605
β-mercaptoethanol	Life Technologies	21985–023	0.1 mM36