This work was funded by NEI Fast-track SBIR grant R44-EY027654-01A1&#160;and SBIR grant 3 R44 EY 027654 - 02 S1 (I.O.N., Lineage Cell Therapeutics; Dr. Petersen-Jones is a co-PI). The authors would like to thank Ms. Janice Querubin (MSU RATTS) for her help with anesthesia and general care for the animals included in this study as well as help with surgical setting and instruments preparation/sterilization. The authors would like to thank Dr. Paige Winkler for the help in receiving the organoids and placing them in media on the day prior to the implantation and for the help on the day of the implantation. The authors are also grateful to Mr. Randy Garchar (LCTX) for diligent shipping of retinal organoids, assembling the shipper, and downloading temperature and G-stress-records after each shipment. This work was performed while author Igor Nasonkin was employed by&#160;Biotime (now Lineage).

Procedures were conducted in compliance with the Association for Research in Vision and Ophthalmology (ARVO) statement for Use of Animals in Ophthalmic and Vision Research. They were also approved by the Michigan State University Institutional Animal Care and Use Committee. Wild-type and CrxRdy/+ cats from a colony of cats maintained at Michigan State University were used in this study. Animals were housed under 12 h : 12 h light-dark cycles and fed a commercial complete cat diet.
<p clas...

Implantation of hPSC-derived retinal tissue (retinal organoids) into the subretinal space is a promising experimental approach for restoring vision for late-stage retinal degenerative diseases caused by PR cell death (profound or terminal blindness). The presented approach builds on an earlier developed and successfully tested experimental therapy based on subretinal grafting of a piece of human fetal retinal tissue23,24,25. It ...

Millions of people around the world are impacted by retinal degeneration (RD) with resulting visual impairment or blindness associated with loss of the light-sensing photoreceptors (PRs). Age-related macular degeneration (AMD) is a major cause of blindness resulting from a combination of genetic risk factors and environmental/lifestyle factors. In addition, over 200 genes and loci have been found to cause inherited RD (IRD)1. Retinitis pigmentosa (RP), the commonest IRD, is genetically heterogenous with more than 3,000 genetic mutations in approximately 70 genes being reported2,3,4. Leber Congenital Amaurosis (LCA), which causes blindness in childhood is also genetically heterogenous5,6. Gene augmentation therapy has been developed and is in clinical trials for treating a small number of IRDs3,7. However, a separate therapy must be developed for the treatment of each distinct genetic form of IRD and thereby only treating a small subset of patients. Furthermore, gene augmentation relies on the presence of a population of rescuable photoreceptors and is, therefore, not applicable for advanced degeneration.
There is, therefore, an urgent and yet unmet clinical need for the development of therapies addressing and treating advanced RDs and profound to terminal blindness. Over the last 2 decades neuroprosthetic implants have been developed and tested in large animal models, such as the cat, prior to human use8,9,10,11,12,13,14. Likewise, in the past 20 years retinal replacement therapies utilizing sheets of embryonic or even mature mammalian retina grafted subretinally have been developed15,16,17,18,19,20,21,22 and even tested successfully in RD patients23,24,25. Both approaches utilize the idea of introducing new sensors (photovoltaic silicon photodiodes in the case of neuroprosthetic devices26,27, and healthy mutation-free photoreceptors organized in sheets, in the case of retinal sheet implantation) into retina with degenerated PRs. Recent studies have investigated the use of stem cells-based approaches such as transplantation of human pluripotent stem cell (hPSC)-derived retinal progenitors28,29, hPSC-photoreceptors30, and hPSC-retinal organoids31,32,33. Retinal organoids enable the formation of retinal tissue in a dish and derivation of photoreceptor sheets with thousands of mutation-free PRs, which resemble the photoreceptor layer in the developing human fetal retina34,35,36,37,38,39,40. Transplanting hPSC-derived retinal tissue (organoids) into the subretinal space of patients with RD conditions is one of the new and promising investigational cell therapy approaches, being pursued by a number of teams31,32,41,42. Compared to transplantation of the cell suspension (of young photoreceptors or retinal progenitors), transplanted sheets of fetal photoreceptors were demonstrated to result in vision improvements in clinical trials23,24.
The protocol presented here describes, in detail, a transplantation procedure for subretinal delivery of the whole retinal organoids (rather than organoid rims33,41) as a potentially better way to introduce intact retinal sheets with PRs, to increase graft survival and improve the sheet preservation. Though procedures for introducing a flat piece of human retina and also RPE patches have been developed43,44,45, transplantation of larger 3D grafts has not been investigated. Stem cell-derived retinal organoids provide an inexhaustible source of photoreceptor sheets for developing vision restoration technologies, are free of ethical restriction, and are considered an excellent source of human retinal tissue for therapies focused on treating advanced RD and terminal blindness46. Development of surgical methods for precise subretinal implantation of retinal organoids with minimal injury to the host retinal niche (neural retina, retinal pigment epithelium and retinal and choroidal vasculature) is one of the critical steps for advancing such therapy toward clinical applications31,32. Large animal models such as cats, dogs, pigs, and monkeys have proven to be good models for investigating surgical delivery methods as well as to demonstrate the safety of implanted sheets of tissue (retinal pigment epithelium (RPE) cells) and investigate the use of organoids41,44,45,47,48,49,50. The large animal eye has a similar globe size to human as well as similar anatomy including the presence of a region of high photoreceptor density, including cones (the area centralis), resembling the human macula6,51,52.
In this manuscript, a technique for the implantation of hPSC-derived retinal tissue (organoids) into the subretinal space of feline large animal models (both wild-type and CrxRdy/+ cats) is described, which, together with promising efficacy results32,53 builds a foundation for further development of such investigational therapy toward clinical applications to treat RD conditions.

<table>
	<tbody>
		<tr>
			<td>0.22 &#181;m pore syringe filter with PES membrane</td>
			<td>Cameo</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>23G subretinal injector with extendable 41 G cannula</td>
			<td>DORC</td>
			<td>1270.EXT</td>
			<td></td>
		</tr>
		<tr>
			<td>250 &#181;L hamilton gas tight luer lock syringe</td>
			<td>Hamilton</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>6-0 Silk suture</td>
			<td>Ethicon</td>
			<td>707G</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0/7-0 polyglactin suture</td>
			<td>Ethicon</td>
			<td>J570G</td>
			<td></td>
		</tr>
		<tr>
			<td>Acepromazine maleate 500mg/5mL (Aceproject)</td>
			<td>Henry Schein Animal Health</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Par Pharmaceutical</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>cSLO + SD-OCT</td>
			<td>Heidelberg Engineering</td>
			<td>Spectralis HRA+ OCT</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclosporine</td>
			<td>Novartis</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Dexamethasone 2mg/mL (Azium)</td>
			<td>Vetone</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Doxycyline 25mg/5mL</td>
			<td>Cipla</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Fatal Plus solution (pentobarnital solution)</td>
			<td>Vortech</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Gentamicin 20mg/2mL</td>
			<td>Hospira</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Glass capillary (Thin-Wall Single-Barrel Standard Borosilicate (Schott Duran) Glass Tubing</td>
			<td>World Precision Instruments</td>
			<td>TW150-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylprednisolone actetate 40 mg/mL</td>
			<td>Pfizer</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT medium (Tissue-Tek O.C.T. Compound)</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympic Vac-Pac Size 23</td>
			<td>Natus</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution</td>
			<td>EMS</td>
			<td>15719</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylephrine Hydrochloride 10% Ophthalmic Solution</td>
			<td>Akorn</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Prednisolone 15mg/5mL</td>
			<td>Akorn</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Propofol 5000mg/50mL (10 mg/mL) (PropoFlo28)</td>
			<td>Zoetis</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>RetCam II video fundus camera</td>
			<td>Clarity Medical Systems</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Triamcinolone 400mg/10 mL (Kenalog-40)</td>
			<td>Bristol -Myers Squibb Company</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Tropicamide 1% ophthalmic solution</td>
			<td>Akorn</td>
			<td>NA</td>
			<td>can be found by various suppliers</td>
		</tr>
		<tr>
			<td>Vitrectomy 23G port</td>
			<td>Alcon</td>
			<td>Accurus systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitrectomy machine</td>
			<td>Alcon</td>
			<td>Accurus systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitreo-retinal vertical 80&#176; scissors with squeeze handle</td>
			<td>Frimen</td>
			<td>FT170206T</td>
			<td></td>
		</tr>
	</tbody>
</table>

subretinal transplantation of human embryonic stem cell-derived retinal tissue in a feline large animal model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

This procedure enables the successful and reproducible implantation of hPSC-derived retinal organoids in the subretinal space of a large eye animal model (demonstrated here using 2 examples: wild-type cats with healthy photoreceptors (PRs) and CrxRdy/+ cats with degenerating PRs and retina). Using the steps indicated in Figure 1 prepare and load the hPSC-derived retinal organoids into the borosilicate glass cannula of the injection device so that the organoids ar...

Watch this Scientific Journal Video about Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model at JoVE.com

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

The current protocol shows in detail subretinal delivery of the whole retinal organoids in a large animal model, aiming at developing cell therapy approaches for retinal degeneration. The main advantage of this technique is that it allows for delivery of organoids directly to the target tissue, the retina. The technique aims to treat advanced blinding retinal degenerative conditions, such as age-related macular degeneration, retinitis pigmentosa, or leber congenital amaurosis.Demonstrating the procedure will be Dr.Peterson Jones. For subretinal implantation of the organoids, use Stevens Tenotomy Scissors to perform a 0.5 to 1 centimeter lateral canthotomy in a four-month-old overnight-fasted anesthetized cat, and place an appropriately sized Barraquer eyelid speculum into the incision to keep the eyelids open with a surgical assistant regularly irrigating the cornea with BSS throughout the procedure. Use 0.5 Castroviejo corneal tying forceps and a small mosquito hemostat to gently grasp the bulbar conjunctiva next to the limbus.Place two 6-0 silk sutures into the conjunctiva immediately adjacent to the limbus at the 4 and 8 o'clock positions to hold the eye in primary gaze and to retract the third eyelid. Clamp the ends of the sutures with small mosquito hemostats to help their manipulation, and place another suture at the 12 o'clock position at the limbus partially through the thickness of the limbus, taking care not to penetrate the eye. Loosely knot the suture, cutting the end short, and place a stay suture in the conjunctiva immediately adjacent to the limbus at the 4 o'clock position to hold the eye and primary gaze.Reflect the bulbar conjunctiva between 10 and 2 o'clock, and use tenotomy scissors to incise the conjunctiva two to three millimeters from the limbus. Undermine the conjunctiva and clear the Tenon's capsule to expose the sclera at the two and 10 o'clock vitrectomy port sites three to five millimeters from the limbus. Use calipers to identify the sites for sclerotomy, avoiding the major scleral vessels that can be prominent in the cat.Planning for a three millimeter region at the 10 o'clock instrument port for a right-handed surgeon, pre-place 6-0 or 7-0 polyglactin cruciate pattern sutures without tying at the sites of the proposed sclerotomies. Once the sutures are in place, have the assistant grasp the 12 o'clock stay sutures to help keep the globe in position, and use 0.12 millimeter Castroviejo forceps to hold the tissue next to the sclerotomy site. Uses a trocar to introduce a 23 gauge vitrectomy port through the sclera at both the 2 and 10 o'clock positions angled toward the optic nerve to avoid contacting the lens, and use tying forceps to gently push the irrigation port to determine whether the tips can be visualized within the vitreous.Have the assistant place a Machemer magnifying irrigating vitrectomy lens onto the cornea to allow visualization of the posterior segment of the eye, and a 23 gauge vitrectomy probe cutter through the instrument port to perform a partial core vitrectomy. Next, introduce the needle of a one milliliter syringe containing one milliliter of triamcinolone solution into the instrument port and inject 250 to 500 microliters of the crystal suspension. When the solution has been delivered, Advance the vitrectomy probe through the instrument port to close to the optic nerve head with the port facing away from the retinal surface.Use high vacuum to help detach the vitreal face from the retina, insert the injector through the instrument port toward the retinal surface, and extrude the cannula tip. Gently press the tip of the retinal surface and have the assistant to give a slight quick push on the syringe plunger to start the retinal detachment, reducing the injection pressure to permit a slow increase of the retinal detachment until the desired size is achieved. Use a 41 gauge cannula of the injector to slightly enlarge the detachment until retinal scissors can be introduced, and remove the 10 o'clock scleral port.Then use a straight 2.850 millimeter slit knife oriented toward the optic nerve to avoid touching the lens to enlarge the sclerotomy at this site. Make the retinotomy on the bleb away from the optic nerve to avoid damaging retinal nerve fibers originating from the transplanted region, and avoid cutting major retinal vessels to reduce the risk of hemorrhage. To implant the organoids, insert a glass capillary loaded with organoids through the enlarged sclerotomy toward the retinotomy site, and use the tip of the capillary to slightly open the retinotomy to allow access to the subretinal bleb opening.Have the assistant slowly press the plunger of the injector again to inject the organoids into the subretinal bleb. BSS should proceed organoids, the organs, and flush the retinotomy open. When all of the organoids have been delivered, leave the glass capillary in place for a few seconds before slowly removing the capillary from the eye, taking care to avoid any sudden release of fluid.When the capillary has been removed, close the sclerotomy using the pre-placed suture in a cruciate pattern. Have the assistant remove the infusion port, and quickly tie the sclerotomy suture and close the peritomy with the polyglactin sutures in a simple continuous pattern. Close the lateral canthotomy with a 6-0 polyglactin suture and figure-eight skin sutures to perform the lateral canthus and use simple interrupted skin sutures to close the rest of the wound.Then administer the appropriate post-surgical steroid and antibiotic cocktail. Human pluripotent stem cell-derived retinal organoid loading can be confirmed within the glass cannula of the injection device and during the surgery by direct visualization. The presence of the organoids within the subretinal space can be confirmed postoperatively by ophthalmic examination and fundus imaging.Prior to euthanasia, confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography imaging can also be performed to assess the position of the organoids. These techniques can be used to demonstrate the persistence of retinal organoids in the subretinal space between the neural retina and the retinal pigment epithelium of the recipient eye. Histological and immunohistochemical analyses can be used to illustrate the survival of the xenogeneic graphs within the subretinal space of a large eye in an immunosuppressed animal.Only those skilled in vitreoretinal surgery, such as trained veterinary ophthalmologists or vitreoretinal surgeons familiar with the species differences in the cat eye compared to human eye should undertake this procedure. The technique presented in this video should be applicable to other large animal models that are used for translating vitreoretinal surgical techniques to the clinics.

subretinal-transplantation-human-embryonic-stem-cell-derived-retinal

Research

JoVE Journal

Neuroscience

1.8K Views.  Michigan State University. The current protocol shows in detail subretinal delivery of the whole retinal organoids in a large animal model, aiming at developing cell therapy approaches for retinal degeneration. The main advantage of this technique is that it allows for delivery of organoids directly to the target tissue, the retina. The technique aims to treat advanced blinding retinal degenerative conditions, such as age-related macular degeneration, retinitis pigmentosa, or leber congenital amaurosis.Demonstra...