Name: subretinal transplantation of human embryonic stem cell-derived retinal tissue in a feline large animal model
Uploaded: 2021-08-05T13:00:00.000+00:00
Duration: 7 min 43 s
Description: 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

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).

Ratnesh K. Singh, Ph.D., Francois Binette, Ph.D., and Igor O. Nasonkin Ph.D. are employees of Lineage Cell Therapeutics, Inc. The authors declare no conflict of interest.

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 presents the use of an alternative, replenishable and ethically acceptable retinal tissue source derived from hPSCs. Demonstrating surgical feasibility and ocular safety of therapy in large eye animal models51,55 is needed for advancement of this promising approach toward clinical applications. This manuscript provides a detailed method for subretinal implantation of hPSC-3D retinal tissue (retinal organoids) in a large animal model with both normal and degenerating retina (model of CrxRdy/+ cats model of LCA). Though procedures for introducing a flat piece of human retina and also RPE patches have been developed43,44,45, transplantation of larger 3D grafts (needed to restore vision in conditions with advanced RD) have not been investigated. The protocol described here in detail is for a transplantation procedure for subretinal delivery of the whole retinal organoids (rather than organoid rims33,41) also carrying some RPE as a potentially better way to introducing intact retinal sheets with PRs, to increase graft survival and improve the sheet preservation. Note that different portions of this protocol are well established. For example, vitrectomy is widely used by vitreoretinal surgeons during retinal reattachment surgery56,57,58,59,60. Subretinal injections are becoming more commonly used, for example, in gene augmentation therapy3,7,61,62,63,64. There are limited descriptions of creation of an adequate retinotomy and the injection of relatively large organoids into the subretinal space.
The critical steps include careful positioning and performance of the sclerotomy to avoid the lens, complete removal of vitreous cortex from the retinal surface over the transplant site, controlled formation of the subretinal bleb, generating retinotomy of the optimal size to accommodate the width of the transplantation cannula, maintaining the defined infusion pressure during different steps, and cannula withdrawal. Choosing the right transplantation cannula with optimized inner and outer diameter (ID and OD) and length, controlling intraocular bleeding, sterility of the whole procedure from organoids to surgical room and instruments, and the duration of surgery (30&#8211;45 min/animal) to ensure optimal results. The authors find that the best results are obtained with 23 G vitrectomy due to the thickness/viscosity of the cat vitreous, creating a bleb with an injector with a 41 G cannula, and then, extending the retinotomy at the edge of the bleb using 80&#176; angle retinal scissors. Other important factors include extending the sclerotomy using a 2.85 mm keratome to fit the borosilicate glass cannula (outer diameter OD 1.52 mm; inner diameter, ID 1.12 mm; and length 10.16 cm) and permitting implantation of larger organoids in a large eye with axial length about 20.5 mm (20.91 mm &#177; 0.53 mm)55. The use of a glass capillary was the current best available for loading and delivering descent size organoids without damaging them during the implantation process. It was found that reducing the infusion pressure from 20&#8211;30 mmHg during the vitrectomy to 10 mmHg during the bleb formation step is optimal for generating retinal detachments, needed for creating space for retinal organoids. In addition, viability of hPSC-3D retinal tissue (organoids) during long-distance shipment and choosing the optimized immunosuppression regimen for maintenance of xenogeneic human grafts are critical as recently reported31,54.
The authors found that due to the highly reflective cat tapetum endo-illumination was not required to perform the surgery. Transplantation in an atapetal species (e.g., pig, nonhuman primate) would require a 3-port vitrectomy that includes endo-illumination. Tamponade (e.g., with perfluorocarbon followed by silicone oil) as performed in routine retinal detachment surgery was not performed because pressure on the bleb would risk extruding the organoids into the vitreous. Future developments could include the use of materials currently being investigated for sealing retinal holes. This could be used to prevent any post-implantation loss of organoids into the vitreous. Additionally, Triescence, which is similar to Kenalog-40 but contains preservative-free triamcinolone, could be used as an alternative to visualize the vitreous.
The smaller globe size in younger animals (e.g., 1&#8211;2 months) presented more surgical challenges compared to the large eye (adult animals). Nevertheless, using the method described here, it is possible to deliver the subretinal grafts. In the CrxRdy/+ LCA model, larger retinal blebs were found to not always reattach. Keeping the bleb to the minimum size needed to allow the retinal organoids to be injected, reduced this complication. Transplanting sooner in RD retina (before the complete loss of PRs when neural retina becomes markedly thinned) is another guideline, in line with the earlier work with human fetal retina grafts20. Another note &#8211; the detailed technique does not depend on placing the retinal sheet in correct orientation because the whole retinal organoids are transplanted. With development of flat hESC-retinal sheets, this will be important. However, it is not the focus of this protocol, which is aimed at delivering intact hESC-retinal tissue sheets and optimizing the subretinal preservation of PR sheets. Once the technique was developed, it was reproducible in both normal and RD retina.
There are many potential complications to this surgical procedure. Only those skilled in vitreoretinal surgery (e.g., trained veterinary ophthalmologists or vitreoretinal surgeons familiar with the species differences in the cat eye compared to human eye) should undertake the procedure. Possible complications include lens touch during trocar placement, scleral bleeding during sclerotomy enlargement, and subretinal or retinal hemorrhages. Other complications such as the host immune response to xenogeneic human organoid grafts leading to destruction of the graft over time or endophthalmitis are possible; the use of oral immunosuppressant and antibiotic medications help prevent these from occurring. It is also important to note there is a difference between implantation in wildtype and CrxRdy/+ cats. When there is advanced retinal degeneration, the retinal bleb tends to spread wider than in the wildtype, and, in some instances, this can prevent complete retinal reattachment after the surgery. The technique presented here is applicable for implantation of organoids in eyes of large animal models of IRDs. Further refinement would be needed once the transplantation is ready for translation to the clinic.
Based on the authors&#8217; experience with performing complex vitreoretinal surgical procedures in large eye models, the technique presented in this manuscript should be applicable to other large animal models (with the inclusion of endo-illumination in atapetal species) that are used for translating vitreoretinal surgical techniques to the clinic43,44,45.

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 &amp;micro;m pore syringe filter with PES membrane</td><td>Cameo</td><td>NA</td><td>can be found through 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 &amp;micro;L hamilton gas tight luer lock syringe</td><td>Hamilton</td><td>NA</td><td>can be found through 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 through various suppliers</td></tr><tr><td>Buprenorphine 0.3 mg/mL</td><td>Par Pharmaceutical</td><td>NA</td><td>can be found through 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 through various suppliers</td></tr><tr><td>Dexamethasone 2mg/mL (Azium)</td><td>Vetone</td><td>NA</td><td>can be found through various suppliers</td></tr><tr><td>Doxycyline 25mg/5mL</td><td>Cipla</td><td>NA</td><td>can be found through various suppliers</td></tr><tr><td>Fatal Plus solution (pentobarbital solution)</td><td>Vortech</td><td>NA</td><td>can be found through various suppliers</td></tr><tr><td>Gentamicin 20mg/2mL</td><td>Hospira</td><td>NA</td><td>can be found through 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 through 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 through 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 through various suppliers</td></tr><tr><td>Prednisolone 15mg/5mL</td><td>Akorn</td><td>NA</td><td>can be found through various suppliers</td></tr><tr><td>Propofol 500mg/50mL (10 mg/mL) (PropoFlo28)</td><td>Zoetis</td><td>NA</td><td>can be found through various suppliers</td></tr><tr><td>RetCam II video fundus camera</td><td>Clarity Medical Systems</td><td>NA</td><td>can be found through 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 through various suppliers</td></tr><tr><td>Tropicamide 1% ophthalmic solution</td><td>Akorn</td><td>NA</td><td>can be found through 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&amp;deg; 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 are not damaged. This can be confirmed by direct visualization during the loading of the organoids (step 2.10) and during the surgery (step 3.16) (Figure 2A,B) as well as by fundus imaging at the end of surgery (step 3.23, Figure 2C). The presence of the organoids in the subretinal space using this technique is confirmed post-operatively by ophthalmic examination and fundus imaging (Figure 3A), which records the position and appearance of the organoids. Knowing the position of the transplant is very important when processing the globes for frozen histology and immunohistochemistry and substantially reduces workload, as sectioning a large eye at 12&#8211;14 &#181;m (thickness of a cryosection) takes time. Prior to euthanasia, confocal scanning laser ophthalmoscopy (cSLO) and spectral domain &#8211; optical coherence tomography (SD-OCT) imaging are also performed to assess the position of the organoids in the subretinal space (Figure 3A-D). These techniques demonstrate the persistence of retinal organoids in the subretinal space (between the neural retina and RPE) of the recipient eye (Figure 3E). Following euthanasia (done humanely following AVMA recommendations) the histology and immunohistochemistry (IHC) is routinely performed (see details in previously published paper31). The histology and IHC demonstrate the survival of xenogeneic grafts (hPSC-derived retinal organoids) in the subretinal space of a large eye when the animals were immunosuppressed (as previously described31), see Figure 4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61683/61683fig01.jpg" /> 
Figure 1: Schematic of the steps for organoids preparation prior to the implantation. 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61683/61683fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61683/61683fig02v2.jpg" /> 
Figure 2: Surgical subretinal implantations of organoids.&#160;(A) Direct visualization of organoids being delivered into the subretinal space through a glass cannula without being damaged, (B) Direct visualization of organoids in the subretinal bleb, (C) Wide-angle fundus color image of the subretinally implanted organoids immediately after surgery. The bleb edges are indicated by the black arrowheads and the retinotomy site by the black stars. 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61683/61683fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61683/61683fig03v2.jpg" /> 
Figure 3: Post-operative assessment of subretinally implanted organoids 3 months post-implantation in a CrxRdy/+ cat.&#160;(A) Fundus color image of the subretinally implanted organoids, (B) cSLO fundus image of the subretinally implanted organoids, (C) 3D volume scan reconstruction of the area containing the organoids, (D) cSLO image of the area containing subretinal organoids, (E) SD-OCT high-resolution, cross-section image of the subretinally implanted organoids. The retinotomy site is indicated by the black stars. 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61683/61683fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61683/61683fig04.jpg" /> 
Figure 4: Human retinal organoid-derived photoreceptor sheets (PR marker RCVRN) in subretinal space of CrxRdy/+ cat, 3 months after grafting.&#160;&#160;*Synaptic boutons (hSYP=Synaptophysin) in cat inner nuclear layer (INL). 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61683/61683fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.9K Views.  Michigan State University. Het huidige protocol toont in detail subretinale toediening van de gehele retinale organoïden in een groot diermodel, gericht op het ontwikkelen van celtherapiebenaderingen voor retinale degeneratie. Het belangrijkste voordeel van deze techniek is dat het organoïden rechtstreeks aan het doelweefsel, het netvlies, kan afleveren. De techniek is gericht op de behandeling van geavanceerde blinderende retinale degeneratieve aandoeningen, zoals leeftijdsgebonden maculaire degeneratie, retinitis p...

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

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Medicine

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

Development of a Surgical Technique for Subretinal Implants in Rats

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.

The present protocol describes the scleral approach for subretinal device implantation, a feasible surgical technique for implementation in animal models of retinal diseases in research.

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been ...

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

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.

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.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Biology

Revitalizing Sight: Stem Cell Therapy for Retinal Degeneration

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

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.

Author Spotlight: Advancing Vision Restoration - Stem Cell-Based Therapy for Retinal Diseases 

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.

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video