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 &#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 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

1.8K Views.  Michigan State University. 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.

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 ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

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 ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

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

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Chapters in this video

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

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

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

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

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders