We thank Wei Sheng Tan, Luanne Chiang Xue Yen, Xinyi Lee, and Yingying Chung for providing technical assistance for the preparation of the day 32 hESC-derived photoreceptor progenitors after cryopreservation. This work was supported in part by grants from the National Medical Research Council Young Investigator Research Grant Award (NMRC/OFYIRG/0042/2017) and National Research Foundation 24th Competitive Research Program Grant (CRP24-2020-0083) to H.G.T.

The in vivo experiments were done in accordance with the guidelines and protocol approved by the Institutional Animal Care and Use Committee of SingHealth (IACUC) and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in Ophthalmic and Vision Research. The pups were immunosuppressed from P17 (pre-transplantation) to P30 (post-transplantation) by feeding them drinking water containing cyclosporine (260 g/L).
1. Preparation of Day 32 hESC-derived photoreceptor progenitors after cryopreservation
<ol>
	<li>Pre-warm photoreceptor differentiation medium (PRDM) in a 37 &#176;C water bath.</li>
	<li>Retrieve a cryovial containing day 32 hESC-derived photoreceptor progenitor cells from liquid nitrogen. Keep on dry ice.</li>
	<li>Thaw the cryovial at 37 &#176;C in a water bath for 3-5 min. Resuspend the day 32 cells in 1 mL of PRDM and centrifuge at 130 x g for 4 min.</li>
	<li>Remove the supernatant and resuspend cells in 1 mL of PRDM.</li>
	<li>Remove 10 &#181;L of the mixture for cell counting. Mix cells using 0.2% trypan blue according to the manufacturer&#39;s instructions. Pipette cell mixture into the cell counting chamber slide. Determine cell number and viability by automated cell counter.</li>
	<li>Proceed to the next step when the cell viability is above 70%. Centrifuge the remaining cell suspension at 130 x g for 4 min. Remove the supernatant and resuspend the cell pellet in PRDM at the concentration of 3 x 105 cells/&#181;L for transplantation.</li>
	<li>Observe for visible cell clumps. Resuspend cell clumps repeatedly using a 10 &#181;L pipette tip in the microfuge tube until no visible cell clump is observed. Load into the 33G injection syringe to observe for extrusion of cell solution through the needle.</li>
</ol>
2. Sub-retinal delivery of the hESCs in rd10 mice
<ol>
	<li>Preparation of the animals
	<ol>
		<li>Anaesthetize the mouse (P20, male/female, 3-6 g) using a combination of ketamine (20 mg/kg body weight) and xylazine (2 mg/kg body weight) in a 1 mL tuberculin syringe attached to a 27G needle by intraperitoneal approach. Administer a subcutaneous injection of buprenorphine (0.05 mg/kg) to the mouse as a pre-emptive analgesic.</li>
		<li>After administering the anesthesia, instill a drop each of 1% tropicamide and 2.5% phenylephrine for pupil dilation. Apply an ophthalmic gel to the cornea to prevent the eye from dryness and anesthesia-related cataract.</li>
		<li>Place the mouse in an empty cage until fully anesthetized. Assess the proper anesthetic level by pinching the paw pad and confirm if the animal does not react to the hard pinch.</li>
		<li>When the mouse is fully anesthetized, place the animal on a warm pad set at 38 &#176;C.</li>
	</ol>
	</li>
	<li>Sub-retinal delivery of the cells
	<ol>
		<li>To use a 1-port trans-vitreal pars plana approach, as done here, perform sub-retinal injection in a sterile environment. Use an upright operating microscope with a direct light path to perform the injection.</li>
		<li>Prepare the 10 &#181;L glass syringe by removing the needle hub. Mount the 33G blunt needle onto the glass syringe. Take the metal hub cover and carefully secure the needle on the syringe.</li>
		<li>Flush with distilled water to check for any signs of leakage and patency of the needle. Empty the syringe and put it at the side carefully.</li>
		<li>Place the anesthetized mouse on a pillow, with the treatment eye looking up straight to the objective of the microscope. Apply the 0.5% proparacaine hydrochloride and wait for 30 s. Apply 150 &#181;L of ophthalmic gel on the eye and place a round cover slip on it.</li>
		<li>Perform a rough examination of the eye by observing the cornea, iris, pupil, lens, and conjunctiva. Through the pupil, visualize the fundus of the mouse eye by adjusting the focal plane. Adjust the head until the optic head is positioned at the center of the pupil and minimize the movement of the head by proper positioning on the pillow.</li>
		<li>Gently tap the base of the tube with the hESCs multiple times to get a uniform cell suspension. By using the 10 &#181;L glass microliter syringe with a 33G blunt needle, withdraw 2 &#181;L of cells/media. Withdraw the cells right before the injection to avoid cell settlement/clumping in the syringe.</li>
		<li>Using a 30G disposable needle, make a sclerectomy wound 2 mm behind the limbus. Keep the angle of the needle at ~45&#176; to avoid touching the lens. Once the tip of the needle is visualized in the eye, gently withdraw the needle. Discard the needle into the sharp bin after use to avoid needle prick injury.</li>
		<li>Take the glass syringe and insert the blunt needle into the sclerectomy wound. Without touching the lens, advance the blunt needle until it reaches the opposite retina of the entry wound. Ensure the injection area is clear of major retinal blood vessels to avoid bleeding.</li>
		<li>Gently penetrate the retina until a pressure branch on the sclera is seen. Keep the blunt end of the needle parallel to the sclera to avoid leaking of the cells into the vitreous space.</li>
		<li>Slowly inject 2 &#181;L of cell suspension or PRDM media (control) into the sub-retinal space while gentle pressure is maintained on the syringe. With a successful injection, a visible bleb (i.e., raised retina with the cell suspension/media in it) should be formed at the injection site. 
		NOTE: Only gentle pressure should be used during injection to avoid retina tearing and blockage of the cells at the needle tip.</li>
		<li>After confirming the bleb, wait for 10 s to let the cells settle down. Gently retract the needle from the eye.</li>
	</ol>
	</li>
	<li>Intraoperative optical coherence tomography (OCT) scanning of the bleb (optional)
	<ol>
		<li>Position the mouse eye to visualize the bleb under the microscope by slowly moving the head. Secure the position by gently holding the head. No additional pupil dilation is necessary. Do not remove the cover slip and the gel on the eye. It gives a clear optical media to visualize the bleb.</li>
		<li>Perform the intra-operative OCT using the built-in iOCT function of the operating microscope.</li>
		<li>Press the Cube option on the OCT screen and position the scanning area on the bleb by pressing the Arrow buttons. Adjust the OCT by sliding Centering and Focus for the best OCT quality. Press Capture/Scan to acquire the OCT scan of the bleb area. Review the images to check the quality of the scans.</li>
	</ol>
	</li>
	<li>Recovery
	<ol>
		<li>Remove the cover slip and clean the gel from the eye with gauze. Apply antibiotic ointment one time after the injection to prevent infection.</li>
		<li>Allow the animal to recover from anesthesia under a warm light until they regain sufficient consciousness to maintain sternal recumbency and return to the home cage. Monitor the animal for at least 3 days post-injection for signs of inflammation, infection, and distress. 
		NOTE: The 150 W warm light should be at least 30 cm away from the animal cage, and caution should be taken to avoid a burn injury.</li>
		<li>Administer a subcutaneous injection of buprenorphine (0.05 mg/kg) 8 hourly for 1 day or per veterinarian&#8217;s recommendation. If inflammation or infection of the eye is observed, consult a veterinary professional for appropriate treatment.</li>
	</ol>
	</li>
	<li>Cleaning and sterilization of the instruments
	<ol>
		<li>Flush the 10 &#181;L glass microliter syringe and the 33G blunt needle with 100% ethanol 10x. Wash away the ethanol by flashing the syringe with distilled water.</li>
		<li>Dissemble the glass syringe and the needle. Dry the syringe for storage.</li>
	</ol>
	</li>
</ol>

Revitalizing Sight: Stem Cell Therapy for Retinal Degeneration

Hwee Goon Tay is a co-founder of Alder Therapeutics AB. Other authors declare no competing interests.

The sub-retinal injection has been used for cell suspension transplantation to treat RPE and retinal diseases23,25,26,27,28,31,40. This approach is highly essential in rodent studies not only for cell transplantation and gene therapy approaches but also to evaluate novel therapeutic compound...

Inherited retinal diseases and age-related macular degeneration lead to photoreceptor cell loss and eventual blindness. The retinal photoreceptor is the outer segment layer of the retina comprised of specialized cells responsible for phototransduction (i.e., conversion of light to neuronal signals). The rod and cone photoreceptor cells are adjacent to the retinal pigmented layer (RPE)1. Photoreceptor cell replacement therapy to compensate the cell loss has been an emerging and developing therapeutic approach. Embryonic stem cells (ESCs)2,3,4, induced pluripotent stem cells (iPSCs)-derived RPE cells, and retinal progenitor cells (RPCs)4,5,6,7,8 were used to restore the damaged photoreceptor cells. Sub-retinal space, a confined space between the retina and the RPE, is an attractive location to deposit these cells to replace damaged photoreceptor cells, RPE, and Mueller cells due to its vicinity9,10,11.
Gene and cell therapies have been utilizing the sub-retinal space for regenerative medicine for various retinal diseases in pre-clinical studies. This includes the delivery of functional copies of the gene or gene editing tools in the form of either anti-sense oligonucleotide therapy12,13 or CRISPR/Cas9 or base editing via adeno-associated virus (AAV) based strategy14,15,16, implantation of materials (e.g., RPE sheet, retinal prosthetics17,18,19) and differentiated stem cell-derived retinal organoids20,21,22 to treat retinal and RPE-related diseases. Clinical trials using hESC-RPE31 in the sub-retinal space to treat RPE65-associated Leber congenital amaurosis (LCA)23,24, CNGA3-linked achromatopsia25, MERTK-associated retinitis pigmentosa26, choroideremia27,28,29,30 have been proven to be an effective approach. Direct injection of cells to the vicinity of the damaged area greatly improves the chance of cell settlement at the appropriate region, synaptic integration, and eventual visual improvement.
Even though sub-retinal injection in human and large-eyed models (i.e., pig32,33,34,35, rabbit36,37,38,39,40, and non-human primate41,42,43) has been&#160;established, such injection in the murine model is still challenging due to the constrain of the eyeball size and enormous lens occupying the mouse eye44,45,46. However, genetically modified models are only readily available in small animals and not in large animals (i.e., rabbits and non-human primates), therefore sub-retinal injection in mice draws attention to investigate novel therapeutic approaches in retinal genetic disorders. Three major approaches are being used to deliver cells or AAVs into the sub-retinal space, namely the trans-corneal route, trans-scleral route, and the pars plana route (See Figure 2). Trans-corneal and trans-scleral routes are associated with cataract formation, synechiae, choroidal bleeding, and reflux from the injection site11,44,45,47,48,49. We adopted the pars plana approach as a direct visualization of the injection process, and the injection site can be achieved in real-time under the microscope.
We recently described a method that can differentiate human embryonic stem cells (hESCs) into photoreceptor progenitors under xenofree, chemically defined conditions using recombinant human retina-specific laminin isoform LN523. Since LN523 was found to be present in the retina, we hypothesized that the extracellular matrix niche of the human retina could be recapitulated in vitro and thereby support photoreceptor differentiation from the hESCs36. Single-cell transcriptomic analysis showed that photoreceptor progenitors co-expressing cone-rod homeobox and recoverin were generated after 32 days. A retinal degeneration 10 (rd10) mutant mouse model that mimics autosomal human retinitis pigmentosa was used to evaluate the efficacy of the day 32 hESC-derived photoreceptor progenitors in-vivo. The hESC-derived photoreceptor progenitor cells were injected into the sub-retinal space of rd10 mice at P20, where photoceptor dysfunction and degeneration are ongoing36. Here, we describe a detailed protocol for the preparation of the post-cryopreserved hESC-derived photoreceptor progenitors and delivery into the sub-retinal space of rd10 mice. This method can also be used to administer AAVs, cell suspensions, peptides, or chemicals into the sub-retinal space in mice.

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

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.

The 10 &#181;L glass syringe was assembled according to the manufacturer&#39;s instructions (Figure 1), and the blunt needle used to deliver the cell suspension/media is shown in Figure 1B. Different approaches for sub-retinal injection are illustrated in Figure 2. We describe the pars plana approach in this protocol (Figure 2C). The blunt needle mounted on a glass syringe was inserted through a sclerot...

Watch this Scientific Journal Video about Advancing Vision Restoration — Stem Cell-Based Therapy for Retinal Diseases at JoVE.com

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.

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 ...

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840 Views.  Singapore Eye Research Institute. Mijn onderzoek richt zich op de ontwikkeling van stamceltherapie voor gevorderde stadia van netvliesaandoeningen die fotoreceptordegeneratie veroorzaken. Het doel van ons onderzoek is om het verlies van fotoreceptoren bij patiënten te vervangen door klinisch veilige en functionele fotoreceptorvoorlopercellen met als doel hun gezichtsscherpte te verbeteren of zelfs hun gezichtsvermogen te herstellen. Het belangrijkste preklinische model zijn muizen, maar hun kleine oogbol maakt het een uitdag...

Video: Auteur in de schijnwerpers: Geavanceerd herstel van het gezichtsvermogen - op stamcellen gebaseerde therapie voor netvliesaandoeningen 

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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 ...

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 ...

Neuroscience

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.

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 ...

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. ...

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, ...

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

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