This study was conducted in accordance with the Tenets of the Declaration of Helsinki and approved by the Institutional Ethics Committee of the Chinese PLA General Hospital. The WA09 (H9) ESC line was obtained from the WiCell Research Institute.
1. Culture media and reagent preparation
<ol>
	<li>Human ESC culture medium and passage solution
	<ol>
		<li>Maintenance medium (MM): Prepare 500 mL of complete MM (Basal Medium + 5x supplement; see Table of Materials) aseptically. Thaw 5x supplement at room temperature (RT) or overnight at 2-8 &#176;C. Prewarm to RT. Stir well before use until the supplement is free of cloudiness.</li>
		<li>1% extracellular matrix (ECM): Use a pre-chilled pipet tip and sterile tubes to dispense 200 &#181;L of ECM (see Table of Materials) per tube on ice. Store the tubes in a -20 &#176;C freezer. Melt ECM on ice and dilute with pre-cooled DMEM/F12 at 1:100.</li>
		<li>0.5 mM pH = 8.0 EDTA: To prepare 500 mL of 0.5 mM EDTA, add 500 &#181;L of 0.5 M EDTA and 0.9 g of NaCl to 500 mL of 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) (see Table of Materials). Mix thoroughly and store at 2-8 &#176;C.</li>
	</ol>
	</li>
	<li>Retinal differentiation medium
	<ol>
		<li>Growth factor-free chemically defined medium (gfCDM): Prepare the gfCDM by combining 45% Iscove&#39;s modified Dulbecco&#39;s medium-GlutaMAX (IMDM-GlutaMAX), 45% Ham&#39;s F12-GlutaMAX (F12-Glutamax), 10% knockout serum replacement (KSR), 1% cholesterol lipid concentrate, and 450 &#181;M thioglycerol (see Table of Materials).</li>
	</ol>
	</li>
	<li>Small molecule compounds
	<ol>
		<li>Y-27632 2HCl: Add 50 mg of Y-27632 powder to 3.122 mL of dimethyl sulfoxide (DMSO). Dispense and store Y-27632 (see Table of Materials) at a concentration of 50 mM at -80 &#176;C for 2 years, keeping away from light. Dilute the stock 2,500 times (20 &#181;M) for use, equivalent to 0.4 &#181;L of Y-27632 per mL of gfCDM.</li>
		<li>IWR1-endo: Add 2.4424 mL of DMSO to dissolve 10 mg of IWR1-endo (see Table of Materials) to obtain a 10 mM stock solution. Dispense aliquots and store them at -80 &#176;C for up to 2 years. Add 0.3 &#181;L of 10 mM IWR1-endo per mL of gfCDM for the 3 &#181;M concentration for induction.</li>
		<li>SB431542: To prepare 50 mM SB431542, add 10 mg of SB431542 (see Table of Materials) to 0.5203 mL of DMSO and mix thoroughly. Store at -80 &#176;C in solvent for a maximum of 2 years. For the preparation of SB431542 at a working concentration of 10 &#181;M, add 2 &#181;L of the 50 mM stock solution to 10 mL of gfCDM.</li>
		<li>LDN-193189 2HCl: Dissolve 5 mg of LDN-193189 2HCl (see Table of Materials) in 10.4297 mL of DMSO to get a 1 mM of stock solution. Store at -20 &#176;C or -80 &#176;C (as recommended by the manufacturer). Add 0.1 &#181;L of the stock to every 10 mL of gfCDM, resulting in 100 nM of LDN-193189 for induction.</li>
		<li>Recombinant Human bone morphogenetic protein 4 (BMP4): Reconstitute at 50-200 &#181;g/mL in 4 mM HCl (see Table of Materials). Store at 2 &#176;C to 8 &#176;C for 1 month or -20 &#176;C for 1 year after reconstitution. Perform induction of NR using 1.5 nM BMP4.</li>
	</ol>
	</li>
	<li>Long-term NR culture medium
	<ol>
		<li>Retinoic acid (RA): Measure 6 mg of RA powder (see Table of Materials) and add to 3.9941 mL of DMSO. Store in aliquots of 5 mM at &#8722;80 &#176;C and use within 3 months. To achieve a working concentration of 0.5 &#181;M, add 10 &#181;L of the master mix to 100 mL of neural retina differentiation medium (NRDM). Add just before use. 
		NOTE: Keep away from light during preparation and storage.</li>
		<li>Taurine: Add taurine powder (see Table of Materials) weighing 200 mg to 7.9904 mL of DMSO to obtain a 200 mM stock solution. Dispense and store at 2-8 &#176;C. A working concentration of 0.1 mM taurine is achieved by adding 50 &#181;L of the stock solution per 100 mL of NRDM.</li>
		<li>NRDM: Compose NRDM with DMEM/F12-GlutaMAX medium, 1% N2 supplement, 10% fetal bovine serum, 0.5 mM RA and 0.1 mM taurine (see Table of Materials). Store at 2-8 &#176;C for up to 2 weeks or 6 months at -20 &#176;C to ensure the activity of the components.</li>
	</ol>
	</li>
	<li>Blocking buffer: Dilute 1:9 with DPBS to obtain a 10% working solution for use (see Table of Materials). Store at -20 &#176;C for up to 5 years. 
	&#8203;NOTE: Perform all other procedures in a Class II Biosafety Cabinet to ensure sterility, except for weighing. If adding weighed reagents during the preparation process is necessary, use 0.22 &#181;m filters for filtration.</li>
</ol>
2. Culturing of H9-ESCs
<ol>
	<li>Thawing of H9-ESCs
	<ol>
		<li>Add 1 mL of 1% ECM to each well of a 6-well plate. Incubate for 1 h in an incubator at 37 &#176;C in a 5% CO2 atmosphere. 
		NOTE: Avoid the addition of ECM along the well walls, because it may stick to the surface.</li>
		<li>Take an H9-ESC cryovial stock from the liquid nitrogen tank and quickly shake it in 37 &#176;C water for 30 s. 
		NOTE: Do not allow the vial to thaw completely.</li>
		<li>Remove the vial and carefully sterilize it using 75% disinfectant alcohol spray. Add the thawed H9-ESC from the cryovial to a 15 mL tube containing 9 mL MM with 10 &#181;M Y-27632.</li>
		<li>Centrifuge the tube at 190 &#215; g for 5 min at RT. Carefully remove most of the supernatant with a 1 mL pipette, leaving approximately 50 &#181;L of supernatant to avoid cell loss.</li>
		<li>Add 1 mL of MM containing 10 &#181;M of Y27632 to the cell sediment and gently resuspend the cell sediment with a 1 mL pipette by pipetting up and down 5-10 times.</li>
		<li>Remove the ECM coating after 1 h of incubation. Add 2 mL of pre-warmed MM containing 10 &#181;M Y-27632 to each well.</li>
		<li>Dispense 0.5 mL of cell suspension per well. Gently shake the plate laterally to ensure even distribution of cells.</li>
		<li>Incubate the plate at 37 &#176;C under 5% CO2 for at least 24 h without touching it.</li>
		<li>Change the medium daily. When clone density reaches 70% and above, passaging is required.</li>
	</ol>
	</li>
	<li>Passaging of H9-ESCs
	<ol>
		<li>Prepare the ECM-coated plate as described above (step 2.1.1) and add 2 mL of MM per well.</li>
		<li>Remove the spent medium from the 6-well plates. Wash each well twice with 1 mL of DPBS.</li>
		<li>Wash each well twice by slowly adding 1 mL of EDTA, and incubating with 1 mL of EDTA for 4-7 min at RT. 
		NOTE: During the incubation, examine the 6-well plate for 3 min under a microscope. Proceed immediately to the next step if most cells are close to detaching from the dish. Avoid long time incubation to reduce negative impact on the cells.</li>
		<li>Discard EDTA and add 1 mL of MM to abort digestion.</li>
		<li>Gently tap the well plate until the majority of the cells are detached.</li>
		<li>Carefully transfer the cell suspension into a 15 mL centrifuge tube with a 5 mL pipette. Gently resuspend the H9-ESC colonies up and down 3-5 times to mix them with a Pasteur pitette. Transfer 20-100 &#181;L of cell suspension in a new ECM-coated 6-well culture dish and shake it back and forth.</li>
		<li>When the cell density is observed as sufficient under a microscope, incubate the plate at 37 &#176;C under 5% CO2 for at least 24 h without touching it.</li>
		<li>Change the media daily. At 70% clone density and above, passage is required.</li>
	</ol>
	</li>
</ol>
3. Generation of human NRs
NOTE: Once the colonies achieve approximately 70% confluence, they can be directed towards differentiation into retinal organoids (ROs) using the procedural steps outlined in Figure 1.
<ol>
	<li>Day 0 - Embryoid body (EB) formation
	<ol>
		<li>After washing the cells with 2 mL of DPBS, add 0.5 mL of CDS (see Table of Materials) containing 20 &#181;M Y27632 to the well. Incubate for 3 min at 37 &#176;C in a humidified 5% CO2 incubator. 
		NOTE: Check under a microscope. Reduce the time of incubation as much as possible to avoid harmful effects on the cells.</li>
		<li>Abort digestion by adding 3 mL of MM containing 20 &#181;M Y27632. Centrifuge at 190 &#215; g for 5 min and discard supernatant.</li>
		<li>Resuspend the cell pellet in 1 mL of gfCDM containing 20 &#181;M Y27632, and count the cells. Add the corresponding volume for 1.2 &#215; 106 cells (1.2 &#215; 104 cells per well) to 10 mL of gfCDM, which is pre-incorporated with 20 &#181;M Y-27632, 3 &#181;M IWR1-endo, 10 &#181;M SB431542, and 100 nM LDN-193189 (see Table of Materials).</li>
		<li>Add 100 &#181;L of cell suspension to each well of 96 V-bottomed conical wells. Place the plate in a humidified 5% CO2 incubator until day 6. 
		NOTE: Do not move the dishes for at least 24 h to enhance the adherence of EBs.</li>
	</ol>
	</li>
	<li>Day 6 - Retina induction
	<ol>
		<li>On day 6, add 10 mL of gfCDM containing 55 ng/mL BMP4 to allow a complete change of culture medium on the EBs. Return the plate to the incubator. 
		NOTE: Pipette toward the walls of the 96 V-bottomed conical well to avoid air bubbles. Avoid taking up any organoids when changing the medium.</li>
		<li>Carry out half-medium change on day 9, day 12 and day 15. Replace half of the medium with fresh gfCDM to gradually dilute the BMP4.</li>
	</ol>
	</li>
	<li>Day 18 - Long term NR culture
	<ol>
		<li>On day 18, carefully transfer the formed EBs to 15 mL centrifuge tubes using 5 mL Pasteur pipettes and gently rinse again with NRDM. Transfer the EBs to low-adsorption 6-well or 24-well plates (see Table of Materials). Return the plate to the incubator.</li>
		<li>Replace the medium with fresh NRDM every 3 days. 
		&#8203;NOTE: Switch off the light during the medium change because the RA is light-sensitive. Remove badly differentiated organoids, and separate adherent organoids under a microscope.</li>
	</ol>
	</li>
</ol>
4. Analysis of human NRs
<ol>
	<li>Mounting of the ROs
	<ol>
		<li>Select different generations of H9-ESCs to induce three batches of ROs. 
		NOTE: Three plates of the number of forming NRs for each of the three assessed methods (Kuwahara et al.12, D&#246;pper et al.27, and the modified method in the present study) are calculated to assess the induction success rate. Induction is considered successful if light microscopy reveals the formation of neuroepithelial-like structures on day 30.</li>
	</ol>
	</li>
	<li>Immunofluorescent staining of ROs
	<ol>
		<li>Transfer 3-5 ROs to 1.5 mL tubes. Pipette off excess medium and wash the ROs once with 1 mL of DPBS at RT. Allow the ROs to sink and carefully remove the DPBS. Add 1 mL of 4% paraformaldehyde (see Table of Materials) per 1.5 mL tube and fix at 4 &#176;C. 
		NOTE: ROs on day 6 and day 18, fix 2 h. Fix for 12 h on day 30 and 14 h on day 60.</li>
		<li>After fixation, dehydrate using gradient alcohol. Leave the ROs to stand for 15 min each in 50%, 60%, and 70% alcohol, and subsequently for 10 min each in 80%, 90%, 95%, 100%, and 100% alcohol. Then, add a 1:1 mix of 100% alcohol and xylene for 10 min, followed by xylene twice for 10 min each.</li>
		<li>Place the ROs in metal molds filled with melted paraplast (see Table of Materials) for 40 min, and then quench the molds on ice to fix the ROs. Place embedding boxes into the molds and freeze them at -20 &#176;C overnight. Subsequently, carefully detach the embedding boxes. Finally, store them at RT.</li>
		<li>Create continuous sections (5 &#181;m thickness) with a paraffin slicer. Fix slices on adhesion microscope slides, dry, and store at RT.</li>
		<li>Perform dewaxing and rehydration before antigen repair12,27.</li>
		<li>Add 10 mL of 20x pH 6.0 Citrate Antigen Retrieval Solution (see Table of Materials) to 190 mL of ddH2O (double distilled H2O). Heat in a microwave on high mode for 4 min until boiling. After adding paraffin sections, heat the sections on low mode for 20 min to complete antigen repair.</li>
		<li>Allow paraffin sections to cool naturally in a ventilated area and then put them into a humid chamber.</li>
		<li>Use a pap pen (see Table of Materials) to outline the sections. Incubate with 0.2% Triton X-100 at RT for 30 min to break the membranes, followed by washing the slides three times with TPBS, for 5 min each.</li>
		<li>Block with 10% donkey serum diluted in DPBS at RT for 1 h in a humid chamber with 10 &#181;L per staining area.</li>
		<li>Add primary antibodies diluted in 10% donkey serum. Incubate overnight at 4 &#176;C in a humidity chamber. Wash samples thrice with TPBS for 10 min each to remove the unbound antibody. 
		NOTE: Primary antibodies are as follows: anti-PAX6 (1: 250), anti-SOX2 (1: 200), anti-KI67 (1: 200), anti-CHX10 (1: 200), anti-&#946; tubulin III (1: 250) and anti-NESTIN (1: 200) (see Table of Materials).</li>
		<li>Incubate with secondary antibodies diluted in DPBS for 1 h at RT in a humidified chamber. Repeat the wash step with TPBS three times for 10 min each. 
		NOTE: Keep in the dark to prevent quenching of the fluorescent secondary antibody. Secondary antibodies are Alexa Fluor 488 conjugated with donkey anti-mouse IgG and Alexa Fluor 568 conjugated with donkey anti-rabbit IgG at a dilution of 1:400 (see Table of Materials).</li>
		<li>Incubate with DPBS containing DAPI (1: 500; see Table of Materials) for 10 min at RT in the dark. Wash three times with TPBS for 10 min each.</li>
		<li>Drop an appropriate amount of anti-fluorescent sealer (see Table of Materials) onto the slide and cover with cover slips.</li>
		<li>Visualize using a flow-like tissue cell quantitative analyzer (see Table of Materials), or similar.</li>
		<li>Store slides at -20 &#176;C in the dark after microscopic analysis.</li>
	</ol>
	</li>
</ol>

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoblastoma+from+human+stem+cell-derived+Retinal+organoids.">Retinoblastoma from human stem cell-derived Retinal organoids.</a> Nat Commun. 12 (1), 4535 (2021).</li></ol>

All authors declare that they have no conflicts of interest.

Human ROs can spatially and temporally recapitulate the development of the fetal retina, and early ROs exhibit a high degree of similarity to the fetal retina at equivalent stages of development15. In terms of tissue morphology and molecular expression, human RO closely mirror the actual growth status of the retinal tissue, providing tremendous and unprecedented opportunities in the fields of disease modeling, drug screening, and regenerative medicine. Currently, several different methods have bee...

The eye serves as the primary source of information among human sensory organs, with the retina being the principal visual sensory tissue in mammalian eyes1. Retinopathy stands as one of the primary global causes of eye diseases, leading to blindness2. Approximately 2.85 million people worldwide suffer from varying degrees of vision impairment due to retinopathy3. Consequently, investigating its pathogenesis is crucial for early diagnosis and timely treatment. Most studies on human retinopathy have primarily focused on animal models4,5,6. However, the human retina is a complex, multi-layered tissue comprising various cell types. Traditional two-dimensional (2D) cell culture and animal model systems typically fail to faithfully recapitulate the normal spatiotemporal development and drug metabolism of the native human retina7,8.
Recently, 3D culture techniques have evolved to generate tissue-like organs from pluripotent stem cells (PSCs)9,10. Retinal organoids (ROs) generated from human PSCs in a 3D suspension culture system not only contain seven retinal cell types but also exhibit a distinct stratified structure similar to the human retina in vivo11,12,13. Human PSC-derived ROs have gained popularity and widespread availability and are currently the best in vitro models for studying the development and disease of the human retina14,15. Over the past few decades, numerous researchers have demonstrated that human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into ROs using various induction protocols. These advancements hold enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies16,17,18.
However, the generation of neural retina (NR) from human pluripotent stem cells (PSCs) is a complex, cumbersome, and time-consuming process. Furthermore, batch-to-batch variations in tissue organoids may lead to lower reproducibility of results19,20. Numerous intrinsic and extrinsic factors can influence the yield of retinal organoids (ROs), such as the number or species of starting cells and the use of transcription factors and small-molecule compounds21,22,23. Since the first human RO was generated by the Sasai laboratory11, multiple modifications have been proposed over the years to enhance the ease and effectiveness of the induction process13,21,24,25. Unfortunately, to date, no gold standard protocol has been established for generating ROs in all laboratories. Indeed, there is a certain degree of discrepancy in ROs resulting from different induction methods, as well as wide variation in the expression of retinal markers and the robustness of their structure22,26. These issues may severely complicate sample collection and the interpretation of study findings. Therefore, a more consolidated and robust differentiation protocol is needed to maximize efficiency with minimal heterogeneity of RO generation.
This study describes an optimized induction protocol based on a combination of Kuwahara et al.12 and D&#246;pper et al.27 with detailed instructions. The new method significantly reduces the probability of organoid vesiculation and fusion, increasing the success rate of generating NR. This development holds great promise for disease modeling, drug screening, and cell therapy applications for retinal disorders.

<table><tbody><tr><td>0.01 M TPBS</td><td>Servicebio</td><td>G0002</td><td>Washing slices</td></tr><tr><td>4% Paraformaldehyde</td><td>Servicebio</td><td>G1101-500ML</td><td>Fix retinal organoids</td></tr><tr><td>5 mL Pasteur pipette</td><td>NEST Biotechnology</td><td>318516</td><td>Pipette retinal organoids</td></tr><tr><td>96 V-bottomed conical wells</td><td>Sumitomo Bakelit</td><td>MS-9096VZ</td><td></td></tr><tr><td>Adhesion Microscope Slides</td><td>CITOTEST</td><td>188105</td><td>Fix slices</td></tr><tr><td>AggreWell medium</td><td>STEMCELL Technologies</td><td>5893</td><td>Medium</td></tr><tr><td>Anhydrous ethanol</td><td>SINOPHARM</td><td>10009218</td><td>Dehydrate&amp;nbsp;</td></tr><tr><td>Anti-CHX10</td><td>Santa Cruz</td><td>sc-365519</td><td>Primary antibody</td></tr><tr><td>Antifade Solution</td><td>ZSGB-BIO</td><td>ZLI-9556</td><td></td></tr><tr><td>Anti-KI67</td><td>Abcam</td><td>ab16667</td><td>Primary antibody</td></tr><tr><td>Anti-NESTIN</td><td>Sigma</td><td>N5413</td><td>Primary antibody</td></tr><tr><td>Anti-Neuronal Class III &amp;beta;-Tubulin(TUJ1)</td><td>Beyotime</td><td>AT809</td><td>Primary antibody</td></tr><tr><td>Anti-PAX6</td><td>Abcam</td><td>ab195045</td><td>Primary antibody</td></tr><tr><td>Cell dissociation solution(CDS)</td><td>STEMCELL Technologies</td><td>7922</td><td>Cell dissociation</td></tr><tr><td>CHIR99021</td><td>Selleckchem</td><td>S2924</td><td>GSK-3&amp;alpha;/&amp;beta; inhibitor</td></tr><tr><td>Cholesterol Lipid Concentrate</td><td>Gibco</td><td>12531018</td><td>250&amp;times;</td></tr><tr><td>Citrate Antigen Retrieval Solution</td><td>Servicebio</td><td>G1202-250ML</td><td>20&amp;times;, pH 6.0</td></tr><tr><td>CS10</td><td>STEMCELL Technologies</td><td>1001061</td><td>Cell Freezing Medium</td></tr><tr><td>DAPI</td><td>Roche</td><td>10236276001</td><td>Nuclear counterstain</td></tr><tr><td>Dimethyl sulfoxide(DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11330032</td><td>Medium</td></tr><tr><td>DMEM/F12-GlutaMAX</td><td>Gibco</td><td>10565018</td><td>Medium</td></tr><tr><td>Donkey anti-Mouse Alexa Fluor Plus 488</td><td>Invitrogen</td><td>A32766</td><td>Secondary Antibody</td></tr><tr><td>Donkey anti-Rabbit Alexa Fluor 568</td><td>Invitrogen</td><td>A10042</td><td>Secondary Antibody</td></tr><tr><td>Ethylene Diamine Tetraacetic Acid (EDTA)</td><td>Biosharp</td><td>BL518A</td><td>0.5 M, pH 8.0, cell dissociation</td></tr><tr><td>Extracellular matrix (ECM)</td><td>Corning</td><td>354277</td><td>Coating plates</td></tr><tr><td>F12-Glutamax</td><td>Gibco</td><td>31765035</td><td>Medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>A5669701</td><td></td></tr><tr><td>Flow-like tissue cell quantitative analyzer</td><td>TissueGnostics</td><td>TissueFAXS Plus</td><td>Scan sections</td></tr><tr><td>IMDM-GlutaMAX</td><td>Gibco</td><td>31980030</td><td>Medium</td></tr><tr><td>IWR1-endo</td><td>Selleckchem</td><td>S7086</td><td>Wnt-inhibitor</td></tr><tr><td>KnockOut Serum Replacement</td><td>Gibco</td><td>10828028</td><td></td></tr><tr><td>LDN-193189 2HCl</td><td>Selleckchem</td><td>S7507</td><td>BMP-inhibitor</td></tr><tr><td>Low-adhesion 24-well Plates</td><td>Corning</td><td>3473</td><td></td></tr><tr><td>Low-adhesion 6-well Plates</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>Maintenance medium (MM)</td><td>STEMCELL Technologies</td><td>85850</td><td>Medium</td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Solarbio</td><td>SL050</td><td>Blocking buffer</td></tr><tr><td>Paraplast</td><td>Leica</td><td>39601006</td><td>Tissue embedding</td></tr><tr><td>PBS pH 7.4 basic (1x)</td><td>Gibco</td><td>C10010500BT</td><td>Without Ca+,Mg+</td></tr><tr><td>Reconbinant human bone morphogenetic protein-4(rhBMP4)</td><td>R&amp;amp;D</td><td>314-BP</td><td>Key protein factor</td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td>Powder, keep out of light</td></tr><tr><td>SB431542</td><td>Selleckchem</td><td>S1067</td><td>ALK5-inhibitor</td></tr><tr><td>SU5402</td><td>Selleckchem</td><td>S7667</td><td>Tyrosine kinase inhibitor</td></tr><tr><td>Super PAP Pen</td><td>ZSGB-BIO</td><td>ZLI-9305</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>T0625-10G</td><td></td></tr><tr><td>Thioglycerol</td><td>Sigma</td><td>M1753</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td>Permeabilization</td></tr><tr><td>WA09 embryonic stem cell line</td><td>WiCell Research Institute</td><td></td><td>Cell line</td></tr><tr><td>Xylene</td><td>SINOPHARM</td><td>10023418</td><td>Dewaxing</td></tr><tr><td>Y-27632 2HCL</td><td>Selleckchem</td><td>S1049</td><td>ROCK-inhibitor</td></tr></tbody></table>

generating neural retina from human pluripotent stem cells

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of retinopathy. Unfortunately, ethical barriers hinder the collection of evidence from humans. Recently, numerous studies have shown that human pluripotent stem cells (PSCs) can be differentiated into retinal organoids (ROs) using different induction protocols, which have enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies. This study describes an optimized induction protocol to generate neural retina (NR) that significantly reduces the probability of vesiculation and fusion, increasing the success rate of production until day 60. Based on the ability of PSCs to self-reorganize after dissociation, combined with certain complementary factors, this new method can specifically drive NR differentiation. Furthermore, the approach is uncomplicated, cost-effective, exhibits notable repeatability and efficiency, presents encouraging prospects for personalized models of retinal diseases, and supplies a plentiful cell reservoir for applications such as cell therapy, drug screening, and gene therapy testing.

A graphical overview of the modified protocol is shown in Figure 1. H9-ESCs were used to generate ROs when the cells were grown to a density of 70%-80%. Single-cell suspensions of H9-ESCs in 96 V-bottomed conical wells aggregated on day 1 and formed well-circumscribed round EBs by day 6. As the culture time increased, the volume of EBs gradually increased. On day 30, neuroepithelial-like structures were clearly formed and thickened during long-term NR differentiation.
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Watch this Scientific Journal Video about Generating Neural Retina from Human Pluripotent Stem Cells at JoVE.com

Author Spotlight: Simple and Efficient Neural Retina Organoid Production for Disease Modeling

The present protocol describes an optimized 3D neural retina induction system that reduces the adhesion and fusion of retinal organoids with high repeatability and efficiency.

Generating Neural Retina from Human Pluripotent Stem Cells

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of ...

generating-neural-retina-from-human-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

1.1K Views.  Medical School of Chinese PLA. The present protocol describes an optimized 3D neural retina induction system that reduces the adhesion and fusion of retinal organoids with high repeatability and efficiency.

Video: Author Spotlight: Simple and Efficient Neural Retina Organoid Production for Disease Modeling

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Biology

Efficient and Scalable Generation of Human Ventral Midbrain Astrocytes from Human-Induced Pluripotent Stem Cells

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal degeneration has diverse causes in Parkinson's, including non-cell autonomous mechanisms mediated by astrocytes. Throughout the CNS, astrocytes are essential for neuronal survival and function, as they maintain metabolic homeostasis in the neural environment. Astrocytes interact with the immune cells of the CNS, microglia, to modulate neuroinflammation, which is observed from the earliest stages of Parkinson's, and has a direct impact on the progression of its pathology. In diseases with a chronic neuroinflammatory element, including Parkinson's, astrocytes acquire a neurotoxic phenotype, and thus enhance neurodegeneration. Consequently, astrocytes are a potential therapeutic target to slow or halt disease, but this will require a deeper understanding of their properties and roles in Parkinson's. Accurate models of human ventral midbrain astrocytes for in vitro study are therefore urgently required.
We have developed a protocol to generate high purity cultures of ventral midbrain-specific astrocytes (vmAstros) from hiPSCs that can be used for Parkinson's research. vmAstros can be routinely produced from multiple hiPSC lines, and express specific astrocytic and ventral midbrain markers. This protocol is scalable, and thus suitable for high-throughput applications, including for drug screening. Crucially, the hiPSC derived-vmAstros demonstrate immunomodulatory characteristics typical of their in vivo counterparts, enabling mechanistic studies of neuroinflammatory signaling in Parkinson's.

Here, we present a method for reproducible generation of ventral midbrain patterned astrocytes from hiPSCs and protocols for their characterization to assess phenotype and function.

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal ...

Neuroscience

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

Medicine

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...