Generating Neural Retina from Human Pluripotent Stem Cells

Summary

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.

Abstract

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.

Introduction

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 models^4,5,6. 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 retina^7,8.

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 vivo^11,12,13. 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 retina^14,15. 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 therapies^16,17,18.

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 results^19,20. 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 compounds^21,22,23. 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 process^13,21,24,25. 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 structure^22,26. 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 Döpper 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.

Protocol

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

1. Human ESC culture medium and passage solution
   1. 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 °C. Prewarm to RT. Stir well before use until the supplement is free of cloudiness.
   2. 1% extracellular matrix (ECM): Use a pre-chilled pipet tip and sterile tubes to dispense 200 µL of ECM (see Table of Materials) per tube on ice. Store the tubes in a -20 °C freezer. Melt ECM on ice and dilute with pre-cooled DMEM/F12 at 1:100.
   3. 0.5 mM pH = 8.0 EDTA: To prepare 500 mL of 0.5 mM EDTA, add 500 µL of 0.5 M EDTA and 0.9 g of NaCl to 500 mL of 1x Dulbecco's phosphate-buffered saline (DPBS) (see Table of Materials). Mix thoroughly and store at 2-8 °C.
2. Retinal differentiation medium
   1. Growth factor-free chemically defined medium (gfCDM): Prepare the gfCDM by combining 45% Iscove's modified Dulbecco's medium-GlutaMAX (IMDM-GlutaMAX), 45% Ham's F12-GlutaMAX (F12-Glutamax), 10% knockout serum replacement (KSR), 1% cholesterol lipid concentrate, and 450 µM thioglycerol (see Table of Materials).
3. Small molecule compounds
   1. 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 °C for 2 years, keeping away from light. Dilute the stock 2,500 times (20 µM) for use, equivalent to 0.4 µL of Y-27632 per mL of gfCDM.
   2. 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 °C for up to 2 years. Add 0.3 µL of 10 mM IWR1-endo per mL of gfCDM for the 3 µM concentration for induction.
   3. 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 °C in solvent for a maximum of 2 years. For the preparation of SB431542 at a working concentration of 10 µM, add 2 µL of the 50 mM stock solution to 10 mL of gfCDM.
   4. 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 °C or -80 °C (as recommended by the manufacturer). Add 0.1 µL of the stock to every 10 mL of gfCDM, resulting in 100 nM of LDN-193189 for induction.
   5. Recombinant Human bone morphogenetic protein 4 (BMP4): Reconstitute at 50-200 µg/mL in 4 mM HCl (see Table of Materials). Store at 2 °C to 8 °C for 1 month or -20 °C for 1 year after reconstitution. Perform induction of NR using 1.5 nM BMP4.
4. Long-term NR culture medium
   1. 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 −80 °C and use within 3 months. To achieve a working concentration of 0.5 µM, add 10 µL 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.
   2. 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 °C. A working concentration of 0.1 mM taurine is achieved by adding 50 µL of the stock solution per 100 mL of NRDM.
   3. 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 °C for up to 2 weeks or 6 months at -20 °C to ensure the activity of the components.
5. Blocking buffer: Dilute 1:9 with DPBS to obtain a 10% working solution for use (see Table of Materials). Store at -20 °C for up to 5 years.
   ​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 µm filters for filtration.

2. Culturing of H9-ESCs

1. Thawing of H9-ESCs
   1. Add 1 mL of 1% ECM to each well of a 6-well plate. Incubate for 1 h in an incubator at 37 °C in a 5% CO₂ atmosphere.
      NOTE: Avoid the addition of ECM along the well walls, because it may stick to the surface.
   2. Take an H9-ESC cryovial stock from the liquid nitrogen tank and quickly shake it in 37 °C water for 30 s.
      NOTE: Do not allow the vial to thaw completely.
   3. 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 µM Y-27632.
   4. Centrifuge the tube at 190 × g for 5 min at RT. Carefully remove most of the supernatant with a 1 mL pipette, leaving approximately 50 µL of supernatant to avoid cell loss.
   5. Add 1 mL of MM containing 10 µM 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.
   6. Remove the ECM coating after 1 h of incubation. Add 2 mL of pre-warmed MM containing 10 µM Y-27632 to each well.
   7. Dispense 0.5 mL of cell suspension per well. Gently shake the plate laterally to ensure even distribution of cells.
   8. Incubate the plate at 37 °C under 5% CO₂ for at least 24 h without touching it.
   9. Change the medium daily. When clone density reaches 70% and above, passaging is required.
2. Passaging of H9-ESCs
   1. Prepare the ECM-coated plate as described above (step 2.1.1) and add 2 mL of MM per well.
   2. Remove the spent medium from the 6-well plates. Wash each well twice with 1 mL of DPBS.
   3. 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.
   4. Discard EDTA and add 1 mL of MM to abort digestion.
   5. Gently tap the well plate until the majority of the cells are detached.
   6. 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 µL of cell suspension in a new ECM-coated 6-well culture dish and shake it back and forth.
   7. When the cell density is observed as sufficient under a microscope, incubate the plate at 37 °C under 5% CO₂ for at least 24 h without touching it.
   8. Change the media daily. At 70% clone density and above, passage is required.

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.

1. Day 0 - Embryoid body (EB) formation
   1. After washing the cells with 2 mL of DPBS, add 0.5 mL of CDS (see Table of Materials) containing 20 µM Y27632 to the well. Incubate for 3 min at 37 °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.
   2. Abort digestion by adding 3 mL of MM containing 20 µM Y27632. Centrifuge at 190 × g for 5 min and discard supernatant.
   3. Resuspend the cell pellet in 1 mL of gfCDM containing 20 µM Y27632, and count the cells. Add the corresponding volume for 1.2 × 10⁶ cells (1.2 × 10⁴ cells per well) to 10 mL of gfCDM, which is pre-incorporated with 20 µM Y-27632, 3 µM IWR1-endo, 10 µM SB431542, and 100 nM LDN-193189 (see Table of Materials).
   4. Add 100 µL 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.
2. Day 6 - Retina induction
   1. 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.
   2. 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.
3. Day 18 - Long term NR culture
   1. 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.
   2. Replace the medium with fresh NRDM every 3 days.
      ​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.

4. Analysis of human NRs

1. Mounting of the ROs
   1. 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, Döpper 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.
2. Immunofluorescent staining of ROs
   1. 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 °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.
   2. 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.
   3. 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 °C overnight. Subsequently, carefully detach the embedding boxes. Finally, store them at RT.
   4. Create continuous sections (5 µm thickness) with a paraffin slicer. Fix slices on adhesion microscope slides, dry, and store at RT.
   5. Perform dewaxing and rehydration before antigen repair^12,27.
   6. 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.
   7. Allow paraffin sections to cool naturally in a ventilated area and then put them into a humid chamber.
   8. 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.
   9. Block with 10% donkey serum diluted in DPBS at RT for 1 h in a humid chamber with 10 µL per staining area.
   10. Add primary antibodies diluted in 10% donkey serum. Incubate overnight at 4 °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-β tubulin III (1: 250) and anti-NESTIN (1: 200) (see Table of Materials).
   11. 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).
   12. 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.
   13. Drop an appropriate amount of anti-fluorescent sealer (see Table of Materials) onto the slide and cover with cover slips.
   14. Visualize using a flow-like tissue cell quantitative analyzer (see Table of Materials), or similar.
   15. Store slides at -20 °C in the dark after microscopic analysis.

Results

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.

Discussion

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...

Materials

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