Summary
Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.
Abstract
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.
Introduction
Pluripotent stem cells (PSCs) are characterized by their self-renewal and ability to differentiate into all kinds of somatic cells. Thus, organoids derived from PSCs have become an important resource in regenerative medicine research. Retinal degeneration is characterized by the loss of photoreceptors (rods and cones) and retinal pigment epithelium. Retinal cell replacement could be an encouraging treatment for this disease. However, it is not feasible to obtain human retinas for disease research and therapy. Therefore, retinal organoids (ROs) derived from PSCs, which effectively and successfully recapitulate multi-layered native retinal cells, are beneficial for basic and translational research1,2,3. Our research focuses on RO differentiation to provide sufficient and quality cells for studying retinal degeneration4.
Methods for differentiating ROs are continuously emerging, with three-dimensional (3D) suspension differentiation pioneered by the Sasai laboratory in 20125. We introduced the CRX-tdTomato tag in the human embryonic stem cells (hESCs) to specifically track the photoreceptor precursor cells and modified the method with the addition of COCO, a multifunctional antagonist of the Wnt, TGF-β, and BMP pathways6. COCO has been shown to efficiently improve the differentiation efficiency of photoreceptor precursors and cones6,7.
Altogether, by modifying the classical differentiation method, we have developed an accessible protocol to harvest abundant photoreceptor precursors and cones from human ROs for analyzing the retinal disease associated with the photoreceptors through laboratory investigations and for further clinical application/transplantation.
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Protocol
This study was approved by the institutional Ethics Committee of Beijing Tongren Hospital, Capital Medical University. H9 hESCs were obtained from the WiCell Research Institute and genetically engineered to tdTomato-tagged cell line.
1. Generation of human ROs
- Culture the hESCs under feeder-free conditions.
- Coat one well of a 6-well plate with 1 mL of 0.1 mg/mL reagent A (Table of Materials) at 37 °C for at least half an hour following the manufacturer's instructions. Thaw an aliquot of 1x106 hESCs.
NOTE: Prepare 3 mL of pre-warmed reagent B (Table of Materials) and transfer the cryopreserved cells (1 mL) into 3 mL of fresh medium. Do not pipette the hESCs to single cells. - Centrifuge at 200 x g for 5 min and remove the supernatant.
- Seed the cells into the reagent A-coated plate with 2 mL of reagent B and change 2 mL of reagent B daily. Passage cells when reaching approximately 80% confluency (usually around 4 days).
- Coat one well of a 6-well plate with 1 mL of 0.1 mg/mL reagent A (Table of Materials) at 37 °C for at least half an hour following the manufacturer's instructions. Thaw an aliquot of 1x106 hESCs.
- Day 0
- Dissociate hESCs to a single-cell suspension using medium I (Table 1). Prepare medium I by mixing the following: 20% (v/v) KnockOut serum replacement (KSR), 0.1 mM MEM non-essential amino acids solution (NEAA), 1 mM pyruvate,3 µM IWR-1-endo (IWR1e), 30 ng/mL COCO, 100 U/mL penicillin, 100 µg/mL streptomycin (PS), 0.1 mM β-mercaptoethanol, and Glasgow's Eagle's minimal essential medium (GMEM).
NOTE: Before the start of the dissociation, prepare medium I and transfer 12 mL of medium I with 20 µM Y-27632 into a 10 cm Petri dish, 500 µL of reagent C (Table of Materials) containing 0.05 mg/mL of reagent D (Table of Materials) and 20 µM Y-27632 in a 1.5 mL tube. Perform the above steps in the dark, as component IWR1e in medium I is light-sensitive. - Wash the hESCs with pre-warmed 1x Dulbecco's phosphate-buffered saline (DPBS) buffer.
- Add the prepared 500 µL reagent (in the 1.5 mL) tube and incubate the hESCs for 3.5 min at 37 °C and 5% CO2.
- Detach the cells by flicking the side and bottom of the plate for a few seconds and add 500 µL of prepared medium I from the Petri dish into the hESC plate.
NOTE: Prepare a 1.5 mL tube with 900 µL of 1x DPBS and use a hemocytometer for cell counting. - Harvest the cells in a new 1.5 mL tube and pipette the cell suspension up and down and then take out 100 µL from the tube and then add into the tube with 900 µL of DPBS for cell counting.
- Disperse the left 900 µL cell suspension and then dilute the cells with prepared medium I in the Petri dish to 9 x 104 cells/mL.
NOTE: The whole volume of the medium is 12 mL; 1.08 x 106 cells are needed in total. - Add 100 µL of cell suspension to each well of a non-adherent, V-bottom, 96-well plate (Table of Materials).
NOTE: Use a multichannel pipette to shorten the time and ensure that each well contains an equivalent cell number. Shake the Petri dish each time before removing 100 µL portions. It is important that the cells are uniformly distributed. - Lightly spin down the 96-well plate in a low-speed shaker for 5 min and then incubate at 37 °C and 5% CO2.
NOTE: Keep the plate in dark. Set the day as day 0.
- Dissociate hESCs to a single-cell suspension using medium I (Table 1). Prepare medium I by mixing the following: 20% (v/v) KnockOut serum replacement (KSR), 0.1 mM MEM non-essential amino acids solution (NEAA), 1 mM pyruvate,3 µM IWR-1-endo (IWR1e), 30 ng/mL COCO, 100 U/mL penicillin, 100 µg/mL streptomycin (PS), 0.1 mM β-mercaptoethanol, and Glasgow's Eagle's minimal essential medium (GMEM).
- Day 2
- Add 1% reagent A (Table of Materials). Prepare reagent A (protein concentration of 10 mg/mL) by adding 133.4 µL of reagent A into 2 mL of medium I.
NOTE: Maintain reagent A at 4 °C overnight before use to achieve complete and uniform melting. Please note the product information and search the catalog number the and the lot number in the official website of the company to have the protein concentration of reagent A as each bottle of reagent A is at a different protein concentration. If the protein concentration is low, an increased volume of reagent A would be helpful. - Add 20 µL of prepared reagent A to each well and pipette twice in the center to scatter the dead cells.
NOTE: Maintain the reagent A and the 96-well plate under cool conditions and complete all the steps on ice. Place the plate in dark.
- Add 1% reagent A (Table of Materials). Prepare reagent A (protein concentration of 10 mg/mL) by adding 133.4 µL of reagent A into 2 mL of medium I.
- Day 2-12
- Clean the bottom of the 96-well plate and incubate at 37 °C and 5% CO2 until day 6. At day 6, change half of the medium by removing 58 µL of the medium from each well and adding 60 µL of medium I.
NOTE: Use a multichannel pipette to complete the half medium change. Conduct this step gently to ensure that no cell pellets are removed from the well. Aspirate the medium into a clean 10 cm Petri dish. If there are cell pellets inside, add them back to the 96-well plate.
- Clean the bottom of the 96-well plate and incubate at 37 °C and 5% CO2 until day 6. At day 6, change half of the medium by removing 58 µL of the medium from each well and adding 60 µL of medium I.
- Day 12- 18
- Change the medium to medium II (Table 1) at day 12. Prepare medium II using 1% (v/v) reagent A by mixing the following: 10% fetal bovine serum (FBS), 0.1 mM NEAA, 1 mM pyruvate,100 U/mL penicillin, 100 µg/mL streptomycin, 0.1 mM β-mercaptoethanol, 100 nM SAG dihydrochloride and GMEM. Store it under cool and dark conditions.
- Harvest the cell pellets in a 15 mL conical tube from the 96-well plate and allow the pellets to settle naturally for 5 min at room temperature.
NOTE: Remove the medium and pellets gently under the surface to avoid bubbles. - Remove the supernatant while taking care of the organoids. Transfer the cell aggregates to a 10 cm suspension dish containing 18 mL of prepared medium II with reagent A.
NOTE: Do not add the prepared medium II into the 10 cm dish in advance, as the reagent A should be at 4 °C. - Incubate at 37 °C and 5% CO2 until day 18.
- From day 18 onwards
- Change the medium to medium III (Table 1). Prepare medium III under dark conditions by mixing the following: 10% FBS, 1xsupplement 1, 0.5 µM retinoic acid (RA), 100 µM taurine, 100 U/mL penicillin, 100 µg/mL streptomycin and 1:1 mixture of Dulbecco's modified eagle medium (DMEM)/nutrient mixture F-12.
- On day 18, when a semitransparent optic vesicle is generated, cut the organoids using a microsurgical knife.
NOTE: Cleave the organoid, usually into four pieces, in a Petri dish with medium II. Each piece should grow into an intact optic cup in the following weeks. - Aggregate all the pellets to the center of the dish. Harvest the cells into a 15 mL conical falcon tube and allow natural settling. Then gently remove the supernatant.
NOTE: Due to their sizes, organoids can be aggregated at the center by rotating the Petri dish in one direction for 90° on a horizontal plane a few times. - Suspend and disperse the pellets in two 10 cm Petri dishes, with 18 mL of medium III per dish, and gently transfer the dishes to the incubator to avoid cell aggregation. Continue culturing in medium III at 37 °C and 5% CO2 and change the medium weekly. The CRX expressed after day 45 and until day 120, we could detect the out-segment of the photoreceptor.
2. Analyzing human ROs
- FACS analysis
- Assemble three CRX-tdTomato and three H9 ES-derived organoids from the dishes by using the cut 1 mL tips. Wash the organoids with 1 mL of pre-warmed (room temperature) DPBS.
- Prepare the digestion buffer by mixing 0.25% trypsin-EDTA Solution with 0.05 mg/mL of reagent D.
- Dissociate the organoids into single cells by using the prepared digestion buffer for 8 min at 37 °C. Then add the same volume of DPBS with 10% FBS and 0.05 mg/mL of reagent D to inactivate the reaction.
- Lightly suspend and scatter the cells and then filter using a 100 µm cell strainer and use a Fluorescence-Activated Cell Sorting (FACS) system at 561 nm excitation laser line and 780/60 filter to analyze the CRX-tdTomato positive signals.
NOTE: CRX initially expresses at day 45 and increases with the maturation of organoids. Ten thousand cells are used for each test, for each timepoint, at least three repeats are completed.
- Fluorescence intensity quantification of ROs
- Prepare the viable organoids randomly for imaging.
NOTE: Set the parameters and use the same filters and parameters for all the fluorescence intensity monitors. - Capture images and analyze the mean fluorescence intensity using suitable software (e.g., ImageJ).
- Import the images and then convert to 8-bit using Image | Type.
- Adjust the threshold by using the default parameters by Image | Adjust | Threshold.
- Determine the measured area and then evaluate the gray value by using Analyze | Measure.
NOTE: The mean values in the result panel are the mean fluorescence intensity of the measured area.
- Prepare the viable organoids randomly for imaging.
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Representative Results
The schematic illustration depicts the differentiation protocol to improve precursor cells with COCO (Figure 1). From PSC to ROs, numerous details could cause result variations. It is recommended to record every step and even the catalog number and lot number of every medium to track the entire procedure.
Herein, we provide bright field images for days 6, 12, 18, and 45 (Figure 2). On day 6, the organoids are usually around 600 µm in diameter in a 96-well plate, with dense connections inside and bright rim (Figure 2A). On day 12, the optic vesicle-like structures initially generate (Figure 2B). From day 12 to day 18, the presence of optic vesicle structures is clear, and they continued to grow after day 18. The organoids without the vesicle-like architecture are discarded (Figure 2C). By day 30, the vesicle-like architecture is more obvious, and it is easier to distinguish the superior ROs from the inferior ones (Figure 2D-E). The organoids that lose the translucent structure (asterisks in Figure 2D-E), should be removed in the following days.
The organoids express the CRX, which is a marker of photoreceptor precursors, from day 45 onwards (Figure 2F,2I). Other photoreceptor precursor markers, such as RCVRN and OTX2, were also positively detected at day 45 (Figure 2G-H). The addition of COCO promotes the generation of photoreceptor precursors.
Figure 1. Timetable for stepwise treatment for RO differentiation from hESC. Please click here to view a larger version of this figure.
Figure 2. Human retinal organoid generation. (A-B) Early-stage optic vesicle-like structure formed in a 96-well plate. The black arrows indicate the optic vesicle-like structures. (C) The first day of suspension culture in a Petri dish on day 18. (D-E) Optic cup structures are observed at this stage. The stars show the inferior organoids (F) The bright field image on day 45. Scale bars = 400 µm. (G-H) Immunostaining results of RCVRN (G) and OTX2 (H) on day 45. Scale bars = 50 µm. (I) TdTomato-positive signals indicate the expression of CRX on day 45 in (F). Scale bars = 400 µm. Please click here to view a larger version of this figure.
| Medium I (50 mL) | ||||||||
| KSR | G-MEM | NEAA | Pyruvate | b-ME | IWR1e | COCO | PS | |
| Percentage % OR final concentration | 20 | 78 | 0.1 mM | 1 mM | 0.1 mM | 3 µM | 30 ng/mL | 1 |
| Volume | 10 mL | 39 mL | 0.5 mL | 0.5 mL | 90.9 µL | 5 µL | 10 µL | 0.5 mL |
| The store concentration of IWR1e is 30 mM. COCO is 150 µg/mL. | ||||||||
| Medium II (50 mL) | ||||||||
| FBS | G-MEM | NEAA | Pyruvate | b-ME | SAG | PS | ||
| Percentage % OR final concentration | 10 | 88 | 0.1 mM | 1 mM | 0.1 mM | 100 nM | 1 | |
| Volume | 5 mL | 44 mL | 0.5 mL | 0.5 mL | 90.9 µL | 2.5 µL | 0.5 mL | |
| The store concentration of SAG is 2 mM. | ||||||||
| Medium III (50 mL) | ||||||||
| FBS | DMEM/F12- Glutamax | Supplement 1 | RA | Taurine | PS | |||
| Percentage % OR final concentration | 10 | 88 | 1 | 0.5 µM | 100 µM | 1 | ||
| Volume | 5 mL | 44 mL | 0.5 mL | 5 µL | 50 µL | 0.5 mL | ||
| The store concentration of RA is 5 mM, and Taurine is 100 mM. | ||||||||
Table 1: Medium I, II, and II recipes
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Discussion
Retinal organoid differentiation is a desirable method for the generation of ample functional retinal cells. The RO is a composite of different retinal cells, such as ganglion cells, bipolar cells, and photoreceptors, generated by pluripotent stems cells toward the neural retina4,5,8,9. Although confluent ROs could be harvested, it is time-consuming, which may require long culturing periods (up to 180 days). However, for photoreceptor transplantation, or studying cone-rod or rod-cone dystrophy, it is advantageous to obtain a relatively high percentage of photoreceptors in the 3D culturing system10.
It is also challenging to monitor the organoids' development without interrupting the normal developmental processes. Therefore, we used CRX, a cone-rod homeobox protein, predominantly expressed in photoreceptor precursors, as a target gene to trace photoreceptor precursor cells during their 3D differentiation. With the tdTomato system, CRX-expressing cells can be spatio-temporally tracked by the red fluorescence without interrupting retinalization during their 3D differentiation. The utilization of CRX-tdTomato system could accelerate the process of drug screening for photoreceptor precursor differentiation in the ROs.
Using our method, around 70% organoids could develop into retinal organoids, which display the vesicle-like structures. Importantly, with the incision of superior organoids, we usually could harvest around 100 retinal organoids from a 96-well plate. Additionally, abundant photoreceptor precursors are generated in the early stage of RO maturation with the COCO culture, which helps progression toward direct differentiation of certain cells through pathway regulation11,12. The protein concentration of reagent A is crucial for the differentiation. Altogether, sufficient reagent A with the aggregates in the early days as well as the cutting of the organoids on day 18 are important to harvest abundant ROs with high quality. This method also promotes the development of directional differentiation of photoreceptor cells in 3D organoids and contributes to the transplantation of photoreceptor cells.
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Acknowledgments
We thank members of 502 laboratory for their technical supports and helpful comments regarding the manuscript. This work was partly supported by the Beijing Municipal Natural Science Foundation (Z200014) and National Key R&D Program of China (2017YFA0105300).
Materials
| Name | Company | Catalog Number | Comments |
| 2-mercaptoethanol | Life Technologies | 21985-023 | |
| COCO | R&D Systems | 3047-CC-050 | DAN Domain family of BMP antagonists |
| DMEM/F-12 | Gibco | 10565-042 | |
| DMSO | Sigma | D2650 | |
| DPBS | Gibco | C141905005BT | |
| EDTA | Thermo | 15575020 | |
| Fetal Bovine Serum (FBS), Qualified for Human Embryonic Stem Cells | Biological Industry | 04-002-1A | |
| GMEM | Gibco | 11710-035 | |
| KnockOut Serum Replacement-Multi-Species | Gibco | A3181502 | |
| MEM Non-essential Amino Acid Solution (100X) | sigma | M7145 | |
| Pen Strep | Gibco | 15140-122 | |
| Primesurface 96 V-plate | Sbio | MS9096SZ | Cell aggregation in 1.2.7 |
| Pyruvate | Sigma | S8636 | |
| Reagent A | BD | 356231 | Matrigel in 1.1.1 |
| Reagent B | StemCell | 5990 | mTeSR- E8 , PSCs basal medium in 1.1.2 |
| Reagent C | Gibco | 12563-011 | TrypLE Express in 1.2 |
| Reagent D | Roche | 11284932001 | DNase I , in 1.2 |
| Retinoic acid | Sigma | R2625-100MG | |
| SAG | Enzo Life Science | ALX-270-426-M001 | |
| Supplement 1 | Life Technologies | 17502-048 | N-2 Supplement (100X), Liquid, supplemet in medum III |
| Taurine | Sigma | T-8691-25G | |
| Trypsin-EDTA (0.25%), phenol red | Gibco | 25200056 | organoids dissociation in 2.1.3 |
| Wnt Antagonist I, IWR-1-endo - Calbiochem | Sigma | 681669 | Wnt inhibitor |
| Y-27632 2HCl | Selleck | S1049 |
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