Assembling Retinal Organoids with Microglia

Summary

Microglia are unique resident immune cells in the retina, playing crucial roles in various retinal degenerative diseases. Generating a co-culture model of retinal organoids with microglia can facilitate a better understanding of the pathogenesis and development progress of retinal diseases.

Abstract

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal the mechanism of retinal development and the occurrence of retinal disease. Microglia (MG) are unique resident macrophages in the retina and central nervous system (CNS), serving crucial immunity functions. However, retinal organoids lack microglia since their differentiation origin is the yolk sac. The specific pathogenesis of microglia in these retinal diseases remains unclear; therefore, the establishment of a microglia-incorporated retinal organoid model turns out to be necessary. Here, we successfully constructed a co-cultured model of retinal organoids with microglia derived from human stem cells. In this article, we differentiated microglia and then co-cultured to retinal organoids in the early stage. As the incorporation of immune cells, this model provides an optimized platform for retinal disease modeling and drug screening to facilitate in-depth research on the pathogenesis and treatment of retinal and CNS-related diseases.

Introduction

As the limited source of the human retina, the differentiation of human stem cells into three-dimensional (3D) retinal organoids represents a promising in vitro model for simulating the retina¹. It contains different cell types in the retina, including photoreceptors, retinal ganglion cells, bipolar cells, Müller cells, horizontal cells, and astrocytes². This model enables the emulation and study of both retinal development mechanisms and the pathogenesis of retinal diseases. However, due to the directional differentiation method, retinal organoids were derived from the neuroectoderm³, lacking many other cell types originating from different germ layers, such as microglia from the yolk sac and perivascular cells from the mesoderm^4,5,6.

At present, many retinal diseases, such as retinitis pigmentosa⁷, glaucoma⁸, and retinoblastoma⁹, have been proven to be closely related to microglia within the retina. However, due to the lack of proper research models, specific mechanisms illustrating the relationship between microglia and these diseases still remain unclear. While mice have served as a favorable model for studying retinal diseases, recent studies have highlighted significant differences between mouse and human microglia in terms of lifespan, proliferation rate, and the absence of human homologous genes^10,11. These findings suggested that conclusions drawn from mouse models may not be entirely reliable, emphasizing the importance of constructing human retinal organoids containing microglia.

Over the past few decades, various methods for the 3D differentiation of retinal organoids have been developed^12,13. To facilitate the co-culture operation of microglia within retinal organoids, we have selected a differentiation method involving a transition from adherent to suspension culture. This approach successfully enables microglia to be incorporated into the retinal organoids, maintaining them for at least 60 days¹⁴.

Protocol

This study was approved by the Institutional Ethics Committee of Beijing Tongren Hospital, Capital Medical University. HESCs cell line H9 was from the WiCell Research Institute. Pre-warm the cell culture medium at room temperature (RT) for 30 min before the experiment.

1. Generation of human microglia

1. Culture the hESCs in stem cell medium until the cell density reaches 80%-90%. Seed at least 1 x 10⁶ cells in each well.
2. Aspirate the stem cell medium and rinse the cells with 1x DPBS (1 mL/well of 6 well plate). The next step requires 2 x 10⁷-1 x 10⁸ cells. If not enough, combine cells of 2-5 wells to do the following steps.
3. Dissociate the stem cell colonies using the 0.5 mM EDTA solution for about 5 min in a 37 °C incubator (1 mL/well of 6 well plate).
4. Check colony morphology after 5 min. When the colony edges are slightly curled up with loose gaps, cells are ready to be differentiated and aspirate the EDTA. If not, incubate cells at 37 °C for another 1-2 min. Do not incubate for longer than 8 min.
5. Gently rinse the cells in each well using 6 mL of Medium A (Table 1) with 6 µL of 10 mM ROCK inhibitor Y-27632 solution. Gently tap the bottom of the dish to help rinse the cells. Set the day as day 0.
   NOTE: Do not pipette the cells. Cells will form embryoid bodies (EBs) the next day.
6. After 24 h, observe the cells under the microscope to confirm the EB formation. Collect the EBs into a 15 mL centrifugal tube with a 10 mL pipet and wait for the EBs to settle to the bottom in 5 min.
7. Carefully aspirate the supernatant and resuspend the EBs with 6 mL of fresh Medium A and transfer them into a new low-adhesion well of 6-well plate. Culture in a 37 °C incubator.
   NOTE: The purpose of the well transferring on day 1 is to reduce the influence of a large number of dead cells during the process of EB formation, which may affect the state of cell growth.
8. Refresh the fresh Medium A daily until day 4 (6 mL/well of 6-well plate).
9. On day 4, coat a 10 cm dish with 3 mL of 0.1% fish gelatin solution for at least 1 h in a 5% CO₂, 37 °C incubator.
   NOTE: If the coating time is less than 1 h, it is difficult to carry out the following steps.
10. Carefully collect the EBs into a 15 mL centrifugal tube with a 10 mL pipet and wait for the EBs to settle to the bottom in 5 min.
11. Remove the 0.1% gelatin solution and rinse the 10 cm dish once with 5-6 mL of 1x DPBS. Remove the DPBS.
12. Gently resuspend the EBs with 15 mL of Medium B (Table 1) in the 15 mL centrifugal tube and transfer them to the coated 10 cm dish. Culture in a 37 °C, 5% CO₂ incubator for 1 week.
    NOTE: In the first 7 days, do not move the dish.
13. Refresh with fresh Medium B once a week until day 49 (15 mL/10 cm dish), and examine the cells under the microscope once a week.
    NOTE: After day 49, several secretory cells can be found in the supernatant in the culture dish.
14. Centrifuge the supernatant cells at 200 x g for 5 min at RT.
15. Carefully aspirate the supernatant and resuspend the cells with 2 mL of fresh Medium B and transfer into a new low-adhesion well of 6-well plate. Incubate in a 5% CO₂, 37 °C incubator.
    NOTE: In the next 7 days, do not move the plate. The microglia could adhere to the bottom of the low-adhesion well.
16. On day 56, replace with Medium C (Table 1) (2 mL/well of 6-well plate). The microglia adhere to the bottom of the low-adhesion well and will look branched under the microscope.
    NOTE: By changing Medium C every 3 days (2 mL/well of 6-well plate), the microglia could be cultivated in Medium C for about 15 days.

2. Generation of human ROs and co-culture the ROs with microglia

1. Cultured the hESCs in stem cell medium until the cell density reaches 80%-90%. Seed at least 1 x 10⁶ cells in each well.
2. Add Dispase (1 mL/well of 6 well plate) to the culture cell. Incubate at 37 °C for 5 min. Aspirate the Dispase solution from the well.
3. Add Medium D (Table 1) (1 mL/well of 6 well plate) to the well. Cut the cell into small pieces with a 10 µL pipette.
4. Gently collect all the cell pieces and medium in a 1.5 mL microcentrifuge tube.
5. Centrifuge at 200 x g for 5 min and remove the supernatant.
6. Gently resuspend the cells in 200 µL of a cold matrix. Move the 1.5 microcentrifuge tube into an incubator for 20 min. Operate fast because the matrix will solidify at room temperature (RT). After 20 min in the incubator, the matrix will solidify.
7. Prepare a 10 cm dish with 15 mL Medium D. Resuspend the Matrix with Medium D and shake the Petri dish gently.
8. Then, put the dish in an incubator for 5 days. Do not move the plate. Set the day as day 0 (ROs). The cells will adhere in 3 days.
9. On day 5 (ROs) and day 10 (ROs), refresh the medium with Medium D.
10. On day 12 (ROs), remove the medium and add 3 mL of Dispase to each dish for 5 min.
11. Aspirate the Dispase and add 15 mL of Medium E (Table 1).
12. Harvest microglia: On day 56 (MGs), gently aspirate the Medium C, add Accutase (1 mL/well of a 6-well plate), and incubate at 37 °C for 3 min.
13. Collect the microglia cells into a 15 mL centrifugal tube with a 5 mL pipet. Centrifuge cells at 200 x g at RT for 5 min.
14. Gently aspirate the supernatant and suspend the microglia with 1 mL of Medium E. Add microglia to digested ROs on day 12.
    NOTE: On day 12 (ROs), the cells are adhesive. On day 13 (ROs), the cells are found suspending in Medium E. If the medium changes yellow from day 12 (ROs) to day 19 (ROs), refresh the medium with Medium E.
15. On day 19 (ROs), aggregate the organoids to the center of the dish by gently swirling the plate. Gently aspirate Medium E and refresh the medium with Medium F.
16. Transfer the organoids with Medium F to a new suspension dish.
17. Change the medium once a week and select organoids for experiments.

Results

The procedure for generating retinal organoids is described in our previous study¹⁵. Here, we show the representative results of microglia and co-culture microglia and retinal organoids.

Here, we demonstrate each stage of microglia differentiation (Figure 1A). Day 0 represents the stage of stem cell culture. Then, the stem cells were digested and cultured for EB formation. In the initial 4 days of the process, cells will form EBs (Figure 1B). Subsequently, we transfer the suspended EBs to adherent 10 cm dishes. After approximately 7 days, cells are similar to that shown in Figure 1C. As the culture progresses, adherent cells secrete hematopoietic progenitors into the supernatant (Figure 1D). This process takes approximately 45 days. At this point, cells can be harvested from the supernatant and transferred to 6-well plates. Over the next 7 days, the microglia will become mature (Figure 1E) and ready for further experimentation.

On day 12 of retinal organoid differentiation, adherent cells are digested to suspension culture, and Medium E containing microglia is added to the retinal organoids. Microglia will migrate into retinal organoids.In order to observe the morphology of microglia clearly, we added EGFP-lentivirus transfection into the hESCs, which made the differentiated microglia express GFP have autofluorescence (Green) (Figure 2A-C). We also examined the tissue structure of retinal organoids co-cultured with microglia using photoreceptor cell marker CRX and microglial cell marker IBA1 by immunofluorescence assay in microglia co-cultured retinal organoids after differentiation for 50 days (Figure 2D).

Figure 1: Schematic diagram and timeline of microglia cell derived from hESC. (A) The protocol begins with the thawing and passaging of hESCs in a 6-well plate. (B) The homogenous EBs formed containing Medium A. (C) EBs are transferred to a coated dish, and hematopoietic progenitors are formed on days 4-56. (D,E) On day 56, the supernatant with cells is collected (D) and transferred to a low-adhesion 6-well plate to form microglia on day 63 (E). Scale bar: 500 µm. Please click here to view a larger version of this figure.

Figure 2: Representative images along the timeline during co-culture of microglia and retinal organoids. (A) Representative images of microglia cells derived from EGFP-hESC. (B) Co-culture of EGFP⁺ microglia with retinal organoids after differentiation for 18 days. (C) Co-culture of EGFP⁺ microglia with retinal organoids after differentiation for 30 days. Scale bar: 300 µm. (D) CRX (photoreceptor cell marker; red) and IBA1 (microglial marker; green) were used to detect the tissue structure of retinal organoids co-cultured with microglia after 50 days of differentiation. DAPI (blue) stains the nucleus. Scale bar: 30 µm. Please click here to view a larger version of this figure.

Table 1: Composition of media used in this study. The components required to prepare 500 mL volume of each medium are listed in the table. Please click here to downnload this Table.

Discussion

Due to the restricted availability of the human retina, our current comprehension of retinal inflammatory responses almost comes from animal models. To overcome this limitation, retinal organoids were differentiated. The development of retinal organoid models has been an active area of research, aiming to recapitulate the complexity of the human retina for disease modeling and therapeutic development. Several studies have reported successfully generating retinal organoids from human pluripotent stem cells^1,2,12,13. However, most of these models lack the presence of microglia, which are known to play crucial roles in retinal development and disease pathogenesis. Recent studies have attempted to integrate microglia into retinal organoids or brain organoid models^16,17,18, but the detailed method is unclear. Here, we provide a detailed step-by-step protocol for co-culturing microglia with retinal organoids in the early stage, both derived from the same hESC line. While some studies have explored the role of microglia in retinal diseases using animal models or cell culture systems^7,8,9, the co-culture model presented here offers a more physiologically relevant human-based system for studying microglia-retina interactions and their implications in disease pathogenesis.

The critical steps are the cell state of human stem cells and the differentiation of microglia and retinal organoids. Microglia and ROs should be differentiated step by step, and correct reagents and dish (tissue cultured treated dish or suspension dish) should be used in different stages. This model can help us understand the process of how microglia contribute to the occurrence of retinal diseases. It holds significant implications for future drug screening and investigations into disease mechanisms. However, the methods we currently publish still have some limitations. Due to the prolonged differentiation period of microglia and the lack of suitable cryopreservation methods, we should plan our time wisely for microglia and retinal organoids differentiation for co-culture.

In addition to retinal organoids, we believe that integrating immune cells into other organoid models can also foster organoid development. Therefore, testing other organoid systems is crucial. The integration of various cell types and organoids to form complete organs represents a promising model for the future.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acctuase	Stemcell Technologies	07920
Advanced DMEM/F12	Thermo	12634-010
Anti-CRX(M02)	abnova	H00001406-M02	Antibody; dilution as per the manufacturer's instructions
Anti-IBA1	Abcam	ab5076	Antibody; dilution as per the manufacturer's instructions
B27	Life Technologies	17105-041
Dispase (1U/mL)	Stemcell Technologies	07923
DMEM basic	Gibco	10566-016
DMEM/F12	Gibco	10565-042
DPBS	Gibco	C141905005BT
EDTA	Thermo	15575020
F12	Gibco	11765-054
FBS	Biological Industry	04-002-1A
Gelatin	Sigma	G7041-100G	Solid
Glutamax	Gibco	35050-061
H9 cell line	WiCell Research Institute
IL-3	RD Systems	203-IL-050
IL-34	PeproTech	200-34-50UG
KSR	Gibco	10828028
Matrix	Corning	356231
M-CSF	RD Systems	216-MC-500
MEM Non-essential Amino Acid Solution	Sigma	M7145
N2	Life Technologies	17502-048
Neurobasal	Gibco	21103-049
Pen/strep	Gibco	15140-122
Stem cell medium	Stemcell Technologies	5990
Taurine	Sigma	T-8691-25G
X-ViVO	LONZA	04-418Q
Y27632	Selleck	S1049
β-mercaptoethanol	Life Technologies	21985-023