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* These authors contributed equally
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.
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.
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
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
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 (
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
This study is supported by the National Natural Science Foundation of China (82101145) and the Beijing Natural Science Foundation (Z200014).
....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 |
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