This study is supported by the National Natural Science Foundation of China (82101145) and the Beijing Natural Science Foundation (Z200014).

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
<ol>
	<li>Culture the hESCs in stem cell medium until the cell density reaches 80%-90%. Seed at least 1 x 106 cells in each well.</li>
	<li>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.</li>
	<li>Dissociate the stem cell colonies using the 0.5 mM EDTA solution for about 5 min in a 37 &#176;C incubator (1 mL/well of 6 well plate).</li>
	<li>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 &#176;C for another 1-2 min. Do not incubate for longer than 8 min.</li>
	<li>Gently rinse the cells in each well using 6 mL of Medium A (Table 1) with 6 &#181;L 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.</li>
	<li>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.</li>
	<li>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 &#176;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.</li>
	<li>Refresh the fresh Medium A daily until day 4 (6 mL/well of 6-well plate).</li>
	<li>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 &#176;C incubator. 
	NOTE: If the coating time is less than 1 h, it is difficult to carry out the following steps.</li>
	<li>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.</li>
	<li>Remove the 0.1% gelatin solution and rinse the 10 cm dish once with 5-6 mL of 1x DPBS. Remove the DPBS.</li>
	<li>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 &#176;C, 5% CO2 incubator for 1 week. 
	NOTE: In the first 7 days, do not move the dish.</li>
	<li>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.</li>
	<li>Centrifuge the supernatant cells at 200 x g for 5 min at RT.</li>
	<li>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 &#176;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.</li>
	<li>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.</li>
</ol>
2. Generation of human ROs and co-culture the ROs with microglia
<ol>
	<li>Cultured the hESCs in stem cell medium until the cell density reaches 80%-90%. Seed at least 1 x 106 cells in each well.</li>
	<li>Add Dispase (1 mL/well of 6 well plate) to the culture cell. Incubate at 37 &#176;C for 5 min. Aspirate the Dispase solution from the well.</li>
	<li>Add Medium D (Table 1) (1 mL/well of 6 well plate) to the well. Cut the cell into small pieces with a 10 &#181;L pipette.</li>
	<li>Gently collect all the cell pieces and medium in a 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge at 200 x g for 5 min and remove the supernatant.</li>
	<li>Gently resuspend the cells in 200 &#181;L 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.</li>
	<li>Prepare a 10 cm dish with 15 mL Medium D. Resuspend the Matrix with Medium D and shake the Petri dish gently.</li>
	<li>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.</li>
	<li>On day 5 (ROs) and day 10 (ROs), refresh the medium with Medium D.</li>
	<li>On day 12 (ROs), remove the medium and add 3 mL of Dispase to each dish for 5 min.</li>
	<li>Aspirate the Dispase and add 15 mL of Medium E (Table 1).</li>
	<li>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 &#176;C for 3 min.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Transfer the organoids with Medium F to a new suspension dish.</li>
	<li>Change the medium once a week and select organoids for experiments.</li>
</ol>

Microglia-Integrated Retinal Organoids: A Model for Studying Retinal Diseases

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(170), e62298 (2021).</li><li>Eiraku, M., 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=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472 (7341), 51-56 (2011).</li><li>Ginhoux, F., 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=Fate+mapping+analysis+reveals+that+adult+microglia+derive+from+primitive+macrophages.">Fate mapping analysis reveals that adult microglia derive from primitive macrophages.</a> Science. 330 (6005), 841-845 (2010).</li><li>Kierdorf, K., 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=Microglia+emerge+from+erythromyeloid+precursors+via+pu.1-+and+irf8-dependent+pathways.">Microglia emerge from erythromyeloid precursors via pu.1- and irf8-dependent pathways.</a> Nat Neurosci. 16 (3), 273-280 (2013).</li><li>Schulz, C., 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=A+lineage+of+myeloid+cells+independent+of+myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</li><li>O'koren, E. G., 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=Microglial+function+is+distinct+in+different+anatomical+locations+during+retinal+homeostasis+and+degeneration.">Microglial function is distinct in different anatomical locations during retinal homeostasis and degeneration.</a> Immunity. 50 (3), 723-737.e7 (2019).</li><li>Margeta, M. A., 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=Apolipoprotein+E4+impairs+the+response+of+neurodegenerative+retinal+microglia+and+prevents+neuronal+loss+in+glaucoma.">Apolipoprotein E4 impairs the response of neurodegenerative retinal microglia and prevents neuronal loss in glaucoma.</a> Immunity. 55 (9), 1627-1644.e7 (2022).</li><li>Xu, J., 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=Enhanced+innate+responses+in+microglia+derived+from+retinoblastoma+patient-specific+IPSCs.">Enhanced innate responses in microglia derived from retinoblastoma patient-specific IPSCs.</a> Glia. 72 (5), 872-884 (2024).</li><li>Gosselin, D., 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=An+environment-dependent+transcriptional+network+specifies+human+microglia+identity.">An environment-dependent transcriptional network specifies human microglia identity.</a> Science. 356 (6344), eaal3222 (2017).</li><li>Galatro, T. 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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=Functional+microglia+derived+from+human+pluripotent+stem+cells+empower+retinal+organ.">Functional microglia derived from human pluripotent stem cells empower retinal organ.</a> Sci China Life Sci. 65 (6), 1057-1071 (2022).</li><li>Zhang, X., Jin, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruct+human+retinoblastoma+in+vitro.">Reconstruct human retinoblastoma in vitro.</a> J Vis Exp. (188), e62629 (2022).</li><li>Park, D. 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The authors are not aware of any affiliations, membership, grants, or financial holding that might affect the objectivity of this study.

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 cells1,2,12,13. 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 models16,17,18, 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 systems7,8,9, 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.

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, M&#252;ller 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 mesoderm4,5,6.
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 genes10,11. 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 developed12,13. 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.

<table><tbody><tr><td>Acctuase</td><td>Stemcell Technologies</td><td>07920</td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Thermo</td><td>12634-010</td><td></td></tr><tr><td>Anti-CRX(M02)</td><td>abnova</td><td>H00001406-M02</td><td>Antibody; dilution as per the manufacturer&#039;s instructions</td></tr><tr><td>Anti-IBA1</td><td>Abcam</td><td>ab5076</td><td>Antibody; dilution as per the manufacturer&#039;s instructions</td></tr><tr><td>B27</td><td>Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>Dispase (1U/mL)</td><td>Stemcell Technologies</td><td>07923</td><td></td></tr><tr><td>DMEM basic</td><td>Gibco</td><td>10566-016</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>10565-042</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>C141905005BT</td><td></td></tr><tr><td>EDTA</td><td>Thermo</td><td>15575020</td><td></td></tr><tr><td>F12</td><td>Gibco</td><td>11765-054</td><td></td></tr><tr><td>FBS</td><td>Biological Industry</td><td>04-002-1A</td><td></td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G7041-100G</td><td>Solid</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>H9 cell line</td><td>WiCell Research Institute</td><td></td><td></td></tr><tr><td>IL-3</td><td>RD Systems&amp;nbsp;</td><td>203-IL-050</td><td></td></tr><tr><td>IL-34</td><td>PeproTech</td><td>200-34-50UG</td><td></td></tr><tr><td>KSR</td><td>Gibco</td><td>10828028</td><td></td></tr><tr><td>Matrix</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>M-CSF</td><td>RD Systems&amp;nbsp;</td><td>216-MC-500&amp;nbsp;</td><td></td></tr><tr><td>MEM Non-essential Amino Acid Solution</td><td>Sigma</td><td>M7145</td><td></td></tr><tr><td>N2</td><td>Life Technologies</td><td>17502-048</td><td></td></tr><tr><td>Neurobasal</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>Pen/strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Stem cell medium&amp;nbsp;</td><td>Stemcell Technologies</td><td>5990</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>T-8691-25G</td><td></td></tr><tr><td>X-ViVO</td><td>LONZA</td><td>04-418Q</td><td></td></tr><tr><td>Y27632</td><td>Selleck</td><td>S1049</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr></tbody></table>

assembling retinal organoids with microglia

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.

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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67016/67016fig01.jpg" /> 
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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67016/67016fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67016/67016fig02.jpg" /> 
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 &#181;m. (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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67016/67016fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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. <a href="https://www.jove.com/files/ftp_upload/67016/Table 1-67016R1.xlsx" target="_blank">Please click here to downnload this Table.</a>

Generation of Human Microglia to Combine Them with Retinal Organoids for Improved Disease Modeling

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.

Assembling Retinal Organoids with Microglia

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal ...

assembling-retinal-organoids-with-microglia

Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.1K Views.  Capital Medical University. 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.

Video: Generation of Human Microglia to Combine Them with Retinal Organoids for Improved Disease Modeling

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

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 ...

Developmental Biology

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 ...

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

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.

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.

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

Generating Human Brain Organoids with Resident Microglia Using iPSC-Derived HPCs

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

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 ...

Optimized Protocol for Retinal Organoid Sample Preparation for TEM

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

Author Spotlight: Analyzing the Synaptic Ultrastructure in Mature Retinal Organoids Using TEM

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

Source: Portela-Lomba, M., et al. <a href="https://app.jove.com/v/61863">Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to isolate retinal ganglion neurons (RGNs) from a rat&#39;s retina and coculture them with olfactory ensheathing glia (OEG) cells. This method allows for studying the neuro-regenerative potential of OEGs after neural injury.

Hodowla neuronów zwojowych siatkówki z glejem osłonki węchowej

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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