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 me...

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

<ol>
	<li>Cowan, C. S., 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=Cell+types+of+the+human+retina+and+its+organoids+at+single-cell+resolution.">Cell types of the human retina and its organoids at single-cell resolution.</a> Cell. 182 (6), 1623-1640.e34 (2020).</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=Directed+induction+of+retinal+organoids+from+human+pluripotent+stem+cells.">Directed induction of retinal organoids from human pluripotent stem cells.</a> J Vis Exp. (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. 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=Transcriptomic+analysis+of+purified+human+cortical+microglia+reveals+age-associated+changes.">Transcriptomic analysis of purified human cortical microglia reveals age-associated changes.</a> Nat Neurosci. 20 (8), 1162-1171 (2017).</li><li>Nakano, T., 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-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10 (6), 771-785 (2012).</li><li>Kim, S., 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=transcriptome+profiling,+and+functional+validation+of+cone-rich+human+retinal+organoids.">transcriptome profiling, and functional validation of cone-rich human retinal organoids.</a> Proc Natl Acad Sci U S A. 116 (22), 10824-10833 (2019).</li><li>Gao, M. 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. S., 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=IPS-cell-derived+microglia+promote+brain+organoid+maturation+via+cholesterol+transfer.">IPS-cell-derived microglia promote brain organoid maturation via cholesterol transfer.</a> Nature. 623 (7986), 397-405 (2023).</li><li>Usui-Ouchi, 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=Integrating+human+ipsc-derived+macrophage+progenitors+into+retinal+organoids+to+generate+a+mature+retinal+microglial+niche.">Integrating human ipsc-derived macrophage progenitors into retinal organoids to generate a mature retinal microglial niche.</a> Glia. 71 (10), 2372-2382 (2023).</li><li>Chichagova, V., 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=Incorporating+microglia-like+cells+in+human+induced+pluripotent+stem+cell-derived+retinal+organoids.">Incorporating microglia-like cells in human induced pluripotent stem cell-derived retinal organoids.</a> J Cell Mol Med. 27 (3), 435-445 (2023).</li></ol>

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<sup class="xref...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;s instructions</td>
		</tr>
		<tr>
			<td>Anti-IBA1</td>
			<td>Abcam</td>
			<td>ab5076</td>
			<td>Antibody; dilution as per the manufacturer&#39;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&#160;</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&#160;</td>
			<td>216-MC-500&#160;</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&#160;</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>&#946;-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.

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

Assembling Retinal Organoids with Microglia

To begin, obtain the human embryonic stem cells in the stem cell medium with a cell density of 80 to 90%After removing the spent medium, rinse the cells in each well with one milliliter of 1X DPBS. Incubate the stem cell colonies in each well with one milliliter of 0.5 millimolar EDTA for approximately five minutes at 37 degrees Celsius. Check the colony morphology to confirm that the edges are slightly curled up with loose gaps.Aspirate the EDTA, and if necessary, incubate cells at 37 degrees Celsius for another one to two minutes, but not exceeding eight minutes. Gently rinse the cells in each well with six milliliters of medium A containing six microliters of 10 millimolar ROCK inhibitor Y-27632 solution. Gently tap the bottom of the dish to help rinse the cells.After 24 hours, observe the cells under the microscope to confirm the embryo body formation. Using a 10 milliliter pipette, collect the embryo bodies into a 15 milliliter centrifugal tube and wait for the embryo bodies to settle to the bottom for five minutes. Then carefully aspirate the supernatant and resuspend the embryo bodies in six milliliters of fresh medium A.Transfer them into a new low adhesion well of a six-well plate and culture in a 37 degrees Celsius incubator.On day four, coat a 10 centimeter dish with three milliliters of 0.1%fish gelatin solution and incubate for at least one hour in a 5%carbon dioxide, 37 degrees Celsius incubator. Then remove the gelatin solution and rinse the dish once with five to six milliliters of 1X DPBS. Carefully collect the embryo bodies into a 15 milliliter centrifugal tube and wait for the embryo bodies to settle to the bottom In five minutes.Gently resuspend the embryo bodies with 15 milliliters of medium B and transfer them to the coated 10 centimeter dish. Culture in a 37 degrees Celsius, 5%carbon dioxide incubator for one week after 49 days. Centrifuge the cells at 200 x g for five minutes at room temperature.Carefully aspirate the supernatant and resuspend the cells with two milliliters of fresh medium C.Transfer into a new low adhesion well of a six-well plate. Incubate the plate in a 5%carbon dioxide, 37 degrees Celsius incubator. On day 56, replace the spent medium with two milliliters of medium C per well.Examine the plate under a microscope to identify the branched microglia adhere to the bottom of the low adhesion well. To harvest microglia for co-culturing, gently replace the medium C in each well with one milliliter Accutase and incubate at 37 degrees Celsius for three minutes. Using a five milliliter pipette, collect the microglia cells into a 15 milliliter tube and centrifuge the cells at 200 x g for five minutes at room temperature.After discarding the supernatant, suspend the microglia with one milliliter of medium E.

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 (<strong c...

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 ...

assembling-retinal-organoids-with-microglia

Research

JoVE Journal

Neuroscience

529 ビュー.  Capital Medical University. まず、幹細胞培地でヒト胚性幹細胞を細胞密度80〜90%で取得します使用済み培地を取り出した後、各ウェルの細胞を1ミリリットルの1X DPBSですすいでください。各ウェルの幹細胞コロニーを1ミリリットルの0.5ミリモルEDTAで摂氏37度で約5分間インキュベートします。コロニーの形態をチェックして、エッジがわずかに丸まって隙間が緩いことを確?...

ビデオ: 疾患モデリングの改善のための網膜オルガノイドと組み合わせたヒトミクログリアの生成

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...

https://cloudfront.jove.com/CDNSource/teasers/67016_b.jpg

generation of human retinal organoids and co-culturing with microglia for disease modeling and drug screening

Generation of Human Retinal Organoids and Co-Culturing with Microglia for Disease Modeling and Drug Screening

generation of human brain organoids for mitochondrial disease modeling

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial...

generation of retinal organoids from healthy and retinal disease-specific human-induced pluripotent stem cells

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

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...

generation of human patient ipsc-derived retinal organoids to model retinitis pigmentosa

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

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.

toxicity screens in human retinal organoids for pharmaceutical discovery

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2...

generation of human 3d lung tissue cultures (3d-ltcs) for disease modeling

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

generation of 3d whole lung organoids from induced pluripotent stem cells for modeling lung developmental biology and disease

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with...

generation of multicellular human primary endometrial organoids

Generation of Multicellular Human Primary Endometrial Organoids

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial...

generation of ipsc-derived human brain organoids to model early neurodevelopmental disorders

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

derivation of a human brain organoid with microglia development

Author Spotlight: Generation of Human Brain Organoids with Integrated Microglia and Neurons Through Co-Culture of Hematopoietic Progenitors

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...

Derivation of a Human Brain Organoid with Microglia Development

generation of hipsc-derived intestinal organoids for developmental and disease modelling applications

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

This protocol allows for the differentiation of human pluripotent cells into intestinal organoids. The protocol mimics normal human development by differentiating cells into a population of definitive endoderm, hindgut endoderm and then intestinal epithelium. This makes the protocol suitable for studying both intestinal development as well as disease modelling...

directed induction of retinal organoids from human pluripotent stem cells

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of...

integrative protocol for generation of ipsc-derived 3d human hepatic organoids

Human iPSC-derived 3D hepatic organoids constitute a potential tool for understanding the thyroid hormone's action on liver development.

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

efficient pam-less base editing for zebrafish modeling of human genetic disease with zspry-abe8e

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

This protocol describes how to perform efficient adenine base editing without PAM limitation to construct a precise zebrafish disease model using zSpRY-ABE8e.

generation of human blood vessel organoids from pluripotent stem cells

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement...

isolation of primary porcine retinal pigment epithelial cells for in vitro modeling

Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models

This protocol outlines the procedure for obtaining and culturing primary retinal pigment epithelial (RPE) cells from locally sourced porcine eyes. These cells serve as a high-quality alternative to stem cells and are suitable for in vitro retinal...

A Cost-Effective Method for Isolating Primary RPE Cells from Porcine Eyes

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

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isolation of human primary valve cells for in vitro disease modeling

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

To begin, obtain human microglia from the human embryonic stem cells and harvest them on day 56. For retinal organoid derivation, culture the human embryonic stem cells in a stem cell medium until the cell density reaches 80 to 90%Add one milliliter dispase per well to the culture cells and incubate at 37 degrees Celsius for five minutes. After removing dispase, add one milliliter of medium D to each well.Cut the cells into small pieces. With a 10 microliter pipette, gently collect all the cell pieces and medium into a 1.5 milliliter microcentrifuge tube. Centrifuge the tube at 200 x g for five minutes.After removing the supernatant, gently resuspend the cells in 200 microliters of a cold matrix. Move the 1.5 milliliter microcentrifuge tube into an incubator for 20 minutes. After 20 minutes in the incubator, the matrix will solidify.Prepare a 10 centimeter dish with 15 milliliters of medium D.Resuspend the matrix with medium D and shake the Petri dish gently. Place the dish in an incubator for five days. On day 12, replace the medium with three milliliters of dispase.After five minutes, aspirate the dispase and add 15 milliliters of medium E.Introduce the harvested microglia to digested retinal organoids on day 12. On day 19, gently swirl the plate to aggregate the organoids to the center of the dish and replace the medium E with medium F.Finally, transfer the organoids with medium F to a new suspension dish. By day 30, retinal organoids co-cultured with microglia showed significant GFP autofluorescence, indicating the presence of microglia.The co-cultured retinal organoids displayed clear immunofluorescence signals for the photoreceptor marker CRX and the microglial marker IBA1.