We would like to acknowledge the technical support from the Confocal and Specialized Microscopy Shared Resource of Herbert Irving Comprehensive Cancer Center at Columbia University, funded in part through NIH Center Grant (P30CA013696). This work is supported by NIH (R21NS133635, Y.-H.L.; UH3TR002151, K. W. L.). Figure 1A was created using BioRender.

Figure 1 presents an overview of the steps involved in this protocol. Detailed information regarding the reagents and materials utilized in this protocol is given in the Table of Materials. It is recommended that all the media should be prewarmed at 37 &#176;C before use unless specified otherwise. All the cell culture materials and supplies should be either autoclaved or sterile, and cell handling should be done in a biosafety cabinet. Additionally, the pH value of the coll...

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

<ol>
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The described protocol includes two key modifications for homogenous and reproducible generation of high-quality vascular organoids. The first modification is the use of a microwell plate to enable better control of the EB uniformity. As a starting point of vascular differentiation, the size of EBs is critical as it regulates the stem cell fate and differentiation efficacy towards different lineages. Variations in EB size usually lead to heterogeneous organoids with inconsistent structure and organization<sup class="xref...

In vitro microphysiological models, including organoid and tissue-on-a-chip systems, have emerged over the past decade1,2,3. These three-dimensional (3D), human cell-derived systems address the limitations in conventional two-dimensional (2D) cell culture and animal models, such as the lack of many components in the physiological microenvironment and genetic differences across species, respectively. They provide more physiological insights and are more suitable for disease modeling and drug screening than conventional models. Among the microphysiological models, organoids derived from human induced pluripotent stem cells (hiPSCs) have been proven to recapitulate the architectural features of native human tissues effectively and have been developed to mimic different human organs, including the brain4,5, kidney6, liver7,8&#160;and retina9. Here, we particularly focus on the generation of vascular organoids from hiPSCs and outline a streamlined protocol that aims to minimize the variations between different organoid samples and batches, thereby improving the overall reproducibility.
Vasculature plays a crucial role in maintaining physiological homeostasis across all human organs. The formation of vasculature involves the processes of vasculogenesis and angiogenesis, orchestrated through the interactions between endothelial and mural (e.g., pericytes and vascular smooth muscle cells) cells and influenced by various stimuli10. Penninger, Wimmer&#160;and colleagues developed the first vascular organoid generation method11,12 that mimics these two processes. With the organoids generated from this method, their group12 and us13,14 have demonstrated the potential of vascular organoids for modeling vascular malfunctions in diseases. For example, in our recent works13,14, distinct phenotypes were observed, such as vessel sprouting and mural cell composition variations, between disease-mimicking vascular organoids and their wild-type counterparts. These examples highlight that the vascular&#160;organoid model could serve as a platform for drug screening by mimicking disease-related vascular malfunctions.
Built upon the initial vascular organoid generation works11,12, a revised protocol is presented that includes the use of microwell to facilitate homogenous embryoid body (EB) formation, a critical factor in minimizing the variations often seen within the same organoid generation batch. Additionally, the CEPT cocktail, a combination of chroman 1, emricasan, polyamines&#160;and the integrated stress response inhibitor (trans-ISRIB)15, is employed&#160;to improve the viability of hiPSCs and differentiated cells during the EB formation process. This modification is mainly to reduce the variations between different organoid samples and batches. Uniform vascular organoids can be produced with this roughly two-week-long protocol, where vasculogenesis is induced to help endothelium organize into primitive tubular networks on Day 1, followed by angiogenesis induction on Day 5 to develop complex vascular networks in organoids. On Day 12-15, the generated organoids should show mature vascular networks composed of both endothelium and mural cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol (1000x)</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma-Aldrich</td>
			<td>A6964</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure F(ab&#39;)2 Fragment Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immuno Research Labs</td>
			<td>711-546-152</td>
			<td>reconstitute with 0.25 mL 50% glycerol,&#160; store at -20 &#176;C for up to 1 year, dilution ratio for use: 1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 AffiniPure F(ab&#39;)2 Fragment Donkey Anti-Goat IgG (H+L)&#160;</td>
			<td>Jackson Immuno Research Labs</td>
			<td>705-606-147</td>
			<td>reconstitute with 0.25 mL 50% glycerol,&#160; store at -20&#176;C for up to 1 year, dilution ratio for use: 1:1000</td>
		</tr>
		<tr>
			<td>B27 supplement minus vitamin A (50x)</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20 &#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>ProSpec</td>
			<td>CYT-081</td>
			<td>reconstitute with sterile ddH2O at a concentration of 100 ng/&#181;L, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>CD31 antibody&#160;</td>
			<td>abcam</td>
			<td>ab28364</td>
			<td>dilution ratio: 1:400</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>022626001</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris Bioscience</td>
			<td>4423/10</td>
			<td>make the stock solution at 12 mM with DMSO, and store at -20 &#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>Chroman 1</td>
			<td>Tocris</td>
			<td>7163/10</td>
			<td>make the stock solution at 100 &#181;M with DMSO, and store at -20 &#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>Collagen I&#160;</td>
			<td>Corning</td>
			<td>40236</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>AXRMP/Ti2</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Elplasia Plates</td>
			<td>Corning</td>
			<td>4441</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL automated cell counter</td>
			<td>Thermo Fisher</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX supplement</td>
			<td>Gibco</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, powder</td>
			<td>Gibco</td>
			<td>12500062</td>
			<td>make 10x stock solution with biograde ddH2O and sterilize it with a 0.2 &#181;m filter. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Edi042A</td>
			<td>Cedars Sinai</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Emricasan</td>
			<td>Medchemexpress LLC</td>
			<td>HY1039610MG</td>
			<td>make the stock solution at 1 mM with DMSO, and store at -20 &#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>ERG antibody</td>
			<td>Cell Singali Technology</td>
			<td>97249</td>
			<td>dilution ratio: 1:400</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Premium</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td>thaw at 4 &#176;C&#160; and aliquot, store at&#160; -20 &#176;C for up to five years, or store at 4 &#176;C for up to a month</td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>ProSpec</td>
			<td>CYT557</td>
			<td>reconstitute with sterile ddH2O at a concentration of 100 ng/&#181;L, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Fluorodish Cell Culture Dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Tocris Bioscience</td>
			<td>1099</td>
			<td>reconstitute with DMSO at a concentration of 12 mM, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL)</td>
			<td>Gibco</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement (100x)</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin, 100 kU</td>
			<td>MilliporeSigma</td>
			<td>375095100KU</td>
			<td>reconstitute with sterile ddH2O at a concentration of 4 kU/mL</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td>13998123</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>Matrigel, GFR Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356230</td>
			<td>thaw at 4 &#176;C, aliquot and store at -80&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (NEAA, 100x)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water</td>
			<td>Corning</td>
			<td>46000CV</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus kit</td>
			<td>STEMCELL Technologies</td>
			<td>1001130</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>N2 supplement (100x)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>NaOH solution (1 M)</td>
			<td>Cytiva&#160;HyClone</td>
			<td>SH31088.01</td>
			<td></td>
		</tr>
		<tr>
			<td>NeuroBasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements, ver 5.42</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson Immuno Research Labs</td>
			<td>17000121</td>
			<td>reconstitute with 10 mL sterile ddH2O, store at 4 &#176;C for up to two weeks</td>
		</tr>
		<tr>
			<td>paraformaldehyde, 20%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713S</td>
			<td>dilute with PBS</td>
		</tr>
		<tr>
			<td>PBST, 20x</td>
			<td>Thermofisher</td>
			<td>28352</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGFR&#946; antibody</td>
			<td>R&#38;D</td>
			<td>AF385</td>
			<td>dilution ratio: 1:400</td>
		</tr>
		<tr>
			<td>polyamine supplement (1000x)</td>
			<td>Sigma-Aldrich</td>
			<td>P8483</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapiclear clearing reagent, 1.49</td>
			<td>SUNJIN LAB</td>
			<td>RC149001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (7.5%)</td>
			<td>Gibco</td>
			<td>25080094</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate monohydrate</td>
			<td>Thermofisher</td>
			<td>J6228822</td>
			<td>dissolve with ddH2O for 1% (wt/v) stock solution</td>
		</tr>
		<tr>
			<td>TBST, 20x</td>
			<td>Thermofisher</td>
			<td>28360</td>
			<td></td>
		</tr>
		<tr>
			<td>trans-ISRIB</td>
			<td>Tocris Bioscience</td>
			<td>5284/10</td>
			<td>make the stock solution at 1 mM with DMSO, and store at -20&#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>Tritin X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Thermo Fisher</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF-A</td>
			<td>ProSpec</td>
			<td>CYT-116</td>
			<td>reconstitute with sterile ddH2O at a concentration of 100 ng/&#181;L, aliquot and store at -20&#176;C</td>
		</tr>
	</tbody>
</table>

vascular organoid generation from human-induced pluripotent stem cells

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

This study introduces a modified method for scalable production of homogeneous vascular organoids from human IPSCs. By using novel ROCK inhibitors and the microwell confinement, the approach enhances reproducibility and minimize batch-to-batch variations, making it suitable for downstream morphological analysis and disease modeling applications. Single-cell sequencing technologies have been extensively utilized in organoid research to unravel the genomic, epigenomic, and the transcriptomic heterogeneity and dynamics within cellular populations.3D imaging technologies such as tissue clearing and the light-sheet microscopy facilitate rapid, high-resolution imaging of cellular morphology and the tissue architecture in a three-dimensional context. The throughput and the standardization are key challenges in organoid research. Manual handling is labor-intensive and limits the throughput, making robotic automation a promising solution.Standardization of organoid culture, including IPSC quality control and the differentiation protocol optimization can significantly enhance reproducibility and minimize variations between batches and cell lines.

Figure 1A presents the scheme of this protocol, including the key components used in each stage. The differentiation started with the EB formation, followed by mesoderm induction and vascular lineage specification (Figure 1B-D). The aggregates grew larger and less spherical over time. Organoids started showing rough edges on Day 5. After embedded in the collagen I-basement membrane matrix mixture, vascular endothelial cells and ...

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

vascular-organoid-generation-from-human-induced-pluripotent-stem-cells