Name: Generation of Functional Endodermal Hepatic Organoids (Video) | JoVE Journal
Uploaded: 2025-05-02T12:30:00.000+00:00
Duration: 8 min 45 s
Description: Learn a fast protocol for generating endodermal hepatic organoids using long-term culture techniques. This method produces functional eHEPOs in 2 weeks for liver disease research.

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5 minutes

This research was supported by The Scientific and Technological Research Council of Turkey (T&#220;B&#304;TAK) via projects SBAG-115S465 and SBAG-213S182. Figure 1 was generated using BioRender.

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</ol>

The authors have nothing to disclose. The authors have no conflicts of interest to declare. Esra Erdal is co-founder of the ORGANO-ID Biotechnology company.

The present protocol describes a comprehensive method for generating, expanding, and freezing/thawing hepatic organoids starting from iPSCs. This protocol covers all the steps, including culturing the iPSCs on the feeder and feeder-free culture, 2-dimensional endoderm differentiation, enrichment of the progenitor cells with FACS and organoid formation, and generating function-gaining organoids. Moreover, detailed instructions for validating and characterizing the organoids are also provided. The major obstacle to the wid...

Organoids are miniaturized organ-like structures grown in 3-dimensional culture conditions that mimic the organ microenvironment and include the intrinsic factors necessary for self-organization and self-renewal in organ development itself. Organoids can be derived either from pluripotent stem cells (PSCs) or adult tissue-derived cells (stem or progenitor cells)1. Although their accurate organ-like organization and functional similarity to the specific organ make them valuable tools for disease modeling, they still need further improvements in terms of standardization in culture. In particular, several protocols have been published for the generation of liver organoids, and they differ in their complexity and reproducibility2. For instance, the liver bud organoids developed by Takebe et al. take the form of dense, multi-cellular structures containing the following induced pluripotent stem cells (iPSCs): hepatic endodermal progenitors, human umbilical vein endothelial cells (HUVECs), and mesenchymal stem cells (MSCs). However, those organoids do not have long-term self-renewal capacity3,4.
From a historical perspective, Huch et al. first reported the production of human hepatic epithelial organoids derived from adult tissue, in which the cells polarize and specialize to reproduce aspects of the native epithelium5. Then, Guan et al. used iPSC-derived hepatic organoids to model Alagille syndrome (ALGS), a rare genetic disorder associated with bile duct reduction within the liver6. Both of these organoids have self-renewal capacity and can gain mature hepatocyte functions, such as bile and albumin secretion, glycogen storage, and liver-specific drug detoxification. In a recent study, Ramli et al. introduced a PSC-derived liver organoid model containing functional bile canaliculi networks between polarized hepatocyte-like cells (HLCs) that empty cholestatic drugs into biliary cysts composed of cholangiocyte-like cells (CLCs)7.
This study presents a unique culture for generating iPSC-derived endodermal hepatic organoids, called eHEPOs. The iPSC culture and differentiation into endoderm are described step by step, and the generation of eHEPOs from enriched EpCAM+ progenitors is demonstrated. Finally, the characterization of the functionality and structural organization of the eHEPOs, as well as the cryopreservation of the organoids, are described.

<table><tbody><tr><td>15 mL conical centrifuge tubes</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>37 &amp;deg;C water bath</td><td>N&amp;uuml;ve</td><td>210.NB9</td><td></td></tr><tr><td>37 &amp;deg;C, 5 % CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Memmert</td><td>INCO 153</td><td></td></tr><tr><td>50 mL conical centrifuge tubes</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>70 &amp;micro;m and 40 &amp;micro;m Cell strainer</td><td>Falcon</td><td>352350/ 352340</td><td></td></tr><tr><td>70% Ethanol</td><td>Sigma</td><td>1009832511</td><td></td></tr><tr><td>A1AT</td><td>Abcam</td><td>ab166610</td><td>Dilution: 1/500 (IF), 1/50 (IHC)</td></tr><tr><td>A-83.01 (TGF-&amp;beta; inhibitor)</td><td>Tocris Biosciene</td><td>2939</td><td></td></tr><tr><td>Acetone</td><td>Isolab</td><td>9,01,026</td><td></td></tr><tr><td>Adhesive Microscope Slide</td><td>Histobond</td><td>C981040</td><td></td></tr><tr><td>Advanced DMEM-F12</td><td>Gibco</td><td>12634-010</td><td></td></tr><tr><td>AFP</td><td>Abcam</td><td>ab3980</td><td>Dilution: 1/25 (IHC)</td></tr><tr><td>Agar</td><td>EMS</td><td>10200</td><td></td></tr><tr><td>ALB</td><td>Abcam</td><td>ab10241</td><td>Dilution: 1/100 (IF), 1/20 (IHC)</td></tr><tr><td>Alexa Flour 488 (Mouse)</td><td>Invitrogen</td><td>A11001</td><td>Dilution: 1/1000 (IF)</td></tr><tr><td>Alexa Flour 488 (Rabbit)</td><td>Invitrogen</td><td>A110034</td><td>Dilution: 1/1000 (IF)</td></tr><tr><td>Alexa Flour 594 (Mouse)</td><td>Invitrogen</td><td>A11005</td><td>Dilution: 1/1000 (IF)</td></tr><tr><td>Alexa flour 594 (Rabbit)&amp;nbsp;</td><td>Invitrogen</td><td>A11037</td><td>Dilution: 1/1000 (IF)</td></tr><tr><td>Ammonia Assay Kit</td><td>Sigma-Aldrich</td><td>MAK-310</td><td></td></tr><tr><td>Ammonium cloride</td><td>Santa Cruz</td><td>sc-202936</td><td></td></tr><tr><td>B27 Supplement 50x</td><td>Gibco</td><td>12587010</td><td></td></tr><tr><td>Base mold</td><td>Sakura</td><td>4216</td><td></td></tr><tr><td>b-FGF</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>Biosafety, CLASS II,SAFETY CABINET</td><td>Thermo</td><td>SAFE 2020</td><td></td></tr><tr><td>Calibrated pipettes</td><td>Gilson</td><td>F167380</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Cholylglycylamido-fluorescein</td><td>Corning</td><td>451041</td><td></td></tr><tr><td>Citrate Buffer pH 6.0</td><td>Bio-optica</td><td>15-M103</td><td></td></tr><tr><td>CK-18</td><td>Santa Cruz</td><td>sc-51582</td><td>Dilution: 1/100 (IF), 1/20 (IHC)</td></tr><tr><td>CK-19</td><td>Santa Cruz</td><td>sc-6278</td><td>Dilution: 1/100 (IF), 1/20 (IHC)</td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM880</td><td></td></tr><tr><td>Cryogenic handling gloves and eye protection</td><td>Cryokit</td><td>5274</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 1950</td><td></td></tr><tr><td>Cryovial tubes</td><td>Corning</td><td>430659</td><td></td></tr><tr><td>DAB</td><td>Roche</td><td>11718096001</td><td></td></tr><tr><td>DAPT</td><td>Sigma-Aldrich</td><td>a5942</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>Dispase&amp;nbsp;</td><td>Stem Cell Technologies</td><td>7923</td><td></td></tr><tr><td>DMEM F12</td><td>Gibco</td><td>31330038</td><td></td></tr><tr><td>E-CAD</td><td>Santa Cruz</td><td>sc-8426</td><td>Dilution: 1/100 (IF), 1/20 (IHC)</td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-020</td><td></td></tr><tr><td>Electron Microscope</td><td>Zeiss</td><td>Sigma500</td><td></td></tr><tr><td>ELISA kit</td><td>Fortis life sciences bethyl</td><td>E88-129</td><td></td></tr><tr><td>Embed 812 Embedding Kit</td><td>EMS</td><td>14121</td><td></td></tr><tr><td>Entellan</td><td>Merck</td><td>107961</td><td></td></tr><tr><td>Eosin Y %1</td><td>Sigma-Aldrich</td><td>HT110332</td><td></td></tr><tr><td>EpCAM</td><td>Miltenyi Biotec</td><td>130-059-901</td><td>Dilution: 1/11 (FACS)</td></tr><tr><td>Ethanol</td><td>Merck</td><td>1,00,98,32,511</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>26010066</td><td></td></tr><tr><td>Forskolin (FSK)</td><td>Tocris Biosciene</td><td>1099</td><td></td></tr><tr><td>Freezing container (Mr. Frosty)</td><td>Thermo</td><td>5100-0001</td><td></td></tr><tr><td>Freezing Medium</td><td>Gibco</td><td>12648010</td><td></td></tr><tr><td>Glass Pasteur pipette</td><td>Isolab</td><td>084.01.001</td><td></td></tr><tr><td>Glutamax 100x</td><td>Gibco</td><td>35050-068</td><td></td></tr><tr><td>Gluteraldehyde %25, EM grade</td><td>EMS</td><td>16210-1L</td><td></td></tr><tr><td>Goat Anti-Mouse HRP</td><td>Thermo Fisher</td><td>62-6520</td><td>Dilution: 1/1000 (IHC)</td></tr><tr><td>Goat Anti-Rabbit HRP</td><td>Thermo Fisher</td><td>31460</td><td>Dilution: 1/1000 (IHC)</td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>162-10-072</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>107209</td><td></td></tr><tr><td>Hematoxylin</td><td>Millipore</td><td>HX86017674</td><td></td></tr><tr><td>HEPES, 1 M</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>HNF-4&amp;alpha;</td><td>Abcam</td><td>ab55223</td><td>Dilution: 1/50 (IHC)</td></tr><tr><td>Ice and dry ice</td><td>homemade</td><td>homemade</td><td></td></tr><tr><td>Incubator (65 &amp;deg;C)</td><td>N&amp;uuml;ve</td><td>EN 400</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>24137</td><td></td></tr><tr><td>Leu15 Gastrin I human</td><td>Sigma-Aldrich</td><td>G9145</td><td></td></tr><tr><td>Luminometer</td><td>Berthold Tech</td><td>LB 960</td><td></td></tr><tr><td>Master mix&amp;nbsp;</td><td>Applied Biosystems</td><td>43676659</td><td></td></tr><tr><td>Matrigel matrix, hESC-Qualified</td><td>Corning</td><td>354277</td><td></td></tr><tr><td>Matrigel matrix, phenol-red-free</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>Methanol</td><td>Merck</td><td>179337</td><td></td></tr><tr><td>Microcentrifuge tubes</td><td>Axygen</td><td>321-02-501</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>AXIO VERT A1</td><td></td></tr><tr><td>Microtome blade</td><td>Feather</td><td>S35, C35</td><td></td></tr><tr><td>mTeSR1</td><td>Stem Cell Technologies</td><td>sc-05850</td><td></td></tr><tr><td>Multi well suspension culture plates</td><td>Sarstedt</td><td>83,39,21,500</td><td></td></tr><tr><td>N2 supplement 100x</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>N-Acetylcysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>Neutral Buffered Formalin %10</td><td>Tekkim</td><td>TK.60161.05001</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636</td><td></td></tr><tr><td>Non-essential Amino Acide (NEAA)</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>OCT</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Osmium tetroxide</td><td>EMS</td><td>19110</td><td></td></tr><tr><td>P450-Glo Assays kit&amp;nbsp;</td><td>Promega</td><td>V9001</td><td></td></tr><tr><td>Pap-pen</td><td>Sigma</td><td>Z377821-1EA</td><td></td></tr><tr><td>Paraffin</td><td>Tekkim</td><td>TK.200661.01002</td><td></td></tr><tr><td>PAS stain kit</td><td>Abcam</td><td>ab150680</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>be17-516</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>Phosphotungustic acid</td><td>Ted Pella</td><td>19402</td><td></td></tr><tr><td>Pipette aid</td><td>Axygen</td><td>Motopet-1</td><td></td></tr><tr><td>Plate reader varioskan flash&amp;nbsp;</td><td>Thermo</td><td>5250040</td><td></td></tr><tr><td>Prolong Antifade Mountant</td><td>Invitrogen</td><td>P36980</td><td></td></tr><tr><td>Propylene Oxide, EM grade</td><td>EMS</td><td>20401</td><td></td></tr><tr><td>Real Time PCR system</td><td>Applied Biosystems</td><td>7500 Fast&amp;nbsp;</td><td></td></tr><tr><td>Recombinant human Activin A</td><td>R&amp;amp;D</td><td>338-Ac-050</td><td></td></tr><tr><td>Recombinant human BMP7</td><td>Peprotech</td><td>120-03</td><td></td></tr><tr><td>Recombinant human EGF</td><td>Peprotech</td><td>af-100-15</td><td></td></tr><tr><td>Recombinant human FGF10</td><td>Peprotech</td><td>100-26</td><td></td></tr><tr><td>Recombinant human FGF19</td><td>Peprotech</td><td>100-32</td><td></td></tr><tr><td>Recombinant human HGF</td><td>Peprotech</td><td>100-39</td><td></td></tr><tr><td>Rho kinase inhibitor, Y-27632 dihydrochloride</td><td>Sigma-Aldrich</td><td>Y0503</td><td></td></tr><tr><td>RNase/DNase free 1.5 mL tube</td><td>Axygen</td><td>31108101</td><td></td></tr><tr><td>RNase/DNase free filter tips</td><td>Sarstedt</td><td>703031255</td><td></td></tr><tr><td>Rotary Microtome</td><td>Leica</td><td>RM 2245</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco</td><td>61870010</td><td></td></tr><tr><td>Rspo1-conditioned medium</td><td>Homemade</td><td></td><td></td></tr><tr><td>Slide master</td><td>Bio-optica</td><td>15-MEQ001</td><td></td></tr><tr><td>Sorenson&amp;rsquo;s Phosphate Buffer</td><td>EMS</td><td>11600-10</td><td></td></tr><tr><td>Spinner</td><td>Thermo</td><td>MY SPIN 6</td><td></td></tr><tr><td>Sterile serological pipettes</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>Tissue Casette</td><td>Leica</td><td>3802240</td><td></td></tr><tr><td>Trimmer</td><td>Leica</td><td>EM TRIM2</td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Scientific</td><td>28314</td><td></td></tr><tr><td>TrypLE Express Enzyme</td><td>Gibco</td><td>12605010</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td>EM UC7</td><td></td></tr><tr><td>Uranylacetate</td><td>EMS</td><td>22400</td><td></td></tr><tr><td>Vortex</td><td>Thermo</td><td>88880018</td><td></td></tr><tr><td>Wnt Surrogate-Fc Fusion Protein</td><td>ImmunoPrecise</td><td>N001</td><td></td></tr><tr><td>Xylene</td><td>Sigma&amp;nbsp;</td><td>16446</td><td></td></tr><tr><td>ZO-1</td><td>Invitrogen</td><td>40-2200</td><td>Dilution: 1/400 (IF)</td></tr></tbody></table>

generation of functional endodermal hepatic organoids

Generation of Functional Endodermal Hepatic Organoids

Organoid technology has allowed us to generate a variety of human organ-like mini structures, such as for the liver, brain, and intestine, in vitro. The remarkable advances in organoid models have recently opened a new experimental era for various applications in disease modeling, developmental biology, and drug discovery. Adult stem cells or induced&#160;pluripotent stem cell (iPSC)-derived liver organoids govern the generation of hepatocytes to use for diverse applications. Here, we present a robust and reproducible protocol for generating hepatic organoids from pluripotent stem cells. This protocol is applicable to healthy and patient-derived cells. To achieve 3D endoderm-derived hepatic organoids (eHEPOs), iPSCs were directly first differentiated into endodermal cells, and then FACS-enriched EpCAM-positive (EpCAM+) cells were used to establish hepatic organoids using the expansion medium. We provide a fast and efficient method to generate hepatic organoids within 2 weeks. The generated organoids mimic the essential properties and functions of hepatocytes, such as albumin secretion, glycogen storage, and cytochrome P450 enzyme activity. Besides the liver-specific gene expression similarities, eHEPOs comprise polarized epithelial cells with bile canaliculi in between. In addition, eHEPOs can be expanded and serial passages long term (1 year) without losing their capacity to differentiate into mature hepatocytes. Thus, eHEPOs provide an alternative source to produce functional hepatocytes.

Firstly, human fibroblast cells or peripheral blood mononuclear cells (PBMC) cells were cultured and converted to iPSCs via episomal reprogramming. The fresh knockout serum was essential for obtaining healthy iPSCs. Then, the iPSCs were seeded into the BMM-coated culture plates with 50%-60% confluency. Having iPSC colonies of a small/medium size improved the differentiation efficiency. Then, the iPSCs were differentiated into definitive endoderm with medium containing Activin A, Wnt3a, and R-spo1 factors for 5 d...

This protocol describes the generation of fast and reproducible endodermal hepatic organoids (eHEPOs). With this protocol, eHEPOs can be produced within 2 weeks and expand long-term (more than 1 year) without losing their differentiation and functionality.

Organoid technology has allowed us to generate a variety of human organ-like mini structures, such as for the liver, brain, and intestine, in vitro. The ...

generation-of-functional-endodermal-hepatic-organoids

Research

JoVE Journal

Biology

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

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Learn a fast protocol for generating endodermal hepatic organoids using long-term culture techniques. This method produces functional eHEPOs in 2 weeks for liver disease research.

Generation of Functional Endodermal Hepatic Organoids

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