This work was supported by the National Natural Science Foundation of China (No. 81870432 and 81570567 to X.L.Z.), (No. 81571994 to P.N.S.); the Natural Science Foundation of Guangdong Province, China (No. 2020A1515010054 to P.N.S.), The Li Ka Shing Shantou University Foundation (No. L1111 2008 to P.N.S.). We would like to thank Prof. Stanley Lin from Shantou University Medical College for useful advice.

1. Stem cell maintenance
NOTE: The cell maintenance protocol described below applies to the hES03 cell line maintained in an adherent monolayer. For all the following protocols in this manuscript, cells should be handled under a biological safety cabinet.
<ol>
	<li>Prepare 1x mTesR stem cell culture medium by diluting 5x supplementary medium to mTesR basic medium.</li>
	<li>Prepare 30x hESC-qualified Matrigel medium by diluting 5 mL of hESC-qualified Matrigel with 5 mL of DMEM/F12 on the ice. Store at -20 &#176;C.</li>
	<li>Prepare 1x hESC-qualified Matrigel medium by diluting 33.3 &#181;L of 30x hESC-qualified Matrigel with 1 mL of DMEM/F12.</li>
	<li>Coat sterile 6 well, tissue culture-treated plates with 1 mL of 1x hESC-qualified Matrigel in each well in advance and store at 4 &#176;C overnight. Leave at room temperature (RT) for at least 30 min before use.</li>
	<li>Thaw the cryopreserved hESCs in a 37 &#176;C water bath for 3 min without shaking. Then immediately transfer the cells by pipetting into a 15 mL centrifuge tube containing 4 mL prewarmed 37 &#176;C mTesR medium, pipet up and down gently 2 times.</li>
	<li>Centrifuge the hESCs at 200 x g for 2 min at RT.</li>
	<li>Aspirate the supernatant and gently resuspend the cells in 1 mL of mTesR medium.</li>
	<li>Aspirate the DMEM/F12 medium from the plate and seed the cells into a well of 6-well plate at a density of 1 x 105 cells in 2 mL of mTesR.</li>
	<li>Incubate in a 5% CO2 incubator and maintain the cells by replacing with preheated mTesR medium daily. Passage the cells at roughly 70-80% confluence or when the cell colonies begin to make contact.</li>
</ol>
2. Stem cell passage and differentiation
<ol>
	<li>For passaging cells, aspirate the medium and incubate the cells with 1 mL per well of enzyme solution (e.g., Accutase&#160;for 5 min at 37 &#176;C. Then transfer the cells by pipetting to a 15 mL centrifuge tube containing 4 mL of DMEM/F12 prewarmed to 37 &#176;C.</li>
	<li>Centrifuge the cells at 200 x g at RT for 2 min.</li>
	<li>Aspirate the medium and gently resuspend the cell pellet in 1 mL of mTesR medium.</li>
	<li>Prepare 24-well plates by coating with 250 &#181;L of 1x hESC-qualified Matrigel medium in advance. Seed hESCs as described above at a density of 1 - 1.5 x 105 cells per well in 500 &#181;L of mTesR medium.</li>
	<li>Allow cells to incubate for 24 h at 37 &#176;C in a CO2 incubator.</li>
</ol>
3. DE formation
<ol>
	<li>Prepare stock solutions of 100 &#181;g/mL Activin A with DPBS containing 0.2% BSA and 3 mM CHIR99021 with DMSO.</li>
	<li>Prepare Stage I differentiation basic medium by supplementing RPMI medium with 1x B27.</li>
	<li>Add Activin A to a final concentration of 100 ng/mL and CHIR99021 to a final concentration of 3 &#181;M in an appropriate volume of preheated Stage I differentiation basic medium.</li>
	<li>Aspirate mTesR medium from the cells and replace with Stage I differentiation media containing added differentiation factors (e.g., 0.5 mL per well of a 24-well plate).</li>
	<li>Allow cells to incubate for 3 days at 37 &#176;C in a CO2 incubator, replacing Stage I media daily with freshly added differentiation factors (Days 0-2). 
	&#8203;NOTE: After 3 days of differentiation, differentiated cells should express markers of DE cells, such as FOXA2, SOX17, GATA4, CRCR4, and FOXA1 (minimum 80% of SOX17 needed for proceeding).</li>
</ol>
4. Differentiation of hepatic progenitor cells
<ol>
	<li>Prepare Stage II differentiation basic media by dissolving knockout serum replacement (KOSR) to a final concentration of 20% in Knock Out DMEM.</li>
	<li>Prepare Stage II differentiation medium containing 1% DMSO, 1x GlutaMAX, 1x non-essential amino acid (NEAA) solution, 100 &#181;M 2-mercaptoethanol and 1x penicillin/streptomycin (P/S) to the appropriate volume of KOSR/knockout DMEM.</li>
	<li>On Day 3 of differentiation, aspirate the medium and replace with Stage II medium (e.g., 0.5 mL per well of a 24 well plate). Then incubate for 24 h.</li>
	<li>On Day 4 of differentiation, aspirate the medium and replace with Stage II medium (e.g., 1 mL per well of a 24 well plate).</li>
	<li>Change the medium every other day (replace medium on Day 6). 
	&#8203;NOTE: After 8 days of differentiation, differentiated cells should express appropriate markers of hepatic progenitor cells, such as HNF4&#945;, AFP, TBX3, TTR, ALB, NTCP, CEBPA (minimum 80% of HNF4&#945; needed for proceeding).</li>
</ol>
5. Hepatocyte differentiation
<ol>
	<li>Prepare 10 &#181;g/mL hepatocyte growth factor (HGF), 20 &#181;g/mL Oncostatin (OSM) stock solutions with DBPS (containing 0.2% BSA) and filter with a 0.22 &#181;m filter membrane.</li>
	<li>Prepare Stage III differentiation basic medium (HepatoZYME-SFM (HZM) medium supplemented with 10 &#181;M hydrocortisone-21-hemisuccinate and 1x P/S).</li>
	<li>Add HGF to a final concentration of 10 ng/mL and OSM to a final concentration of 20 ng/mL to the appropriate volume of preheated Stage III differentiation medium.</li>
	<li>On Day 8 of differentiation, aspirate Stage II medium from cells and replace with Stage III medium (e.g., 1 mL per well of a 24 well plate).</li>
	<li>Allow cells to incubate for 10 days at 37 &#176;C in a CO2 incubator, changing Stage III media every other day with freshly added differentiation factors (Days 8-18). 
	&#8203;NOTE: After 18 days of differentiation, differentiated cells should express characteristic markers of hepatocytes, such as AAT, ALB, TTR, HNF4&#945;, NTCP, ASGR1, CYP3A4. The percentage of Albumin-positive cells usually is more than 90%.</li>
</ol>
6. RNA isolation, cDNA synthesis and RT-qPCR analysis
<ol>
	<li>Aspirate medium from cells and add the proper amount of RNAiso Plus reagent, (e.g., 0.3 mL per well of a 24-well plate). Collect the supernatant in a 1.5 mL tube and let stand at RT for 5 min.</li>
	<li>Add 60 &#181;L of chloroform reagent and leave it at RT for 5 min. Then centrifuge at 12,000 x g for 15 min.</li>
	<li>Collect the 125 &#181;L supernatant and transfer it to another centrifuge tube. Add 160 &#181;L of isopropanol, mix well, and stand at RT for 10 min.</li>
	<li>Centrifuge at 12,000 x g for 10 min.</li>
	<li>Remove the supernatant, add 75% ethanol to the precipitated, and centrifuge at 7,500 x g for 5 min.</li>
	<li>After drying at RT, add 40 &#181;L of DEPC-treated water to dissolve. For qPCR, reverse transcribe 2 &#181;g of total RNA with a reverse transcription kit according to the manufacturer&#39;s protocol.</li>
	<li>Perform RT-qPCR using a commercial kit according to the manufacturer&#39;s protocol.</li>
	<li>Normalize gene expression relative to non-induced stem cells and determine fold variation following normalization to GAPDH.</li>
</ol>
7. Immunofluorescence Validation of Differentiation
<ol>
	<li>Prepare 1x PBST washing buffer by adding 0.1% Tween-20 to PBS.</li>
	<li>Aspirate medium from adherent cells and wash briefly with PBS at RT on a shaker.</li>
	<li>Aspirate PBS and incubate cells with 500 &#181;L of ice-cold methanol per well of a 24-well plate to fix the cells at -20 &#176;C overnight.</li>
	<li>Remove the methanol and wash the cells 3 times with PBS on a shaker for 10 min each time at RT.</li>
	<li>Block cells with 500 &#181;L of 5% BSA prepared in PBS for 1-2 h at RT on a shaker.</li>
	<li>Dilute primary antibody at 1: 200 in the same solution used for blocking.</li>
	<li>Incubate the fixed cells in 300 &#181;L primary antibody solution overnight at 4 &#176;C on a shaker.</li>
	<li>After overnight incubation, wash cells 3 times with PBST for 10 min each time at RT on a shaker.</li>
	<li>Prepare secondary antibody solution in blocking solution at 1:1000.</li>
	<li>Incubate in 300 &#181;L of secondary antibody solution protected from light for 1 h at RT on a shaker.</li>
	<li>Aspirate secondary antibody solution and wash cells 3 times with PBST for 10 min each time at RT on a shaker.</li>
	<li>Incubate cells with 300 &#181;L of 1 &#181;g/mL DAPI for 3-5 min, and then wash twice with PBST.</li>
	<li>After aspirating PBST, add an appropriate amount of PBS for taking pictures under a fluorescence microscope.</li>
</ol>
8. Western blot analysis
<ol>
	<li>Prepare 1x TBST washing buffer by adding 0.1% Tween-20 to Tris-buffered saline (TBS).</li>
	<li>Prepare SDS-PAGE electrophoresis buffer and western transfer buffer by using a commercial kit according to the manufacturer&#39;s protocol.</li>
	<li>Resolve total cell protein (40 &#181;g) on a 10% sodium dodecyl sulfate polyacrylamide gel and run in SDS-PAGE electrophoresis buffer at 15 mA constant current for about 2 h.</li>
	<li>Transfer the gel to a polyvinylidene difluoride (PVDF) membrane at 300 mA constant current for 2 h at low temperature. 
	NOTE: The running time and transfer time may vary depending on the equipment used and the type and percentage of the gel.</li>
	<li>Block the membrane with 3% BSA prepared in TBST for 1 h at RT on a shaker.</li>
	<li>Incubate in primary antibody solution (1:1000 dilution) overnight at 4 &#176;C on a shaker.</li>
	<li>After overnight incubation, wash blotting membrane 3 times with TBST for 15 min per time at RT on a shaker.</li>
	<li>Incubate in secondary antibody solution (1:2000 dilution) for 2 h at RT on a shaker.</li>
	<li>Discard secondary antibody solution and wash the membrane 3 times with TBST, for 15 min each time, at RT on a shaker.</li>
	<li>Visualize immunoreactive bands using a chemiluminescence reagent followed by autoradiography. Use &#946;-actin as the loading control.</li>
</ol>
9. Indocyanine green uptake
<ol>
	<li>Prepare 200 mg/mL indocyanine green stock solution in DMSO.</li>
	<li>Prepare a working solution by supplementing Stage III differentiation medium with indocyanine green solution to a final concentration of 1 mg/mL.</li>
	<li>Incubate in a 37 &#176;C, 5% CO2 incubator for 1-2 h.</li>
	<li>Aspirate medium and wash the cells 3 times with PBS.</li>
	<li>Photograph under a microscope.</li>
</ol>
10. Periodic Acid-Schiff (PAS) staining and Hematoxylin-Eosin (H&#38;E) staining
<ol>
	<li>Aspirate medium from adherent cells and briefly wash twice with PBS.</li>
	<li>For cell fixation, aspirate PBS and incubate cells with ice-cold methanol at -20 &#176;C overnight.</li>
	<li>Visualize glycogen storage by PAS staining and observe binucleated cells by H &#38; E staining using a kit following the manufacturer&#39;s instructions.</li>
	<li>Take images with a fluorescence microscope.</li>
</ol>
11. Assay for CYP Activity
<ol>
	<li>Measure CYP450 activity using commercially available cell-based assays (P450-Glo Assays) according to the manufacturer&#39;s instructions.</li>
	<li>Use three independent repeats for testing. Data are represented as the mean &#177; SD.</li>
</ol>

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The authors have nothing to disclose.

Here, we present a stepwise method that induces HLCs from hESCs&#160;in three stages. In the first stage, Activin A and CHIR99021 were used to differentiate hESCs into DE. In the second stage, KO-DMEM and DMSO were used to differentiate DE into hepatic progenitor cells. In the third stage, HZM plus HGF, OSM and hydrocortisone 21-hemisuccinate sodium salt were used to continue to differentiate hepatic progenitor cells into HLCs.
The following critical steps need to be taken into consideration w...

The liver is a highly metabolic organ that plays several roles, including deoxidation, storing glycogen, and secretion and synthesis of proteins1. Various pathogens, drugs and heredity can cause pathological changes in the liver and affect its functions2,3. Hepatocytes, as the main functional unit of the liver, play an important role in artificial liver support systems and drug toxicity elimination. However, the resource of primary human hepatocytes is limited in cell-based therapy, as well as in liver disease research. Therefore, developing new sources of functional human hepatocytes is an important research direction in the field of regenerative medicine. Since 1998 when hESCs have been established4, hESCs have been widely considered by researchers because of their superior differentiation potential (they can differentiate into various tissues in a suitable environment) and high degree of self-renewability, and thus provide ideal source cells for bioartificial livers, hepatocyte transplantation and even liver tissue engineering5.
Currently, the hepatic differentiation efficiency can be greatly increased by enriching the endoderm6. In the lineage differentiation of stem cells into endoderm, levels of the transforming growth factor&#160;&#946;&#160;(TGF-&#946;)&#160;signaling and WNT signaling pathways are the key factors in the node of the endoderm formation stage. Activation of a high-level of TGF-&#946;&#160;and WNT signaling can promote the development of endoderm7,8. Activin A is a cytokine belonging to the TGF-&#946;&#160;superfamily. Therefore, Activin A is widely used in endoderm induction of human induced pluripotent stem cells (hiPSCs) and hESCs9,10. GSK3 is a serine-threonine protein kinase. Researchers have found that CHIR99021, a specific inhibitor of GSK3&#946;, can stimulate typical WNT signals, and can promote stem cell differentiation under certain conditions, suggesting that CHIR99021 has potential for inducing stem cell differentiation into endoderm 11,12,13.
Here we report an efficient and reproducible method for effectively inducing the differentiation of hESCs into functional HLCs. The sequential addition of Activin A and CHIR99021 produced about 89.7&#177;0.8% SOX17 (DE marker)-positive cells. After being further maturated in vitro, these cells expressed hepatic specific markers and exerted hepatocyte-like morphology (based on hematoxylin-eosin staining (H &#38; E)) and functions, such as uptake of indocyanine green (ICG), glycogen storage and CYP3 activity. The results show that hESCs can be successfully differentiated into mature functional HLCs by this method and can provide a basis for liver disease-related research and in vitro drug screening.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>488 labeled goat against mouse IgG</td>
			<td>ZSGB-BIO</td>
			<td>ZF-0512</td>
			<td>For IF,second antibody</td>
		</tr>
		<tr>
			<td>488 labeled goat against Rabbit IgG</td>
			<td>ZSGB-BIO</td>
			<td>ZF-0516</td>
			<td>For IF,second antibody</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Stem Cell Technologies</td>
			<td>7920</td>
			<td>For cell passage</td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>peproTech</td>
			<td>120-14E</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>Anti - Albumin (ALB)</td>
			<td>Sigma-Aldrich</td>
			<td>A6684</td>
			<td>For IF and WB, primary antibody</td>
		</tr>
		<tr>
			<td>Anti - Human Oct4</td>
			<td>Abcam</td>
			<td>Ab19587</td>
			<td>For IF, primary antibody</td>
		</tr>
		<tr>
			<td>Anti - &#945;-Fetoprotein (AFP)</td>
			<td>Sigma-Aldrich</td>
			<td>A8452</td>
			<td>For IF and WB, primary antibody</td>
		</tr>
		<tr>
			<td>Anti -SOX17</td>
			<td>Abcam</td>
			<td>ab224637</td>
			<td>For IF, primary antibody</td>
		</tr>
		<tr>
			<td>Anti-Hepatocyte Nuclear Factor 4 alpha (HNF4&#945;)</td>
			<td>Sigma-Aldrich</td>
			<td>SAB1412164</td>
			<td>For IF, primary antibody</td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Beyotime</td>
			<td>ST023-200g</td>
			<td>For cell blocking</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Sigma-Aldrich</td>
			<td>SML1046</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime</td>
			<td>C1006</td>
			<td>For nuclear staining</td>
		</tr>
		<tr>
			<td>DEPC-water</td>
			<td>Beyotime</td>
			<td>R0021</td>
			<td>For RNA dissolution</td>
		</tr>
		<tr>
			<td>DM3189</td>
			<td>MCE</td>
			<td>HY-12071</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>H&#38;E staining kit</td>
			<td>Beyotime</td>
			<td>C0105S</td>
			<td>For H&#38;E staining</td>
		</tr>
		<tr>
			<td>Hepatocyte growth factor (HGF)</td>
			<td>peproTech</td>
			<td>100-39</td>
			<td>For hepatocyte differentiation</td>
		</tr>
		<tr>
			<td>HepatoZYME-SFM (HZM)</td>
			<td>Gibco</td>
			<td>17705-021</td>
			<td>For hepatocyte differentiation</td>
		</tr>
		<tr>
			<td>Hydrocortisone-21-hemisuccinate</td>
			<td>Sigma-Aldrich</td>
			<td>H4881</td>
			<td>For hepatocyte differentiation</td>
		</tr>
		<tr>
			<td>Indocyanine</td>
			<td>Sangon Biotech</td>
			<td>A606326</td>
			<td>For Indocyanine staining</td>
		</tr>
		<tr>
			<td>Knock Out DMEM</td>
			<td>Gibco</td>
			<td>10829-018</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>Knock Out SR Multi-Species</td>
			<td>Gibco</td>
			<td>A31815-02</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified</td>
			<td>Corning</td>
			<td>354277</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>MEM NeAA</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>mTesR 5X Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>85852</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>mTesR Basal Medium</td>
			<td>Stem Cell Technologies</td>
			<td>85851</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Oncostatin (OSM)</td>
			<td>peproTech</td>
			<td>300-10</td>
			<td>For hepatocyte differentiation</td>
		</tr>
		<tr>
			<td>P450 - CYP3A4 (Luciferin - PFBE)</td>
			<td>Promega</td>
			<td>V8901</td>
			<td>For CYP450 activity</td>
		</tr>
		<tr>
			<td>PAS staining kit</td>
			<td>Solarbio</td>
			<td>G1281</td>
			<td>For PAS staining</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>For cell differentiation</td>
		</tr>
		<tr>
			<td>Peroxidase-Conjugated Goat anti-Mouse IgG</td>
			<td>ZSGB-BIO</td>
			<td>ZB-2305</td>
			<td>For WB,second antibody</td>
		</tr>
		<tr>
			<td>Primary Antibody Dilution Buffer for&#160;Western Blot</td>
			<td>Beyotime</td>
			<td>P0256</td>
			<td>For primary antibody dilution</td>
		</tr>
		<tr>
			<td>ReverTraAce qPCR RT Kit</td>
			<td>TOYOBO</td>
			<td>FSQ-101</td>
			<td>For cDNA Synthesis</td>
		</tr>
		<tr>
			<td>RNAiso Plus</td>
			<td>TaKaRa</td>
			<td>9109</td>
			<td>For RNA Isolation</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>Sangon Biotech</td>
			<td>A600669</td>
			<td>For second antibody preparation</td>
		</tr>
		<tr>
			<td>SYBR Green Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>A25742</td>
			<td>For RT-PCR Analysis</td>
		</tr>
		<tr>
			<td>Torin2</td>
			<td>MCE</td>
			<td>HY-13002</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>Tween</td>
			<td>Sigma-Aldrich</td>
			<td>WXBB7485V</td>
			<td>For washing buffer preparation</td>
		</tr>
	</tbody>
</table>

an efficient method for directed hepatocyte-like cell induction from human embryonic stem cells

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

The schematic diagram of HLC induction from hESCs and representative bright-field images of each differentiation stage are shown in Figure 1. In Stage I, Activin A and CHIR99021 were added for 3 days to induce stem cells to form endoderm cells. In Stage II, the endoderm cells differentiated into hepatic progenitor cells after being treated with differentiation medium for 5 days. In Stage III, early hepatocytes had matured and differentiated into HLCs after 10 days in HGF and OSM (<strong cla...

Watch this Scientific Journal Video about An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells at JoVE.com

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug ...

an-efficient-method-for-directed-hepatocyte-like-cell-induction-from

Research

JoVE Journal

Immunology and Infection

2.7K Views.  Shantou University Medical College. This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

Video: An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.