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

Summary

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).

Abstract

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, HNF4α 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.

Introduction

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 functions^2,3. 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 β (TGF-β) signaling and WNT signaling pathways are the key factors in the node of the endoderm formation stage. Activation of a high-level of TGF-β and WNT signaling can promote the development of endoderm^7,8. Activin A is a cytokine belonging to the TGF-β superfamily. Therefore, Activin A is widely used in endoderm induction of human induced pluripotent stem cells (hiPSCs) and hESCs^9,10. GSK3 is a serine-threonine protein kinase. Researchers have found that CHIR99021, a specific inhibitor of GSK3β, 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±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 & 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.

Protocol

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.

1. Prepare 1x mTesR stem cell culture medium by diluting 5x supplementary medium to mTesR basic medium.
2. 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 °C.
3. Prepare 1x hESC-qualified Matrigel medium by diluting 33.3 µL of 30x hESC-qualified Matrigel with 1 mL of DMEM/F12.
4. 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 °C overnight. Leave at room temperature (RT) for at least 30 min before use.
5. Thaw the cryopreserved hESCs in a 37 °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 °C mTesR medium, pipet up and down gently 2 times.
6. Centrifuge the hESCs at 200 x g for 2 min at RT.
7. Aspirate the supernatant and gently resuspend the cells in 1 mL of mTesR medium.
8. 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.
9. 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.

2. Stem cell passage and differentiation

1. For passaging cells, aspirate the medium and incubate the cells with 1 mL per well of enzyme solution (e.g., Accutase for 5 min at 37 °C. Then transfer the cells by pipetting to a 15 mL centrifuge tube containing 4 mL of DMEM/F12 prewarmed to 37 °C.
2. Centrifuge the cells at 200 x g at RT for 2 min.
3. Aspirate the medium and gently resuspend the cell pellet in 1 mL of mTesR medium.
4. Prepare 24-well plates by coating with 250 µL 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 µL of mTesR medium.
5. Allow cells to incubate for 24 h at 37 °C in a CO₂ incubator.

3. DE formation

1. Prepare stock solutions of 100 µg/mL Activin A with DPBS containing 0.2% BSA and 3 mM CHIR99021 with DMSO.
2. Prepare Stage I differentiation basic medium by supplementing RPMI medium with 1x B27.
3. Add Activin A to a final concentration of 100 ng/mL and CHIR99021 to a final concentration of 3 µM in an appropriate volume of preheated Stage I differentiation basic medium.
4. 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).
5. Allow cells to incubate for 3 days at 37 °C in a CO₂ incubator, replacing Stage I media daily with freshly added differentiation factors (Days 0-2).
   ​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).

4. Differentiation of hepatic progenitor cells

1. Prepare Stage II differentiation basic media by dissolving knockout serum replacement (KOSR) to a final concentration of 20% in Knock Out DMEM.
2. Prepare Stage II differentiation medium containing 1% DMSO, 1x GlutaMAX, 1x non-essential amino acid (NEAA) solution, 100 µM 2-mercaptoethanol and 1x penicillin/streptomycin (P/S) to the appropriate volume of KOSR/knockout DMEM.
3. 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.
4. 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).
5. Change the medium every other day (replace medium on Day 6).
   ​NOTE: After 8 days of differentiation, differentiated cells should express appropriate markers of hepatic progenitor cells, such as HNF4α, AFP, TBX3, TTR, ALB, NTCP, CEBPA (minimum 80% of HNF4α needed for proceeding).

5. Hepatocyte differentiation

1. Prepare 10 µg/mL hepatocyte growth factor (HGF), 20 µg/mL Oncostatin (OSM) stock solutions with DBPS (containing 0.2% BSA) and filter with a 0.22 µm filter membrane.
2. Prepare Stage III differentiation basic medium (HepatoZYME-SFM (HZM) medium supplemented with 10 µM hydrocortisone-21-hemisuccinate and 1x P/S).
3. 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.
4. 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).
5. Allow cells to incubate for 10 days at 37 °C in a CO₂ incubator, changing Stage III media every other day with freshly added differentiation factors (Days 8-18).
   ​NOTE: After 18 days of differentiation, differentiated cells should express characteristic markers of hepatocytes, such as AAT, ALB, TTR, HNF4α, NTCP, ASGR1, CYP3A4. The percentage of Albumin-positive cells usually is more than 90%.

6. RNA isolation, cDNA synthesis and RT-qPCR analysis

1. 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.
2. Add 60 µL of chloroform reagent and leave it at RT for 5 min. Then centrifuge at 12,000 x g for 15 min.
3. Collect the 125 µL supernatant and transfer it to another centrifuge tube. Add 160 µL of isopropanol, mix well, and stand at RT for 10 min.
4. Centrifuge at 12,000 x g for 10 min.
5. Remove the supernatant, add 75% ethanol to the precipitated, and centrifuge at 7,500 x g for 5 min.
6. After drying at RT, add 40 µL of DEPC-treated water to dissolve. For qPCR, reverse transcribe 2 µg of total RNA with a reverse transcription kit according to the manufacturer's protocol.
7. Perform RT-qPCR using a commercial kit according to the manufacturer's protocol.
8. Normalize gene expression relative to non-induced stem cells and determine fold variation following normalization to GAPDH.

7. Immunofluorescence Validation of Differentiation

1. Prepare 1x PBST washing buffer by adding 0.1% Tween-20 to PBS.
2. Aspirate medium from adherent cells and wash briefly with PBS at RT on a shaker.
3. Aspirate PBS and incubate cells with 500 µL of ice-cold methanol per well of a 24-well plate to fix the cells at -20 °C overnight.
4. Remove the methanol and wash the cells 3 times with PBS on a shaker for 10 min each time at RT.
5. Block cells with 500 µL of 5% BSA prepared in PBS for 1-2 h at RT on a shaker.
6. Dilute primary antibody at 1: 200 in the same solution used for blocking.
7. Incubate the fixed cells in 300 µL primary antibody solution overnight at 4 °C on a shaker.
8. After overnight incubation, wash cells 3 times with PBST for 10 min each time at RT on a shaker.
9. Prepare secondary antibody solution in blocking solution at 1:1000.
10. Incubate in 300 µL of secondary antibody solution protected from light for 1 h at RT on a shaker.
11. Aspirate secondary antibody solution and wash cells 3 times with PBST for 10 min each time at RT on a shaker.
12. Incubate cells with 300 µL of 1 µg/mL DAPI for 3-5 min, and then wash twice with PBST.
13. After aspirating PBST, add an appropriate amount of PBS for taking pictures under a fluorescence microscope.

8. Western blot analysis

1. Prepare 1x TBST washing buffer by adding 0.1% Tween-20 to Tris-buffered saline (TBS).
2. Prepare SDS-PAGE electrophoresis buffer and western transfer buffer by using a commercial kit according to the manufacturer's protocol.
3. Resolve total cell protein (40 µg) on a 10% sodium dodecyl sulfate polyacrylamide gel and run in SDS-PAGE electrophoresis buffer at 15 mA constant current for about 2 h.
4. 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.
5. Block the membrane with 3% BSA prepared in TBST for 1 h at RT on a shaker.
6. Incubate in primary antibody solution (1:1000 dilution) overnight at 4 °C on a shaker.
7. After overnight incubation, wash blotting membrane 3 times with TBST for 15 min per time at RT on a shaker.
8. Incubate in secondary antibody solution (1:2000 dilution) for 2 h at RT on a shaker.
9. Discard secondary antibody solution and wash the membrane 3 times with TBST, for 15 min each time, at RT on a shaker.
10. Visualize immunoreactive bands using a chemiluminescence reagent followed by autoradiography. Use β-actin as the loading control.

9. Indocyanine green uptake

1. Prepare 200 mg/mL indocyanine green stock solution in DMSO.
2. Prepare a working solution by supplementing Stage III differentiation medium with indocyanine green solution to a final concentration of 1 mg/mL.
3. Incubate in a 37 °C, 5% CO₂ incubator for 1-2 h.
4. Aspirate medium and wash the cells 3 times with PBS.
5. Photograph under a microscope.

10. Periodic Acid-Schiff (PAS) staining and Hematoxylin-Eosin (H&E) staining

1. Aspirate medium from adherent cells and briefly wash twice with PBS.
2. For cell fixation, aspirate PBS and incubate cells with ice-cold methanol at -20 °C overnight.
3. Visualize glycogen storage by PAS staining and observe binucleated cells by H & E staining using a kit following the manufacturer's instructions.
4. Take images with a fluorescence microscope.

11. Assay for CYP Activity

1. Measure CYP450 activity using commercially available cell-based assays (P450-Glo Assays) according to the manufacturer's instructions.
2. Use three independent repeats for testing. Data are represented as the mean ± SD.

Results

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 (

Discussion

Here, we present a stepwise method that induces HLCs from hESCs 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...

Materials

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Sigma	M7522	For hepatic progenitor differentiation
488 labeled goat against mouse IgG	ZSGB-BIO	ZF-0512	For IF,second antibody
488 labeled goat against Rabbit IgG	ZSGB-BIO	ZF-0516	For IF,second antibody
Accutase	Stem Cell Technologies	7920	For cell passage
Activin A	peproTech	120-14E	For definitive endoderm formation
Anti - Albumin (ALB)	Sigma-Aldrich	A6684	For IF and WB, primary antibody
Anti - Human Oct4	Abcam	Ab19587	For IF, primary antibody
Anti - α-Fetoprotein (AFP)	Sigma-Aldrich	A8452	For IF and WB, primary antibody
Anti -SOX17	Abcam	ab224637	For IF, primary antibody
Anti-Hepatocyte Nuclear Factor 4 alpha (HNF4α)	Sigma-Aldrich	SAB1412164	For IF, primary antibody
B-27 Supplement	Gibco	17504-044	For definitive endoderm formation
BSA	Beyotime	ST023-200g	For cell blocking
CHIR99021	Sigma-Aldrich	SML1046	For definitive endoderm formation
DAPI	Beyotime	C1006	For nuclear staining
DEPC-water	Beyotime	R0021	For RNA dissolution
DM3189	MCE	HY-12071	For definitive endoderm formation
DMEM/F12	Gibco	11320-033	For cell culture
DMSO	Sigma-Aldrich	D5879	For hepatic progenitor differentiation
DPBS	Gibco	14190-144	For cell culture
GlutaMAX	Gibco	35050-061	For hepatic progenitor differentiation
H&E staining kit	Beyotime	C0105S	For H&E staining
Hepatocyte growth factor (HGF)	peproTech	100-39	For hepatocyte differentiation
HepatoZYME-SFM (HZM)	Gibco	17705-021	For hepatocyte differentiation
Hydrocortisone-21-hemisuccinate	Sigma-Aldrich	H4881	For hepatocyte differentiation
Indocyanine	Sangon Biotech	A606326	For Indocyanine staining
Knock Out DMEM	Gibco	10829-018	For hepatic progenitor differentiation
Knock Out SR Multi-Species	Gibco	A31815-02	For hepatic progenitor differentiation
Matrigel hESC-qualified	Corning	354277	For cell culture
MEM NeAA	Gibco	11140-050	For hepatic progenitor differentiation
mTesR 5X Supplement	Stem Cell Technologies	85852	For cell culture
mTesR Basal Medium	Stem Cell Technologies	85851	For cell culture
Oncostatin (OSM)	peproTech	300-10	For hepatocyte differentiation
P450 - CYP3A4 (Luciferin - PFBE)	Promega	V8901	For CYP450 activity
PAS staining kit	Solarbio	G1281	For PAS staining
Pen/Strep	Gibco	15140-122	For cell differentiation
Peroxidase-Conjugated Goat anti-Mouse IgG	ZSGB-BIO	ZB-2305	For WB,second antibody
Primary Antibody Dilution Buffer for Western Blot	Beyotime	P0256	For primary antibody dilution
ReverTraAce qPCR RT Kit	TOYOBO	FSQ-101	For cDNA Synthesis
RNAiso Plus	TaKaRa	9109	For RNA Isolation
RPMI 1640	Gibco	11875093	For definitive endoderm formation
Skim milk	Sangon Biotech	A600669	For second antibody preparation
SYBR Green Master Mix	Thermo Fisher Scientific	A25742	For RT-PCR Analysis
Torin2	MCE	HY-13002	For definitive endoderm formation
Tween	Sigma-Aldrich	WXBB7485V	For washing buffer preparation