Human embryonic stem cell-derived hepatocyte-like cells are useful in disease modeling and drug screening. This is an efficient and reproducible method for effectively inducing the differentiation of human embryonic stem cells into functional hepatocyte-like cells. An undifferentiated status and appropriate confluence of human embryonic stem cells are critical for a successful differentiation to hepatocyte-like cells.
For stem cell maintenance, coat sterile six-well tissue culture-treated plates with one milliliter of 1X human embryonic stem cell-qualified Matrigel per well, and store them at four degrees Celsius overnight. Leave the plates at room temperature for 30 minutes before use. Thaw the cryo-preserved HESEs in a 37 degrees Celsius water bath for three minutes without shaking, then immediately transfer the stem cells by pipetting them into a 15-milliliter centrifuge tube containing four milliliters of pre-warmed, 37 degrees Celsius mTESR medium.
Centrifuge the HESEs and aspirate the supernatant, then gently re-suspend the cells in one milliliter of mTESR medium. Aspirate the DMEM F-12 medium from the plate. Seed the cells into a six-well plate at a density of 1 x 10 to the 5th cells in two milliliters of mTESR, and incubate in a 5%carbon dioxide incubator.
Maintain the cells by replacing the medium with preheated mTESR medium daily. Passage the cells at roughly 70 to 80%confluence, or when the cell colonies begin to make contact. For cells passaging, aspirate the medium and incubate the cells with one milliliter per well of enzyme solution for five minutes at 37 degrees Celsius.
Transfer the cells by pipetting them into a 15-milliliter centrifuge tube containing four milliliters of DMEM F-12 pre-warmed to 37 degrees Celsius. Prepare 24-well plates by coating them with 250 microliters of 1X hESC-qualified Matrigel medium. Seed hESCs at a density of 1 to 1.5 times 10 to the 5th cells per well in 500 microliters of mTESR medium.
Add both activin A to a final concentration of 100 nanograms per milliliter and CHIR99021 to a final concentration of three micromolar in an appropriate volume of preheated stage one differentiation basic medium. After three days of differentiation, differentiated cells should express markers of definitive endoderm cells, such as FOXA2, SOX17, GATA4, CXCR4, and FOXA1. On day three of differentiation, aspirate the medium, replace it with stage two medium, and incubate for 24 hours.
Change the medium every other day on days four through eight. After eight days of differentiation, differentiated cells should express appropriate markers of hepatic progenitor cells, such as HNF4 alpha, AFP, TBX3, TTR, ALB, NTCP, CEBPA. On day eight, aspirate the stage two medium from the cells, and replace it with stage three medium.
Allow cells to incubate for 10 days at 37 degrees Celsius in a carbon dioxide incubator, and change the media every other day with freshly-added differentiation factors. After 18 days of differentiation, differentiated cells should express characteristic markers of hepatocytes, such as AAT, ALB, TTR, HNF4 alpha, NTCP, ASGR1, CYP3A4. The schematic diagram of hepatocyte-like cells from hESCs and bright field images of each differentiation stage are shown.
In stage one, activin A and CHIR99021 were added for three days to induce stem cells to form endoderm cells. In stage two, the endoderm cells differentiated into hepatic progenitor cells after being treated with a differentiation medium for five days. In stage three, after 10 days, early hepatocytes had matured and differentiated into hepatocyte-like cells in hepatocyte growth factor and oncostatin.
In the final stage of differentiation, cells showed a typical hepatocyte phenotype. RT-PCR, immunofluorescence staining, and western blotting were used to detect markers of endoderm cells, hepatic precursor cells, and mature hepatocytes. The differentiated cells showed high expression levels of differentiation-related genes and proteins in each stage, such as endoderm markers SOX17 at day three, hepatic precursor marker HNF4 alpha at day eight, AFP at day 14, and mature hepatocyte marker ALB at day 18.
The HLCs exhibited green staining and extensive cytoplasmic acid shift staining. The hepatocytes showed typical bi-nucleated morphology. The enzymatic activity of CYP3A4, the essential metabolic CYP in the liver in HLCs, was confirmed with an enzyme assay.
Siting of human embryonic stem cells at an appropriate density and starting differentiation on the following date are critical. In stage one, touch the media gently because the cells are prone to float up. The combination of activin A and other small molecules may further improve the differentiation efficiencies, and produce more mature hepatocyte-like cells in this procedure.
This technique can provide a platform to explore hepatocyte transplantation, liver tissue engineering, bioartificial livers, and disease modeling.
