REAGENT SETUP
Preparation of growth factor and inhibitor stocks
Activin (50 µg ml-1)
• Reconstitute 10 µg in 200 µl DPBS supplemented with 0.2 % (w/v) BSA. Store at -20 °C in 10 µl aliquots for up to 6 months. Once defrosted, store at 4 °C and use within 3 days.
FGF-2 (10 µg ml-1)
• Reconstitute 1 mg in 100 ml DPBS supplemented with 0.2 % (w/v) BSA. Filter solution using 0.22 µm filter unit. Store at -20 °C in 1 ml aliquots for up to 12 months. Once defrosted, store at 4 °C and use within 1 month.
BMP2 (20 µg ml-1)
• Reconstitute 2 µg in 100 µl DPBS supplemented with 0.2 % (w/v) BSA. Store at -20 °C in 10 µl aliquots for up to 6 months. Once defrosted, store at 4 °C and use within 3 days.
Insulin (10 mg ml-1)
• Dissolve 100 mg in 10 ml sterile 0.01 M HCl solution supplemented with 0.2 % (w/v) BSA overnight at 4 °C. Store at -20 °C in 10 µl aliquots for up to 6 months. Once defrosted, store at 4 °C and use within 3 days.
Hydrocortisone-21-hemisuccinate (100 mM)
• Dissolve 1 g in 20 ml cell culture grade water. Store at -20 °C in 1 ml aliquots for up to 12 months. Once defrosted, store at 4 °C and use within 2 weeks.
Ascorbic acid (50 mg ml-1)
• Dissolve 0.5 g in 10 ml cell culture grade water. Store at -20 °C in 1 ml aliquots for up to 12 months. Once defrosted, store at 4 °C and use within 2 weeks.
Critical Step: Stability of ascorbic acid in solution is adversely affected by light. Ensure defrosted aliquots are stored in the dark or wrapped in aluminium foil.
HGF (10 µg ml-1)
• Reconstitute 10 µg in 1 ml DPBS supplemented with 0.2 % (w/v) BSA. Store at -20 °C in 50 µl aliquots for up to 6 months. Once defrosted, store at 4 °C and use within 2 weeks.
OSM (10 µg ml-1)
• Reconstitute 10 µg in 1 ml DPBS supplemented with 0.2 % (w/v) BSA. Store at -20 °C in 50 µl aliquots for up to 6 months. Once defrosted, store at 4 °C and use within 2 weeks.
Torin-2 (10 mM)
• Dissolve 10 mg in 2.3 ml DMSO Hybri-MaxTM. Store at -20 C in 100 µl aliquots for up to 12 months. Once defrosted, store at 4 °C and use within 1 month.
Caution: DMSO readily penetrates the skin and can carry across any dissolved inhibitors, handle with care.
Culture medium and regents
Knockout Serum Replacement (KSR) medium (500 ml)
• In a cell culture hood, add 400 ml KO-DMEM supplemented with 100 ml KSR (20%), 2.5 ml M L-glutamine (1 m), 5 ml non-essential amino acids (1X), and 1 ml β-mercaptoethanol (0.1 M) into 500 ml PES vacuum filter (0.22 µm) unit. Store the medium after filtering at 4 °C for up to 1 week.
Critical Step: Aliquot KSR upon first use to avoid freeze-thaw and store at -80 °C.
Plating medium after Accutase/ROCKi split
• To an appropriate volume of MEF-CM, add ROCK inhibitor and FGF-2 to a final concentration of 10 µM and 10 ng ml-1 respectively.
Collagenase (50 ml)
• Using lypophilised collagenase powder at 275 U mg-1, dissolve 36.4 mg in 50 ml of KO-DMEM to obtain a solution containing 200 U ml-1 collagenase. Filter sterilise using 0.22 µm filter unit, aliquot and store at -20 °C. Thaw the aliquot just before use at 37 °C.
Critical Step: Collagenase activity varies between lots, therefore adjust either the volume or amount of collagenase powder accordingly to achieve 200 U ml-1 activity.
DE induction medium (50 ml)
• Under sterile conditions, add 1 ml B27 supplement to 50 ml RPMI AQmedia. Filter sterilise using 0.22 µm filter unit and store at 4 °C for up to a week. Add Activin and Torin to a final concentration of 100 ng ml-1 and 15 nM respectively before use.
Hepatoblast specification medium
• Under sterile conditions, to an appropriate volume of KSR medium, add BMP-2 and FGF-2 to a final concentration of 20 ng ml-1 and 10 ng ml-1 respectively before use. Base medium can be stored at 4 °C for up to 2 weeks.
Hepatocyte maturation medium (500 ml)
• Under sterile conditions, prepare 500 ml L-15 medium supplemented with 8.4% tryptose phosphate broth, 8.3% FBS, 2 mM L-Glutamine, 10 µM hydrocortisone-21-hemisuccinate and 1 µM insulin. Store medium at 4 °C for up to 2 weeks. Supplement with 50 µg ml-1 ascorbic acid, 20 ng ml-1 OSM and 10 ng ml-1 HGF immediately before use. Base media can be stored for up to 2 weeks.
EQUIPMENT SETUP
Matrigel coated plates
• Thaw stock Matrigel® overnight on ice at 4 °C and dilute 1 in 2 with chilled KO-DMEM before aliquoting into 15 ml tubes at 1 ml per tube and store at -20 °C. To make plates, thaw aliquots at 4 °C overnight before diluting 1 in 15 with chilled KO-DMEM. Coat each well of one 6 well plate with 1 ml of the new dilution. Allow plates to set at 4 °C overnight and warm for at least 20 min at room temperature before use. Seal plates with parafilm to prevent evaporation before storage. Upon use, aspirate off excess solution and replace with the appropriate volume of MEF-CM prior to hESC passaging. In emergency circumstances, plates can be made on the day of passaging and allowed to set for a minimum of 4 hours at room temperature before use. Homogeneity of the coating can be verified using a phase contrast light microscope. Plates stored at 4 °C should be used within 2 weeks.
Critical Step: Matrigel sets rapidly at room temperature, to avoid gelling and clumping, ensure that Matrigel solutions are defrosted slowly at 4 °C. Do not let the Matrigel-coated wells dry out after excess is removed.
PROCEDURE
Stage 0: Preparation of hESCs for DE differentiation
Steps 1 – 2: Generation of MEF-CM from KSR medium
Timing: 10 – 14 d
1 Thaw frozen MEFs (passage 0) and grow in DMEM AQmedia containing 10% FBS. Passage 3 to 4 times in order to obtain sufficient number of vigorously growing cells. Trypsinise the cells and irradiate for 40 Gy using a cell irradiator to abolish their replicative potential. Seed irradiated MEFs at a density of 2.5 x 107 cells into T225 flasks that have been pre-coated with 0.5% gelatin for 20 min and allow cells to plate down overnight. The following day, rinse cells once with DPBS and replace growth medium with 150 ml KSR medium supplemented with 4 ng ml-1 FGF-2. Allow the MEFs to condition the media for 24 hours and collect into 150 ml sterile storage bottles. Immediately freeze at -80 °C to maintain quality of the MEF-CM. Repeat collection for up to 7 days.
2 Thaw bottles of frozen MEF-CM in the water bath until fully defrosted. Supplement with a further 1 mM of L-Glutamine and filter through a 0.22 µm filter unit to remove residual MEF cell debris. Take out the required volume for required for daily feed to warm and store the leftover medium at 4 °C for up to a week. Supplement with 10 ng ml-1 FGF-2 immediately prior to addition to hESCs.
Step 3: Culture and expansion of hESCs
Timing: 5 – 7 d
3 Culture and maintain hESCs as colonies with daily exchange of complete MEF-CM for 5-7 days by which they should be almost confluent. For each well this would be 3 ml daily. Prior to passaging, ensure appropriate Matrigel plate has been warmed to room temperature for a minimum of 20 min and contains 2 ml per well MEF-CM. Passage 1 well of hESCs by treating with 1 ml collagenase IV for 5 to 10 min followed by a DPBS rinse. Replace DPBS with 1 ml MEF-CM and detach colonies by gently scraping with the tip of a 5 ml pipette. Add a further 2 ml MEF-CM and break up the colonies by gentle pipetting 3-4 times using a 5 ml pipette. Transfer 1 ml of the cell solution into 3 wells of the new plate to achieve a 1:3 split.
Stage 1: DE differentiation in a Chemically Defined Medium
Steps 4 – 9: Accutase/ROCKi split of hESCs for DE conversion
Timing: 1-2 h
4 Culture and maintain hESCs as in Step 3 until wells are confluent and are typically ready for passaging.
5 Defrost and warm Accutase solution and an appropriate volume of KO-DMEM to 37 °C. Warm appropriate Matrigel gel plate to room temperature and prepare aliquot of MEF-CM.
6 Aspirate off MEF-CM and rinse cells once with DPBS. For one well, exchange DPBS with 1 ml warm Accutase solution and incubate for 5 to 10 min until colonies are disassociated into single cells.
7 Neutralise Accutase solution with 2 ml of KO-DMEM per well, pipette cell suspension through a 1 ml Gilson pipette tip to further dissociate any remaining clumps. Flush the wells and transfer into a 15 ml tube. Centrifuge cell suspension at 60g for 5 min to pellet the cells. During centrifugation, prepare the Matrigel plates for seeding by transferring 2 ml MEF-CM into each well. Ensure that MEF-CM contains both 10 ng ml-1 FGF-2 and 10 µM ROCK inhibitor.
8 After centrifugation, aspirate supernatant and resuspend cell pellet in 3 ml MEF-CM supplemented with 10 ng ml-1 FGF-2 and 10 µM ROCK inhibitor. Dispense 1 ml per well of the new plate to achieve a 1:3 split. Allow cells to plate down overnight and verify formation of a homogenous cell monolayer the following day.
9 To remove residual ROCK inhibitor, exchange medium with fresh MEF-CM supplemented with FGF-2 only and incubate overnight. The cells are now ready for DE induction.
Steps 10 – 13: Induction of DE using Activin and Torin
Timing: 2 – 3 d
10 Warm an appropriate aliquot of DE induction medium to 37 °C. Supplement with 100 ng ml-1 and 15 nM Torin and exchange with MEF-CM to induce the DE differentiation. Observe the cells daily to note key phenotypic changes that reflect the progress of differentiation.
11 The following day, cells will start to adopt a spiky morphology that is reminiscent of cells from the primitive streak and appear as spiked clusters. However, cell death should largely be minimal. Exchange with the same medium as Step 10 for another day to continue the differentiation.
12 By day 2 of differentiation the majority of the cells will adopt a spiky morphology. Clusters will also begin to disperse as the cells proceed through EMT and gain mobility. Again, exchange with the same medium to complete the DE differentiation.
13 By day 3 differentiation should largely be complete. Clusters should be minimal or absent and the predominant cell type are cells that possess a sharp and well-defined polygonal appearance. Cells can be lysed for analysis at this stage or subjected to further differentiation.
Stage 2: Hepatic Specification
Steps 14 – 15: Hepatoblast specification from AA/Torin induced DE
Timing: 3 – 5 d
14 To initiate hepatoblast specification, exchange DE induction medium with hepatoblast specification medium supplemented with 20 ng ml-1 BMP-2 and 10 ng ml-1 FGF-2. Since the cytotoxicity of Torin is rather low, cultures can at this stage become too confluent and compacted. If this arises, split the cells onto Matrigel-coated plates using Accutase at a 1:2 ratio and continue the culture.
Critical Step: Avoid splitting the cells at a large ratio as hepatoblast maturation requires cell-cell contacts to be maintained.
15 Exchange media daily and observe the phenotypic changes to track the progress of the differentiation. Cells initially increase in size but maintain a rough border between each other which gradually becomes more pronounced as the differentiation proceeds. After 3-5 days the cells largely adopt a polygonal morphology that is characteristic of hepatoblasts, however the more mature features such as prominent nucleus and binucleated cells remain absent.
Stage 3: Hepatic Maturation
Steps 16 – 17: Hepatocyte maturation from hepatoblasts
Timing: 3 – 5 d
16 By day 5 of hepatoblast specification, the cells are largely fated to become hepatocyte-like cell morphology, however they require further culture to adopt a phenotype that functionally mimics that of primary hepatocytes. To initiate the maturation step, exchange the hepatoblast specification medium with hepatocyte maturation medium supplemented with 20 ng ml-1 OSM, 10 ng ml-1 HGF and 50 µg ml-1 ascorbic acid.
17 Exchange media daily and observe phenotypic changes to track the progress of the differentiation. Cells visibly increase in size and the cell-cell borders become more reinforced and prominent. Mononucleated and binucleated cells appear with increasing frequency and by the latter stages, cells begin to accumulate lipid droplets. At this stage, cells can be taken for functional assays or lysed for gene expression or protein analysis. Cells can be kept in this state for no longer than 3 days.