Modeling human hepatic steatosis in pluripotent stem cell-derived hepatocytes

Summary This protocol describes the production of hepatocyte-like cells (HLCs) from human pluripotent stem cells and how to induce hepatic steatosis, a condition characterized by intracellular lipid accumulation. Following differentiation to an HLC phenotype, intracellular lipid accumulation is induced with a steatosis induction cocktail, allowing the user to examine the cellular processes that underpin hepatic steatosis. Furthermore, the renewable nature of our system, on a defined genetic background, permits in-depth mechanistic analysis, which may facilitate therapeutic target identification in the future. For complete details on the use and execution of this protocol, please refer to Sinton et al. (2021).


SUMMARY
This protocol describes the production of hepatocyte-like cells (HLCs) from human pluripotent stem cells and how to induce hepatic steatosis, a condition characterized by intracellular lipid accumulation. Following differentiation to an HLC phenotype, intracellular lipid accumulation is induced with a steatosis induction cocktail, allowing the user to examine the cellular processes that underpin hepatic steatosis. Furthermore, the renewable nature of our system, on a defined genetic background, permits in-depth mechanistic analysis, which may facilitate therapeutic target identification in the future. For complete details on the use and execution of this protocol, please refer to Sinton et al. (2021).

BEFORE YOU BEGIN
Coat culture plates with Laminin-521 Timing: 30 min 1. Thaw a 100 mg/mL stock of Laminin-521 (LN-521) for 16-18 h at 4 C. 2. Thawed LN-521 should be diluted in ice-cold 1X DPBS (containing Ca 2+ and Mg 2+ ), to generate a 5 mg/mL stock solution. 3. Add sufficient LN-521 to each well of a culture plate to coat. For a 6-well plate, add 1 mL of LN-521 to each well. For a 10 cm petri dish, add 5 mL of LN-521 to coat the plate. 4. After coating, incubate the plates at 4 C for 16-18 h on a flat surface. Alternatively, plates can be incubated in a 37 C/5% CO 2 cell culture incubator for 2 h. 5. Plates should be sealed to prevent evaporation of LN-521, stored at 4 C, and used within 2 weeks. 6. Prior to use, allow the plates to equilibrate to 18 C-25 C by placing on a flat surface for 2 h. Alternatively, warm the plates in a 37 C/5% CO 2 cell culture incubator for 30 min.
CRITICAL: It is critical that the LN-521 does not evaporate. Seal plates with parafilm to avoid evaporation. Following incubation, LN-521 coating, tip plates 45 and observe the growing surface. If the growing surface plastic can be seen when tipping, then coating has not been successful and must be repeated. If repeated coating is necessary, the addition of an extra 1 mL DPBS can reduce the chance of this occurring.

Preparation of differentiation media, growth factors, and steatosis induction cocktail
Timing: 4 h 7. Stem cell media a. Add 100 ml mTeSR1ä 5X supplement to the 400 ml mTeSR1ä basal medium. Store for up to 1 month at 4 C. 8. Differentiation media a. To prepare endoderm differentiation medium, add 1% penicillin streptomycin and 1X B27 supplement to 500 mL RPMI 1640 medium. Store for up to 1 month at 4 C. b. To prepare hepatic progenitor differentiation medium, mix 400 ml knockout (KO)-DMEM, 100 ml KOSR Serum Replacement, 1% penicillin/streptomycin, 1% DMSO, 1% non-essential amino acids, 0.5% Glutamax and 0.2% b-mercaptoethanol. Store for up to 1 month at 4vC. This media formulation was previously optimized (Wang et al., 2017). c. To prepare hepatocyte maturation media, add 1% Glutamax, 1% penicillin streptomycin, 20 ng/mL hepatocyte growth factor, 10 ng/mL oncostatin M and 10 mM hydrocortisone 21-hemisuccinate sodium salt to HepatoZYME medium. Store for up to 1 month at 4 C. 9. Preparation of growth factors and other reagents a. Prepare a 1,000X stock of human activin A, by dissolving the lyophilized protein in sterile 0.2% bovine serum albumin (BSA; mixed with DPBS), giving a final concentration of 100 mg/mL. Prepare 30 mL aliquots and store at À20 C. Use at a 1:1000 dilution. b. Prepare a 1,000X stock of Wnt3A. Dissolve lyophilized mouse Wnt3A protein in sterile 0.2% BSA/DPBS, to a final concentration of 10 mg/mL. Prepare 30 mL aliquots and store at À20 C. Use at a 1:200 dilution. c. Prepare hydrocortisone 21-hemisuccinate sodium salt solution (HCC) by dissolving HCC in DPBS to a final concentration of 10 mM. Sterile filter the solution using a 22 mm filter and store in 5 mL aliquots at À20 C. Use at a 1:100 dilution. d. Prepare a 1,000X stock of human hepatocyte growth factor. Dissolve lyophilized HGF in sterile BSA/DPBS, to a final concentration of 10 mg/mL. Prepare 30 mL aliquots and store at À20 C. Use at a 1:1000 dilution. e. Prepare a 1,000X stock of oncostatin M. Dissolve lyophilized Oncostatin M (OSM) in sterile BSA/DPBS, to a final concentration of 20 mg/mL. Prepare 30 mL aliquots and store at À20 C. Use at a 1:1000 dilution. f. Prepare a 1,000x stock solution of Rho-associated kinase (ROCK) inhibitor Y-27632. Dissolve in sterile 0.2% BSA/DPBS to a final concentration of 10 mM. Prepare 10 mL aliquots and store at À20 C. Use at a 1:1000 dilution. 10. Prepare components of steatosis induction cocktail 24 h prior to use. a. Dissolve sodium L lactate in DPBS to a concentration of 1 M. Sterile filter the solution with a 22 mm filter. Store in 1 mL aliquots at À20 C for up to 1 week. b. Dissolve sodium pyruvate in DPBS to a concentration of 100 mM. Sterile filter the solution with a 22 mm filter. Store in 1 mL aliquots at À20 C for up to 1 week. c. Prepare octanoic acid to a 100 mM concentration in sterile water. Add lyophilized octanoic acid to sterile deionized water. Adjust pH to 7.0 -7.9 by addition of 4 M NaOH solution with constant stirring. When solution becomes clear, store in 1 mL aliquots at À20 C for up to 1 week.
CRITICAL: Steatosis induction cocktail compounds stored for longer than 1 week become decreasingly effective and should be discarded.
11. Prepare steatosis induction cocktail on the day of use. a. Add lactate, pyruvate, and octanoic acid to HepatoZYME media, to a final concentration of 10mM, 1mM and 2mM, respectively. Sterile filter the solution prior to use.

STEP-BY-STEP METHOD DETAILS Passaging human pluripotent stem cells (hPSCs) in preparation for differentiation
Timing: 1 day This step describes the preparation of hPSCs for differentiation to hepatocyte-like cells (HLCs) ( Figure 1). The protocol requires that hPSCs are at a confluence of 75%-80%. For passaging, hPSCs must be in a single cell suspension. This protocol was adapted from (Lyall et al., 2018;Meseguer-Ripolles et al., 2018;Wang et al., 2017). CRITICAL: Do not allow the Laminin-521 to dry out at any point. If the Laminin-521 pools and exposes a dry plastic growing surface, then coating has not been successful and must be repeated prior to starting the differentiation process.
5. Immediately after aspirating, add 1 mL of supplemented mTeSR1ä, pre-warmed to 37 C, containing 10 mM Rho-associated kinase (ROCK) inhibitor Y-27632 to each well of a 6-well plate. This is half of the media that will be added to each well.
Note: ROCK inhibitor Y-27632 is used to enhance hPSC attachment and survival in single cell suspensions.
6. Incubate the LN-521 coated plates, with ROCK-supplemented media, at 37 C/5% CO 2 until the hPSCs are dissociated and ready for plating. 7. To determine whether hPSCs have formed a single cell suspension, observe the plates under a standard light microscope. If the cells are not ready, increase the incubation step, to a maximum of 10 min. Cells will start to look round when their edges are detaching from the growing surface. 8. Aspirate the Gentle Cell Dissociation Reagent and immediately add 1 mL of pre-warmed supplemented mTeSR1ä with 10 mM ROCK inhibitor Y-27632 to each well. 9. Detach cells from the growing surface using a cell scraper. 10. Collect the media, containing suspended hPSCs, into a 15 mL Falcon tube and centrifuge at 200 3 g for 5 min, at 18 C-25 C. 11. Aspirate the media without disturbing the cell pellet and then tap the tube to ensure that there are no clumps. 12. To the Falcon tube, add 10 mL of supplemented mTeSR1ä with 10 mM ROCK inhibitor Y-27632.
Slowly pipette up and down to resuspend the cell pellet. Count the cells using an automatic cell counter and use Trypan Blue to exclude dead cells. Count cells three times and calculate the mean number of cells.
Note: Manual counting is not recommended as it introduces user-to-user variation 13. Seed the cells at a density of 5.0 3 104 cells per cm2 growing surface. Seeding density may require optimization for different well sizes or shapes. 14. Place the plate on a flat surface and gently shake the plate up and down, then left to right, for 10 times each. 15. Incubate the hPSCs at 37 C/5% CO2 for 24 h before starting the differentiation protocol.

Hepatocyte-like cell differentiation
Timing: 17 days This protocol describes how to differentiate hPSCs to hepatocyte-like cells (HLCs) and generates homogeneous populations. This protocol below was previously described (Wang et al., 2017). 16. Once hPSCs reach 40% confluence (Figure 2A), normally 1 day after seeding, begin the differentiation process. This timing is variable and it can take between 18 and 24 h for cells to reach 40% confluence if cell counts are not accurate. 17. Aspirate the supplemented mTeSR1ä and replace with 2 mL RPMI 1640 supplemented with 100 ng/mL Activin A and 50 ng/mL Wnt3A. Incubate the cells at 37 C/5% CO 2 and refresh the media every 24 h for 3 days. 18. On day 3, H9 cells should achieve an endodermal morphology ( Figure 2B). Aspirate endoderm differentiation medium and replace with hepatic progenitor differentiation medium. Incubate the cells at 37 C/5% CO 2 and refresh the media every 48 h for 5 days. 19. On day 9, H9 cells should achieve a hepatoblast morphology ( Figure 2C). Aspirate the hepatic progenitor differentiation medium and replace with hepatocyte maturation medium, supplemented with 10 ng/mL hepatocyte growth factor and 20 ng/mL oncostatin M. Incubate the cells at 37 C/5% CO 2 and refresh the media every 48 h for 9 days. 20. On day 17, H9 cells should achieve a hepatocyte morphology ( Figure 2D). HLCs are now mature and ready to be used in downstream assays. 21. To determine that cells are functioning as mature hepatocytes, measure CYP3A4 activity on day 17, using the P450-Gloä assay (Promega), as per the manufacturer's instructions (https://www. promega.co.uk/products/cell-health-assays/adme-assays/ p450-glo-cyp3a4-assay-and-screening-system/?catNum=V9001#protocols). Results should be normalized to protein content of the well analyzed. Measure protein using the Pierceä BCA Protein Assay Kit as per the manufacturer's instructions (https://www.thermofisher.com/order/ catalog/product/23225#/23225).
Note: At days 0, 3, 9 and 17, collect cells to measure mRNA of the pluripotency marker NANOG, and the hepatocyte markers HNF4A and ALB (Figure 3). This enables the tracking of the changing cell phenotype throughout the differentiation process.

Timing: [2 days]
This step leads to the accumulation of intracellular lipids within the HLCs, and mimics human hepatic steatosis. This protocol was adapted from (Lyall et al., 2018).
Note: Following induction of steatosis, cells from a single well can be used for either imaging or mRNA collection, but the same well cannot be used for both purposes.

EXPECTED OUTCOMES
This protocol generates stem cell-derived hepatocyte-like cells, which are phenotypically similar to mature human hepatocytes. Figure 3 demonstrates that following differentiation, cells acquire expression of markers typically associated with hepatocytes, which they lack in the pluripotent state. HNF4A activity is understood to be crucial for hepatic progenitor specification (Wang et al., 2019) and, therefore, it is essential to confirm that cells are expressing this marker prior to hepatocyte maturation. Figure 4 highlights that these cells also acquire functional activity that is representative of cytochrome activity associated with primary hepatocytes. Following treatment with lactate, pyruvate, and octanoic acid, HLCs accumulate greater volumes of intracellular lipid, as demonstrated by BODIPY staining in Figure 5. This is accompanied by an increase in expression of markers associated with lipid droplet biogenesis -PLIN2, PLIN4 and PLIN5, highlighted in Figure 6. Recently developed in silico tools enable mathematical modeling of oxygen gradients within liver tissue, providing insight into how these gradients will impact on cell phenotype (Leedale et al., 2021). This platform can be scaled and provides an excellent tool to study liver biology and disease (Lucendo-Villarin et al., 2020a, 2020bSinton et al., 2021).

LIMITATIONS
Although HLCs are morphologically and functionally similar to hepatocytes, they do not necessarily recapitulate the entire transcriptome of human hepatocytes (Godoy et al., 2015). Whilst LPO-treated HLCs express a substantial number of steatosis-related genes (Sinton et al., 2021), it is unclear how expression patters are impacted by the lack of co-culture with other liver-resident cells, such as

TROUBLESHOOTING Problem 1
Following coating of tissue culture plastic with Laminin-521, the coating does not fully cover the well, exposing the growing surface.

Potential solution
Coating of plates with Laminin-521 is vital prior to cell seeding. Check that the correct concentration of Laminin-521 was used and that the plates were incubated for the correct length of time. Repeat the coating process and if the problem persists, increase the volume of Laminin-521 in the well and ensure that plates are being stored on a completely flat surface.

Problem 2
Following seeding of H9s onto wells coated with Laminin-521, there are high levels of cells death, leading to sparse seeding of the well.

Potential solution
When preparing a single cell suspension of H9 cells, media must contain ROCK inhibitor Y-27632 to prevent excessive cell death. Prior to addition of endoderm differentiation medium, H9 cells that are seeded for differentiation should be incubated in mTeSR1ä basal medium containing ROCK inhibitor Y-27632.

Problem 3
When seeding single cells prior to differentiation, cells may not be distributed homogeneously, leading to heterogeneous patterns of differentiation within the well. If this occurs, by day 9 of the differentiation process, cells may not reach confluence and morphology will not appear consistent across the well.

Potential solution
Attachment of the pluripotent stem cells to Laminin-521-coated plates occurs rapidly. To ensure that cells are distributed evenly across the growing surface of the well or plate, gently agitate the plate up and down, and side-to-side, at least 10 times prior to transfer to the incubator. Problem 4 Differentiation of H9 cells to the endoderm, hepatoblast or HLC specification are unsuccessful as determined by qPCR analysis of pluripotency of hepatocyte markers.

Potential solution
Check that the components of each differentiation media have been prepared to the correct concentration and stored under the correct conditions.

Problem 5
Induction of steatosis may be unsuccessful, as determined by BODIPY staining and immunofluorescence.

Potential solution
Components of the steatosis-induction cocktail have a limited shelf life. It is strongly recommended that storage conditions are checked and that fresh solutions are prepared if cells are not developing steatosis following 48 h of incubation with this cocktail.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, David C. Hay (davehay@talktalk.net).

Materials availability
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact.
Data and code availability Datasets will be made available upon request to the lead contact.