Functionally Enhanced Human Stem Cell Derived Hepatocytes in

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Functionally enhanced human stem cell derived hepatocytes in galactosylated cellulosic sponges for hepatotoxicity testing Farah Tasnim, Yi-Chin Toh, Yinghua Qu, Huan Li, Derek Phan, Balakrishnan Chakrapani Narmada, Abhishek Ananthanarayanan, Nikhil Mittal, Ryan Q. Meng, and Hanry Yu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00119 • Publication Date (Web): 08 May 2016 Downloaded from http://pubs.acs.org on May 14, 2016

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Molecular Pharmaceutics

Functionally enhanced human stem cell derived hepatocytes in galactosylated cellulosic sponges for hepatotoxicity testing Farah Tasnim1, +, Yi-Chin Toh1, a, Yinghua Qu1, b, Huan Li1, Derek Phan1, Balakrishnan C. Narmada1, c , Abhishek Ananthanarayanan1, b, Nikhil Mittal1, Ryan Q Meng7 and Hanry Yu1, 2, 3, 4, 5, 6,* 1. Institute of Bioengineering and Nanotechnology, #04-01, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore 2. Department of Physiology, Yong Loo Lin School of Medicine, National University Health System, MD9-03-03, 2 Medical Drive, Singapore 117597, Singapore 3. NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences, #0501, 28 Medical Drive, Singapore 117576, Singapore 4. Mechanobiology Institute, T-Labs, #05-01, 5A Engineering Drive 1, Singapore 117411, Singapore 5. Singapore-MIT Alliance for Research and Technology, 3 Science Drive 2, S16-05-08, Singapore 117543, Singapore 6. Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA 7. Preclinical Development and Safety, Asia Pacific, Janssen Research & Development, 999 South Pudong Road, Shanghai, 200120, China a

Current address: Department of Biomedical Engineering, Faculty of Engineering , 9, Engineering Drive 1, EA #03-12, Singapore 117575, Singapore

b

Current address: InvitroCue Pte Ltd, 11 Biopolis Way, Helios #12-07/08, Singapore 138667, Singapore

c

Current address: Institute of Molecular and Cell Biology, #05-07, 61 Biopolis Way, Proteos, Singapore 138673, Singapore * Corresponding author: Tel: +6568247103, Fax: +68749526 +

Co-corresponding author: Tel: +6568247243, Fax: +64789080

E-mail address: [email protected]; [email protected]

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Abstract Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs, especially for long term studies. If 3D culture systems were to be used for such purposes, it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time, and can also be directly deployed in liver drug testing assays. We report the use of 3-Dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSCHLCs in 3D cellulosic scaffolds increases functionality, as demonstrated by improved urea production and hepatic marker expression. In addition, hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays, making this system highly attractive for drug testing applications. Keywords: Hepatocytes, Stem cells, Hepatotoxicity, Scaffolds 1. Introduction Hepatocyte-like cells (HLCs) generated from human embryonic stem cells (hESCs) 1-14 or human induced pluripotent stem cells (hiPSCs) 15-18 provide an attractive alternative cell source to primary human hepatocytes (PHHs) for cell-based therapies and hepatotoxicity testing. The resulting HLCs (hPSC-HLCs) exhibit typical hepatocyte morphology, express a variety of hepatocyte markers and perform a wide range of hepatocyte functions. However, their functionality is lower than in PHHs.2, 4, 14, 19, 20 This suggests the need for culture conditions, which could enhance and maintain the functionality of hPSC-HLCs. One of the critical factors which might be responsible for compromised functions in hPSC-HLCs is the lack of cellular interactions on two dimensional (2D) substrates, which have been more commonly used for differentiation studies.21 Compactness of cells contained in three dimensional (3D) spheroids in vitro preserve complex in vivo cell phenotypes which are otherwise absent in conventional 2D cultures.22, 23 3D hepatocyte cultures have been reported to better maintain tight cell-cell junctions and liver-specific functions. 24, 25 Likewise, various 3D culture configurations, including co-culture on feeder cell sheets, 3D bioreactors and microwells 2 ACS Paragon Plus Environment

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have been shown to be useful in maintaining or enhancing functions of primary human 26-29 and rat 30-35 hepatocytes, HepG2 36 and HepaRG 37, 38cultures, and enhancing the differentiation of hPSC-HLCs.12, 15, 39-45 However, these methods focus on the formation of 3D hPSC-HLC spheroids and lack features for their long-term maintenance and eventual deployment in drug testing assays. For instance, spheroids in suspension may aggregate over time, which would impede mass transfer of oxygen, nutrients, metabolites and drugs in the inner core of the spheroid.46 The use of co-cultures or 3D bioreactors poses a challenge to utilize the hPSC-HLCs for subsequent drug metabolism and toxicity testing assays. Hence, it would be desirable to functionally mature hPSC-HLCs in a 3D culture system that can not only form and maintain optimal-sized spheroids for long periods of time, but can also be directly deployed in liver drug testing assays. To this aim, we investigated the functional maturation of hPSC-HLCs in a previously developed 3D galactosylated cellulosic sponge, which could maintain liver specific functions and drug metabolism performance of primary rat hepatocytes better than collagen sandwich culture47. The conjugated galactose ligands of the sponge promoted the formation of primary hepatocyte spheroids while the macroporous network provided physical constraints to maintain constant spheroid size for extended period of culture.47 We demonstrated that nascent hPSC-HLCs could be harvested from 2D hepatic induction cultures, functionally matured in the galactosylated cellulosic sponges as 3D spheroids, and the HLC-sponge constructs could be directly used in cytochrome P450 induction and hepatotoxicity testing assays.

2. Materials and Methods 2.1 hESCs and hiPSCs cell culture hESC cell line, H9 and iPSC cell lines iPS (IMR90)-4 and iPS (Foreskin)-4 (iPSCF-4) (WiCell Research Institute, Madison, WI) were cultured and propagated on tissue culture plates coated with matrigel (BD Biosciences, San Jose, CA, USA) in mTeSRTM1 media (Stem Cell Technologies, Vancouver, BC, Canada) and maintained as described previously.48 2.2 PHHs culture Cryopreserved PHHs were obtained from Life Technologies (Carlsbad, CA, USA) and BD Biosciences (Franklin Lakes, NJ, USA). The cells were thawed and cultured as described in 48. Culture medium was changed daily. Three different lots of hepatocytes were used for the experiments. For gene expression analysis, freshly thawed PHHs were used. PHHs cultured for 24 hours were used for albumin and urea production. For assessing CYP basal and induced activities, PHHs were cultured for 48 hours in order to allow time for addition of inducers.



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2.3 In vitro differentiation Human pluripotent stem cells (hPSCs) were allowed to grow in feeder-conditioned medium as described in Section 2.1. The differentiation was initiated when hPSCs reached a confluency of approximately 80%. All differentiations were done in 6 well plates (Nunc) pre-coated with matrigel diluted in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, Gibco) for 1–2 h at 37°C, in a 5% CO2 atmosphere. To induce differentiation, expansion medium was switched to basal differentiation medium, sequentially supplemented with growth factors as described in Figure 1 for 20 days, and 2% fetal bovine serum (FBS, HyClone, Thermo Scientific, Logan, Utah) between days 1-6 and 0.5% FBS between days 6-20. This growth factor based differentiation was adopted from the protocol developed by Roelandt et al.4 Briefly, basal differentiation medium was composed of 60% DMEM-low glucose (Gibco, Carlsbad, CA, USA), 40% MCDB-201-water (Sigma), 0.25X Linoleic acid – Bovine serum albumin (LA-BSA) (Sigma), 0.25× Insulin-transferrin-selenium(ITS) (Sigma), 100 IU/mL Penicillin and 100 µg/mL Streptomycin (Cellgro), 0.1 mM L-Ascorbic Acid (Sigma), 1 µM Dexamethasone (Sigma) and 110 µM 2-mercaptoethanol (Gibco). For hiPSC-derived cell HLC differentiation, a 1:1 mixture of basal differentiation media and APEL (Stemcell Technologies, Vancouver, BC, Canada) was used. The following cytokines and growth factors (all from R & D Systems, Minneapolis, MN, USA) were added at different steps for differentiation: rh/m/r Activin-A (100 ng/ml), rmWnt3a (50ng/ml), rhFGF2 (10ng/ml), rhBMP4 (50ng/ml), rhFGF1 (50ng/ml), rmFGF8b (25ng/ml), rhFGF4 (10ng/ml), rmFollistatin-288 (100ng/ml), rhOncostatin M (40ng/ml), rhHGF( 20ng/ml). Media was changed every other day and half of the media was replaced with fresh media containing growth factors within the same step. At least three independent batches of differentiated HLCs from each hPSC type were used for all assays. 2.4 Harvesting of HLCs and formation of HLC-spheroids Following differentiation, the cells were washed with 1 X PBS and incubated in 2X TrypLE (Life Technologies) for 5 min at 37°C. This was repeated three times and the cells were collected, diluted with 1X PBS and pass through a 70 µm strainer. The flow-through was collected and centrifuged at 1000 rpm for 5 min at 4°C. The cell pellet was resuspended in basal differentiation media (constitution described in section 2.3) containing rmFollistatin-288 (100ng/ml), rhOncostatin M (40ng/ml) and 10 µM ROCK inhibitor (ROCki) Y27632 (Calbiochem, Billerica, MA, USA). Cells were seeded at a density of 300,000 cells/scaffold in 48 well plates and 100,000/cm2 on 2D monolayer controls (48 well plates coated with matrigel). Media was changed the following day and replaced with media without ROCK inhibitor. HLCs were cultured for 12 days in the scaffolds and 2D controls with media changes every other day. 2.5 Spheroid size distribution


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Spheroid size distribution was quantified using ImageJ software from phase contrast images of hepatocyte spheroids formed in 3D cellulosic scaffolds. A minimum of 100 spheroids in representative images from three independent batches of differentiation for hESC-H9_HLCs, hiPSC-IMR90 HLCs and hiPSC-F4_HLCs were used for the analysis. 2.6 Immunostaining Immunostaining of albumin was performed as previously described 47 with a goat polyclonal antibody against Human Albumin (Abcam, Cambridge, U.K.). A secondary Alexa Fluor 488conjugated anti-goat antibody (Invitrogen) was applied. Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Imaging was performed using LSM 510 META confocal microscope (Carl Zeiss). 2.7 Quantitative real-time PCR (qPCR) RNA isolation was performed using RNeasy Micro-kit (Qiagen, Hilden, Germany). Total amount of RNA was determined using a NanoDropTM ND-1000 Spectrophotometer and converted to cDNA using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). 7000 Fast RealTime PCR System (Applied Biosystems, Foster City, CA, USA) was used for qPCR. Primers were obtained commercially from GeneCopoeia, Inc. (Rockville, MD, USA). The expression levels of all marker genes were normalized to the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to account for differences in cell numbers. 2.8 Albumin secretion and urea Production Albumin secretion was measured using a human albumin enzyme-linked immunosorbent assay (ELISA) quantitation kit (Bethyl Laboratories, Inc., Montgomery, TX, USA) as per manufacturer’s protocol. Urea production was measured using Direct Urea Nitrogen Color Reagent and Direct Urea Nitrogen Acid Reagent (Standbio Laboratory, Boerne, TX, USA) as described previously.48 All functional data was normalized to the number of cells seeded which was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies). 2.9 Cytochrome P450 activity Basal activity and induction of three cytochrome P450 (CYP) enzymes, i.e. CYP1A2, CYP2B6 and CYP3A4 was determined. Following the 20-day differentiation period and further 12 day maturation in 3D scaffolds, cells were incubated with media containing inducers (40 µm βnaphthaflavone for CYP1A2, 1 mM phenobarbitol for CYP2B6 and 20 µm rifampicin for CYP3A4). After 48 hours of induction, medium was removed and the cells were incubated for 2 h at 37°C with Krebs-Henseleitbicarbonate (KHB) buffer (118 mM NaCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4.7 mM KCl, 26 mM NaHCO3, and 2.5 mM CaCl2) containing specific substrates (200 µM 5 ACS Paragon Plus Environment


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phenacetin for CYP1A2, 200 µM bupropion for CYP2B6 and 5 µM midazolam, for CYP3A4). The inducers and substrates were obtained from Sigma. The drug metabolite product, (acetaminophen (APAP) for CYP1A2, hydroxy bupropion for CYP2B6 and hydroxy midazolam for CYP3A4) in the supernatant of induced and non-induced samples (basal activity) was analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS Finnigan LCQ Deca XP Max, Agilent 1100 series) according to procedures described previously.47 The same procedure for detection of basal activity and induction was repeated for HLCs in scaffolds and 2D controls at day 32 (20 days differentiation plus 12 days in scaffolds/2D controls). 2.10

Cell viability assays

Cell viability of HLCs was measured 24 hours after treatment with drugs using CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT) (Promega, Madison, WI, USA) according to manufacturer’s instructions. The following drugs were used for the cell viability assays: Acetaminophen, Troglitazone and Methotrexate (all drugs from Sigma). Stock solutions of drugs were prepared in DMSO and controls were treated with DMSO alone (in absence of test compounds) and considered as 100% viability value. The viability was also normalized to cell numbers in scaffolds before drug treatment to account for slight differences in cell numbers between scaffolds. 2.11

Statistical analysis

Statistical comparisons were undertaken using paired two-tailed student t test. Results are expressed as mean ± standard error of the mean (s.e.m). 3. Results 3.1 Optimization of Spheroid Formation by HLCs Monolayer culture conditions have been reported to efficiently differentiate hPSCs into definitive endoderm (DE) 3, 49 and eventually HLCs.1, 3, 4, 10, 11, 16, 18, 50, 51 3D culture provides a better cell–cell contact, which enhances hepatocyte differentiation and function.52 We hypothesized that a combination of 2D hepatic induction and further maturation in 3D environment could greatly enhance the generation of functional hPSC-HLCs. In order to test this hypothesis, we first differentiated hESCs and hiPSCs using monolayer culture for 20-days using a 4-step differentiation protocol and conditions adopted from Roelandt et al 4 (Schematic, Fig. 1) and then seeded them into cellulosic scaffolds for an additional 12 days. All hPSC lines evaluated were able to differentiate into HLCs with vesicles and cobblestone morphology (Fig. 1B-D). HLCs need to be harvested after undergoing 20 days of differentiation before they can be seeded into the 3D cellulosic scaffolds. The harvesting protocol was optimized for sufficient 6 ACS Paragon Plus Environment

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dissociation into single cells and high cell viability (Supplementary Fig. 1A). This was done to ensure that cells can be effectively seeded into the scaffold and can remodel into 3D spheroids. Oncostatin (OSM, 40ng/ml) was added 2 days prior to harvesting (Schematic, Supplementary Fig. 1A) to upregulate the E-cadherin expression, which is important for the formation of 3D spheroids.53 Various proteolytic enzymes were evaluated for their efficacy in dissociating the hPSC-HLCs into single cells, including 0.5% trypsin, TrypLE, and Accutase. We found that treatment with 2X TrypLE Express for 15 min (5 min incubation with fresh TrypLE for 3 times), followed by mechanical trituration and passing through a 70 µm sieve (BD-Falcon) gave the highest yield of single, viable cells. The typical cell yield was between 0.7 million - 1.5 million cells per 6-well, depending on the hPSC line. The dissociated cells were resuspended in differentiation medium supplemented with 10 µM of ROCKi Y27632 (Schematic, Supplementary Fig. 1A). Following treatment with ROCKi, hPSC-HLCs seeded in cellulosic scaffolds (density of 300,000 cells/scaffold) organized into 3D spheroids within 1-2 days of culture (Fig. 1F) and maintained similar sizes for at least 14 days (Supplementary Fig. 1B). The spheroid diameters were y 104 ± 25 µm, 91 ± 14 µm and 91 ± 18 µm for hESC-H9-HLCs, hiPSC-F4-HLCs and hiPSCIMR90-HLCs respectively (Supplementary Fig. 1C). The spheroids were well constrained by the sponge pores and did not detach or disintegrate throughout the 12 day maturation period. In contrast, hPSC-HLCs cultured on matrigel monolayer (2D control) were flat and slowly started to spread out after initial day of cell seeding (Fig. 1E). Altogether, these results suggest that hPSCHLCs cells can form uniform spheroids in 3D scaffolds and require the inhibition of the ROCK signalling pathway for the formation of such spheroids (without these optimizations, hESC and hiPSC-derived HLCs did not form intact spheroids, Supplementary Fig. 1D). Importantly, the spheroids were separated from each other by the macroporous structure of the scaffold. This ensured that optimal sizes were maintained and spheroids did not aggregate to form larger spheroids which would reduce the nutrition/oxygen accessibility of the cells found at the centre and affect the survival of these cells. 3.2 Improvement of hepatic marker expression in HLC-spheroids We first compared the gene expression of liver-specific markers after 20 day differentiation of hPSCs to HLCs in 2D (referred to as d20) across the different cell lines using qPCR (Fig. 2A). The gene expression levels were benchmarked to the averaged expression from 3 different lots of freshly thawed induction-qualified cryopreserved PHHs. Our results showed that hepatic marker genes were expressed in HLCs derived from all three different hPSCs (hESCs, hiPSC-F4 and hiPSC-IMR90). The expression profile indicated that all the hPSC-HLCs exhibited a more fetal phenotype than PHHs. This was shown by higher fetal markers, AFP (>6 ×103 fold compared to PHHs) and CYP1A1 (3 fold for hESC-H9-HLCs compared to PHHs) and lower expression levels of mature markers, such as ALB, CYP3A4, CYP1A2, ASGPR and MRP2. ALB was 9 fold lower compared to PHHs in hiPSC-F4-HLCs; CYP3A4 and CYP1A2 were ≥ 50 fold lower 7 ACS Paragon Plus Environment


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compared to PHHs in hESC-H9-HLCs, hiPSC-IMR90-HLCs and hiPSC-F4-HLCs; ASGPR was 24, 27 and 15 fold lower compared to PHHs in hESC-H9-HLCs, hiPSC-IMR90-HLCs and hiPSC-F4-HLCs respectively; MRP2 was 13 and 4 fold lower in hiPSC-IMR90-HLCs and hiPSC-F4-HLCs respectively. The different hPSC lines exhibited variability in terms of their hepatic differentiation potential. hiPSC-IMR90-HLCs had similar expression levels to hESC-H9-HLCs, whereas expression of ALB, CYP3A4, CYP3A7 and MRP2 were lower in hiPSC-F4-HLCs. Next, we examined if the hepatic marker expression could be improved from a fetal-like phenotype to a more mature phenotype by extending the culture for another 12 days as HLCspheroids in 3D cellulosic scaffolds (referred to as d32-3D). The expression levels were compared to cultures extended for 12 days in 2D monolayer (referred to as d32-2D) and expression levels at d20 (Fig. 2B). The gene expression levels were benchmarked to the averaged expression from 3 different lots of freshly thawed induction-qualified cryopreserved PHHs. HLC-spheroids derived from all 3 hPSC lines exhibited similar trends in changes in hepatic gene expression upon 3D culture. Extending the differentiation period for another 12 days led to a decrease in expression of the fetal marker, AFP in both 2D and 3D configurations. Increase in expression levels of mature hepatic markers compared to d20 hPSC-HLCs was observed in 3D configurations (CYP3A4:3, 11 and 3 fold; CYP1A2: 10, 9 and 7 fold and MRP2: 1.5, 1.5 and 3 fold for hESC-H9-HLCs, hiPSC-F4-HLCs and hiPSC-IMR90-HLCs respectively). This increase was not observed in the 2D controls. A decrease in ALB, AAT and HNF4α expression levels were observed compared to d20. Again, this decrease was a lot lower in d32-3D (e.g. ALB: 2-3 fold decrease) compared to d32-2D (e.g: ALB: >16 fold decrease). This suggests that the culture configuration (i.e., 2D or 3D culture) in which the hPSC-derived hepatocytes were maintained during the maturation phase was important in determining the extent of increase or decrease in the gene expressions post 2D induction. Ratios of ALB and AFP, which are critical for assessing maturity of HLCs were compared. HLCs from all three hPSCs showed an enhanced ALB: AFP ratio in 3D culture configurations compared to both d20 and d32-2D (Fig. 2C). In addition,3D maturation in the cellulosic scaffolds consistently showed higher levels of hepatic gene expression (8 fold for most genes across three different hPSC-HLCs) as compared to 2D cultures (d32-2D) (Fig. 2D). 3.3 Maintenance of albumin production and improvement of urea production in HLC spheroids Albumin and urea production by the hPSC-HLCs was measured via sandwich ELISA after 2D induction (d20) as well as after 2D or 3D maturation (d32-2D and d32-3D). After 2D induction, albumin production in hPSC-HLCs was 0.3 pg/cell/48 h (30% of PHH’s production level) in hESCH9-HLCs and 1-1.3 pg/cell/48 h for hiPSC-F4/IMR90-HLCs (similar to PHH’s production level) (Fig. 3A). After culturing d20 HLCs in 3D scaffolds (d32-3D), hESC-H9-HLCs maintained albumin production while hiPSC-F4/IMR90-HLCs produced slightly lower levels of albumin (this decrease 8 ACS Paragon Plus Environment

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was not statistically significant). In contrast, d20 HLCs cultured in 2D configuration (d32-2D) produced undetectable or very low levels of albumin (0.02-0.07 pg/cell/48 h). This trend in albumin production is consistent with ALB gene expression levels reported in section 3.2. After 2D induction of hESC-H9-HLCs, hiPSC-F4-HLCs and hiPSC-IMR90-HLCs produced 15, 8 and 19 pg/cell/48 h urea respectively at d20, which was 8-19% of urea production by PHHs (100 pg/cell/48 h) (Fig 3B). When these HLCs were cultured in 3D scaffolds, the urea production by HLCs from all three hPSCs significantly increased to approximately 72 pg/cell/48 h (72% of PHHs). In contrast, the HLCs in 2D controls produced much lower levels of urea (1-22 pg/cell/48 h). Overall, these two functional assays suggest that the cellulosic scaffold is superior to the 2D control in maintaining albumin production and improving urea secretion for hPSC-HLCs. 3.4 Improvement of CYP activity and induction Basal CYP1A2 and CYP3A4 activities were measured by the enzymatic conversion of phenacetin and midazolam into their respective metabolic products. The basal activity levels were measured in order to confirm if the enhancement of CYP gene expression levels observed upon 3D maturation (section 3.2) can be translated into enhancement in metabolic activities. There was indeed an enhancement in the CYP1A2 activity of hESC-H9-HLCs (d32-3D: 7.8 pmol/million cells/min vs d20: 2.4 pmol/million cells/min) and hiPSC-F4-HLCs (3D: 0.5 pmol/million cells/min vs d20: undetectable levels) upon 3D culture; while activity of hiPSC-IMR90-HLCs remained constant at 0.3 pmol/million cells/min (Fig. 4A). The CYP1A2 activity levels of hPSC-HLCs at d20 were 3-8% of PHHs levels-HLCs and this increased to 12% for hiPSC-F4-HLCs and 200% for hESCH9-HLCs after culturing in the 3D cellulosic scaffolds. CYP3A4 activity was significantly increased after 3D culture for hESC-H9-HLCs (d32-3D: 0.3 pmol/million cells/min vs d20: 0.03 pmol/million cells/min) and hiPSC-IMR90-HLCs (3D: 0.06 pmol/million cells/min vs d20: 0.04 pmol/million cells/min) (Fig. 4A). The activity levels of the 3D cultured hPSC-derived hepatocytes (d32-3D) ranged from 29% of PHHs (hiPSC-IMR90-HLCs) to 142% of PHHs (hESC-H9-HLCs). Such increment in activity upon 3D maturation compared to d20 HLCs was not observed when the culture was extended in 2D configuration (d32-2d). Since hESC-H9-HLCs and hiPSC-IMR90-HLCs showed better gene expression, albumin production and basal CYP3A4 activity in 3D cultures compared to hiPSC-F4-HLCs, these two cell types were further characterized in their cytochrome induction potential and response to paradigm hepatotoxicants. Induction was analyzed for CYP1A2, CYP3A4 and CYP2B6 using specific inducers detailed in section 2.7. Our results showed that 2D induction of hESC-H9-HLCs and hiPSC-IMR90-HLCs (d20) and further culture in 2D (d32-2D) resulted in almost no induction of CYP1A2 and CYP2B6 as shown by the absolute basal and induced metabolite production levels (Fig. 4B) and fold induction levels (Fig. 4C). In contrast, when these cells were cultured in 3D cellulosic sponges (d32-3D), both CYP1A2 and CYP2B6 could be induced to at least 9 ACS Paragon Plus Environment


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approximately 2 fold (for both hESC-H9-HLCs and hiPSC-IMR90-HLCs; Fig. 4B, C). In the case of hESC-H9-HLCs, 8 fold induction of CYP2B6 was observed, which is similar to induction levels in PHHs (Fig. 4C). hESC-H9-HLCs showed no CYP3A4 induction at d20, but induction was observed in d32-2D cultures, which was similar to the levels observed in d32-3D. hiPSC-IMR90-HLCs showed CYP3A4 induction in all 3 configurations (d20, d32-2D and d32-3D) but the highest levels were observed in the 3D configuration (4.7 fold) which was approximately 17 % of the induction observed in PHHs (Fig 4B, C). The basal and induction levels of the CYPs indicate that hPSC-HLCs cultured in cellulosic scaffolds exhibit a more mature phenotype compared to their 2D counterparts. 3.5 Response to paradigm hepatotoxicants To examine the applicability of the mature hPSC-HLCs in cellulosic scaffolds to drug testing and to analyze if they offer a potential advantage over 2D configurations, we treated hESC-H9-HLCs and hiPSC-IMR90-HLCs in both 2D and 3D configurations (d32-2D and d32-3D) with paradigm hepatotoxicants. These drugs included acetaminophen, troglitazone and methotrexate. The response to these drugs was compared to that of PHHs. Our results showed that upon treatment with methotrexate, both hESC-H9-HLCs and hiPSC-IMR90-HLCs in 3D configurations exhibited toxicity similar to PHHs, but 2D cultures showed no toxicity even upto concentrations of 10 mM treatment (Fig. 5). Treatment with acetaminophen and troglitazone showed similar response in d32-2D and d32-3D cultures with the exception that hiPSC-IMR90-HLCs in 3D configuration was more sensitive to acetaminophen at concentrations above 10 mM and less sensitive to troglitazone at concentrations higher than 50 µM (Fig. 5). Importantly, the drug responses were similar to that of PHHs, suggesting the potential use of these cells as an alternative to PHHs for drug testing. These results suggest that 3D matured hPSC-derived HLCs show similar or better responses to paradigm hepatotoxicants in terms of their similarity to PHHs compared to their 2D counterpart. 4. Discussion We have used 3D cellulosic scaffolds for the long term culture of hPSC-derived HLCs. Our results overall show that the 3D cellulosic scaffolds allow spheroid formation and maintenance for a prolonged time period, owing to the mechanical and chemical cues provided by the scaffold. The mechanical cues provided by the scaffold are related to the stiffness. The elastic modulus of the sponges is 5.6 kPa (measured using atomic force microscopy)47. This modulus is considered to be soft and close to the modulus of native rat and human livers54. Studies in our lab have shown that stiffness affects maturation of stem-cell derived hepatocytes: a soft substrate close to liver stiffness promotes maturation (unpublished data). The chemical cues are provided by the galactose conjugated to the scaffold; galactose has been reported to promote hepatocyte attachment and self-assembly of hepatocytes into three dimensional 10 ACS Paragon Plus Environment

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spheroids55-57. However, these reports using galactose as a substrate don’t employ additional measures to prevent spheroid disintegration or aggregation. The galactosylated scaffold used in this study combined with it’s macroporous network provides the chemical cues and at the same time contains the spheroids within the pores without aggregation or disintegration. Two major optimizations which helped in spheroid formation and retention was the addition of OSM two days prior to seeding the cells into the scaffold and addition of ROCKi during cell seeding. These conditions might have resulted in the most optimal harvesting conditions due to 1) apoptosis being induced by ROCK-dependent hyper activation of actin–myosin contraction following dissociation of hESCs into single cells 58, 59 and 2) ability of OSM to induce E-cadherin expression and promote spheroid formation.53 We compared our 3D culture system to other reported 3D culture systems. Although these studies have advanced the field of 3D liver cultures, some of these systems consist of loose spheroids15, 41 which might make it challenging for prolonged cultures and manipulation of the spheroids for drug testing applications. In addition, these studies did not report spheroid size distribution and hence it is difficult to judge if uniform homogenous spheroids are maintained in culture without aggregation or disintegration. These challenges might be addressed by scaffold based systems including Algimatrix scaffolds12, collagen scaffolds 7 and poly lactic-coglycolic acid(PLGA) scaffolds60, 61 However, these studies focused mostly on hepatic gene expression, glycogen storage and metabolic functions such as albumin and urea production. These systems would need to be further characterized for CYP activity and induction, efficient penetration of drugs (drug absorption properties of the scaffold) and dose response to paradigm hepatotoxicants to determine their suitability for drug testing applications. In light of these requirements, we tested our scaffolds for all these parameters. Drug absorption property of the scaffolds used in our study has been previously analyzed and reported47. We also compared the functional activity of hPSC-HLCs in our system to reported values in literature to understand how our system performs in comparison to the state of the art. hPSCHLCs cultured in our system produced 0.2-1 pg/cell/48 h albumin, which is similar or higher than albumin levels reported in literature where hESCs were differentiated using a three-step protocol1 or on a synthesized basement membrane13. Similarly, urea production in hPSC-HLCs cultured in our system (8-19 pg/cell/48 h in d20 and 2D conditions and 72 pg/cell/48 h in 3D culture conditions) is also similar or higher than reported values in 3D spheroid culture systems deploying Nanopillar plates 15 or Algimatrix scaffolds12. CYP activity in hPSC-HLCs range has been reported to be 10-30% of PHHs. 4, 15, 43 hPSC-HLCs cultured in our system could produce almost equivalent levels of CYP1A2 activity and CYP3A4 activity compared to PHHs. In contrast to CYP activity, CYP induction in hPSC-HLCs is not well-documented in literature. Studies which report CYP induction show very low levels of induction in hPSC-HLCs (approximately 1.5-3 fold), including cells cultured in 3D conditions including Algimatrix scaffolds and Nanopillar plates.12, 11 ACS Paragon Plus Environment


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13, 15

This was also observed in our study when hPSC-HLCs were cultured in 2D conditions. However, when cultured in 3D scaffolds, the induction levels could be increased to 2-8 folds depending on specific CYP and hPSC cell source used. Limited studies compare the effect of hepatotoxicants on hPSC-HLCs to PHHs. Takayama at al. reported a much lower sensitivity of the drugs in hPSC-HLCs when compared to PHHs.15 In their study, when PHHs and hPSC-HLCs (3D cultures) were treated with troglitazone, the IC50 were approximately 35 µM and 70 µM respectively.15 It would be important to generate and maintain hPSC-HLCs which not only express hepatic markers and functions, but also show drug toxicity responses comparable to PHHs. When we studied the toxicity responses of the differentiated hepatocytes to hepatotoxicants, hPSC-HLCs in 3D scaffolds showed similar dose responses to hepatotoxicants when compared to PHHs. Interestingly, slightly higher sensitivity to acetaminophen and slightly lower sensitivity to troglitazone was observed in 3D hiPSCIMR90-HLCs compared to PHHs. This trend is consistent with previously reported results obtained from HepaRG cultures.37 Further studies of the drugs and the metabolites in the culture medium would be required to shed light on the reason behind this trend. Treatment with methotrexate showed toxicity response similar to PHHs only when hPSC-HLCs were cultured in 3D scaffolds. hPSC-HLCs cultured in 2D conditions showed no toxicity to methotrexate. Our results showed that hPSCs-HLCs differentiated for 20 days (before additional culture in the 3D scaffolds) showed fetal phenotype. Extending the culture in 2D configuration led to a further decline in function. This decline could be prevented when the hPSC-HLCs were cultured as spheroids in the scaffolds, which not only maintained but further enhanced their functions and maturation in certain aspects. Importantly, this was shown using three different sources of hPSC-HLCs (i.e. hESC-H9-HLCs, hiPSC-IMR90-HLCs and hiPSC-F4-HLCs), which is important because studies often use either hESCs or hiPSCs. It is not surprising that significant efforts have been put into 3D culturing protocols to improve performance of hPSC-HLCs.4, 14, 40 However, these studies use either loose spheroids or gels composed of either hydrogel or collagen for their cultures.12, 15, 39-45 A means of physical spheroid constraint during extended culture which doesn’t interfere with hepatocyte functions and assays (such as drug absorption) would be an added advantage to a 3D culture system. This will also help to overcome additional challenges such as difficulty in controlling spheroid sizes and in manipulating floating spheroids for accurate drug testing studies. hPSC-HLCs cultured in our 3D scaffolds are able to satisfy these criteria and at the same time improve the functional performance of the cells. Future studies to validate the application of this model for chronic toxicity testing, including repeated dosing studies for upto four weeks will help to further


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highlight the advantages of this system. Including a larger number of paradigm hepatotoxicants at physiological concentrations will help to strengthen such validation studies. 5. Conclusion We have shown that the functional performance of hPSC-HLCs can be enhanced by culturing the cells in cellulosic scaffolds. Enhancement of CYP activity and induction indicate that hPSCHLCs in the scaffolds exhibit a more mature phenotype. In addition, the cells can be maintained in the scaffolds for weeks due to the physical properties of the scaffold. Altogether, the results suggest that hPSC-HLCs maintained in cellulosic scaffolds are an attractive alternative to PHHs for drug toxicity testing. 6. Acknowledgments This work is supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore) and grants from Janssen (LT/SW/432/0611/IBN) and Joint Council Office (JCO, A*STAR) Development Program (1334i00051). Supporting Information Schematics for optimization of harvesting protocol; Phase contrast images of spheroids in scaffolds for 14 days before and after optimization; Spheroid size distribution

Supplementary Figure 1: (A) Schematics of optimization of protocol for seeding hPSC-HLCs into cellulosic scaffolds.(B) Phase contrast images of 3D hPSC-HLCs spheroids maintained within the scaffold for 14 days; image taken at day 14 after seeding into scaffold. Scale bar: 100 m. (C) Spheroid size distribution using ImageJ software from phase contrast images of hepatocyte spheroids formed in 3D cellulosic scaffolds. Table shows number of spheroids counted from representative images from three independent batches of differentiation for hESC-H9-HLCs, hiPSC-IMR90-HLCs and hiPSC-F4_HLCs respectively and average diameter ± s.e.m of the spheroids. (D) Phase contrast images showing sub-optimal spheroid formation when optimized conditions were not used. Scale bar: 100 m.



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59. Ohgushi, M.; Matsumura, M.; Eiraku, M.; Murakami, K.; Aramaki, T.; Nishiyama, A.; Muguruma, K.; Nakano, T.; Suga, H.; Ueno, M.; Ishizaki, T.; Suemori, H.; Narumiya, S.; Niwa, H.; Sasai, Y. Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 2010, 7, (2), 225-39. 60. Li, J.; Tao, R.; Wu, W.; Cao, H.; Xin, J.; Li, J.; Guo, J.; Jiang, L.; Gao, C.; Demetriou, A. A. 3D PLGA Scaffolds Improve Differentiation and Function of Bone Marrow Mesenchymal Stem Cell–Derived Hepatocytes. Stem cells and development 2010, 19, (9), 1427-1436. 61. Levenberg, S.; Huang, N. F.; Lavik, E.; Rogers, A. B.; Itskovitz-Eldor, J.; Langer, R. Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proceedings of the National Academy of Sciences 2003, 100, (22), 12741-12746.

Figure Legends: Figure 1: Differentiation of hPSCs into HLCs and generation of HLCs spheroids in cellulosic scaffolds. (A) Schematics of the step-wise protocol for differentiation of hPSCs into HLCs for 20 days. hPSCs-HLCs differentiated using this method were harvested and seeded into cellulosic scaffolds or in 2D as control. (B, C, D) Phase contrast images showing morphology of hPSC-HLCs generated after 20 days differentiation from 3 different stem cell sources: hESC-H9 (B), hiPSC-IMR90 (C) and hiPSC-F4 (D). hPSCHLCs harvested after differentiation were seeded either in 2D monolayer cultures (E) or into cellulosic scaffolds where they organized into 3D spheroids within the macroporous network (F).Both E and F are phase contrast images taken 2 days after cell seeding. (G) Immunofluorescent image of HLCs-spheroid stained with albumin after 12 days of culture in the scaffold. (H) Immunofluorescent image of HLCsspheroid in the same field of cells as in (G) showing overlay of albumin and cell nuclei counterstained with DAPI. Scale bars: 20 µm (B-D, G, H) 100 µm (E, F). hPSC-HLCs: hepatocyte-like cells derived from human pluripotent stem cells. Figure 2: qPCR analysis showing hepatic marker expression in hPSC-HLCs after 20 days of differentiation and after further maturation for 12 days in cellulosic scaffolds (3D) or on 2D monolayer control. (A) Gene expression of hepatic markers after 20 days of differentiation in hESCH9-HLCs (white bars), hiPSC-F4-HLCs (light grey bars) and hiPSC-IMR90-HLCs (dark grey bars). The black bars represent hepatic marker genes in freshly thawed primary human hepatocytes (PHHs) for comparison with these cell lines. mRNA levels of hESC-HLCs, hiPSC-F4-HLCs and hiPSC-IMR90-HLCs are expressed relative to PHHs (PHHs values were set to 1). Significant differences between any of the 3 cell lines and PHHs are detonated by * (p

Functionally Enhanced Human Stem Cell Derived Hepatocytes in

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