Abstract
Liver-on-a-Chip technology holds considerable potential for applications in drug screening and chemical-safety testing. To establish such platforms, functional hepatocytes are required; however, primary hepatocytes are commonly used, despite problems involving donor limitations, lot-to-lot variation, and unsatisfactory two-dimensional culture methods. Although human pluripotent stem cells (hPSCs) may represent a strong alternative contender to address the aforementioned issues, remaining technological challenges include the robust, highly efficient production of high-purity hepatic clusters. In addition, current Liver-on-a-Chip platforms are relatively complicated and not applicable for high-throughput experiments. Here, we develop a very simple Liver-on-a-Chip platform with mature and functional hepatocyte-like cells derived from hPSCs. To establish a method for hepatic differentiation of hPSCs, cells were first treated by inhibiting the phosphoinositide 3-kinase-and Rho-associated protein kinase-signaling pathways to stop self-renewal and improve survival, respectively, which enabled the formation of a well-defined endoderm and facilitated hepatocyte commitment. Next, a simple microfluidic device was used to create a three-dimensional (3D) culture environment that enhanced the maturation and function of hepatocyte-like cells by increasing the expression of both hepatic maturation markers and cytochrome P450. Finally, we confirmed improvements in hepatic functions, such as drug uptake/excretion capabilities, in >90% of 3D-matured hepatocyte-like cells by indocyanin green assay.
Abstract
These results indicated that the incorporation of hPSC-derived hepatocytes on our Liver-on-a-Chip platform may serve to enhance the processes involved in drug screening and chemical-safety testing.
Abstract
All rights reserved. No reuse allowed without permission.
Abstract
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
Introduction
Organs-on-a-Chip (OoC) platforms hold considerable potential for pre-clinical trials of drug development 1-3 as well as toxicological tests. 4 To date, such testing has relied upon animal models;
Introduction
however, large challenges remain with regard to predicting the safety and efficacy of drug candidates for human clinical use. Therefore, new in vitro models need to be developed as an alternative to animal models to predict treatment effects on humans with high predictability. OoCs represent the most suitable platforms to fulfill these requirements, as these platforms were developed to recapitulate human physiological conditions in vitro. Currently, numerous OoC platforms have been reported as representative of organs, such as the brain, 5 lung, 6, 7 heart, 8,9 gut, 10, 11 intestine, 12 liver [13] [14] [15] [16] or multiple tissues. [17] [18] [19] Considering that the liver constitutes the largest organ in the human body and has many critical roles for body maintenance as well as drug metabolism and detoxification, we focused on optimizing the Liver-on-a-Chip platform, as the reported platform still retains two critical issues involving cell sources and cell culture methods.
Introduction
In terms of cell source, hepatocytes, which constitute the majority of cells in the liver, are mainly used for current drug screening and toxicological testing as well as Liver-on-a-Chip platforms. Primary hepatocytes can be obtained from healthy donors; however, these cells cannot proliferate under standard cell-culture conditions and often experience loss of function. As an alternative, established cell lines are used for cell culture and in vitro drug testing, although these cells were often originally harvested from tumors and do not represent healthy liver functions. Therefore, obtaining sufficient amounts of proper and functional hepatocytes remains challenging.
Introduction
To meet this requirement, human pluripotent stem cells (hPSCs), such as embryonic (hESCs 20 ) and induced pluripotent stem cells (hiPSCs 21 ) hold considerable potential as sources to generate hepatocytes.
Introduction
hPSCs exhibit two distinctive capabilities: unlimited self-renewal without karyotypical abnormality and the ability to differentiate into almost any cell type. These capabilities allow for the generation of embryoid bodies or cell aggregates, 22 or 2) introduction of exogenous genes (e.g., HHEX 23 and SOX17 24 ).
Introduction
Such methods have high potential to cause quality control difficulties involving contamination with other differentiated cells; moreover, the hepatic functionality of the resultant cells still has considerable room for improvement. Therefore, it is necessary to establish a novel method enabling the efficient and robust differentiation of hPSCs into functional hepatocytes.
Introduction
In addition to cell sources, cell culture methods also require new innovation. Although two-dimensional (2D) culture of hepatocytes has been carried out for many years, this method is also problematic; for example, primary hepatocytes often lose their function under 2D culture. Therefore, a number of reports have suggested the use of three-dimensional (3D) culture methods to render hepatocytes more functional. 25 Moreover, recent studies have shown that organoids [26] [27] [28] hold promise to obtain micro live tissues with greater functionality than those of 2D culture. However, the described cell aggregates and organoids exhibited varying size and structure as they are reliant upon only the capacity of the stem cells for tissue formation, resulting in low reproducibility. Therefore, these methods have yet to reach an acceptable stage for use in drug screening. Similarly, the majority of conventional
Introduction
Liver-on-a-Chip platforms were based on 2D cell culture. Moreover, as most previously developed
Introduction
Liver-on-a-Chip platforms are both sophisticated and complicated and require special instruments to control the procedures, they are not user-friendly, limiting their use by general biology laboratories and their applicability for drug screening owing to the lack of high-throughput screening capability. Here, we developed a novel Liver-on-a-Chip platform by 1) establishing a protocol enabling the efficient differentiation of hepatocyte-like cells from hPSCs, resulting in high degrees of purity and reproducibility, and 2) applying a simple microfluidic 3D cell-culture platform 29, 30 (Fig. 1) 
Results and discussion
Cell survival and robustness of definitive-endoderm (DE) differentiation is enhanced by phosphoinositide 3-kinase (PI3K) and Rho-associated protein kinase (ROCK) inhibition
Results and discussion
Induction of DE differentiation is the most critical step required to promote further differentiation (Fig. 2) .
Results and discussion
In this process, transforming growth factor (TGF)-, 31,32 bone morphogenetic protein (BMP)-4,
33
-catenin, 34, 35 and basic fibroblast growth factor (bFGF) 32, 36 -signaling pathways play critical roles.
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Moreover, the PI3K-signaling pathway is critical for the maintenance of hPSC self-renewal, 37, 38 with inhibition of this pathway resulting in hPSC differentiation. Previously, Hannan et al. established a method to effectively achieve hPSC differentiation to DE using a cocktail of activin A, BMP-4, bFGF, LY294002, and CHIR99021, but without the use of exogenous genes. 39 However, this method results in massive cell death and inefficient and irreproducible outcomes. Furthermore, the use of hPSC aggregates is unlikely to allow improved robustness, owing to the challenge associated with size control, which is a critical factor for lineage determination during hPSC differentiation. Although the use of dissociated hPSCs may improve robustness, the related processes continue to result in large amounts of cell death. are also commonly used as key factors to introduce DE differentiation. We observed that hPSCs showed unstable behavior following introduction of the new culture conditions; therefore, for establishing robust differentiation protocols, the initial use of self-renewing culture medium was beneficial, followed by gradually changing to the differentiation medium. Toward this end, activin A, BMP-4, LY294002, and CHIR99021 were subsequently added into the mTeSR-1 medium for DE differentiation.
33
To confirm increases in cell survival during DE differentiation, we counted H9 hESCs 20 following 1 day of treatment ( Fig. 2A) . Cells treated with both 10 µM Y27632 and 10 µM LY294002 showed significant increases in cell number, as compared with levels observed in cells treated with only LY294002. This result indicated that inhibition of the ROCK-signaling pathway improved cell survival to a degree greater than that observed following inhibition of the PI3K-signaling pathway.
33
We then investigated differences in outcomes based on initiating differentiation from either dissociated or clumped hPSCs according to cell number and doubling time ( Fig. 2B and C) . We observed that following a 5-day incubation with 10 µM Y27632 and 10 µM LY294002 and DE differentiation, both dissociated and clumped hESCs increased cell numbers with similar doubling times. These results suggested that Y27632 improves cell survival even during DE differentiation, but not cell proliferation.
33
To confirm cell status following DE differentiation, the expression of C-X-C-motif chemokine receptor-4 (CXCR4), a cell surface DE-differentiation marker, was quantitatively analyzed by flow cytometry (Fig. 2D ) and compared with levels measured in previously-reported chemically defined medium (CDM-PVA) 39 (Supplementary Table S2 ). Both H9 and 253G1 cells treated with mTeSR-based medium showed higher degrees of purity and enhanced CXCR4 expression (99.6%) compared to those of cells cultured in CDM-PVA medium (81.5%). Then, we investigated the effects of initial cell-seeding density on the induction of DE differentiation (Fig. 2E) . During 5-day DE differentiation, cell numbers in all cell densities increased approximately 10 fold (Fig. 2B) ; however, the increase in initial cell number revealed bimodal cell populations with regard to CXCR4 expression (Fig.   2E ). These results indicated that the use of a smaller initial cell-seeding density resulted in a more homogeneous cell population with higher CXCR4 expression.
33
All rights reserved. No reuse allowed without permission.
33
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
33
The copyright holder for this preprint . http://dx.doi.org/10.1101/232215 doi: bioRxiv preprint first posted online Dec. 13, 2017; We also measured expression of the sex-determining region Y box-17 (SOX17) transcription factor and endoderm-lineage marker by quantitative reverse transcription-polymerase chain reaction (RT-PCR) (Fig.   2F) . The use of cell clumps resulted in higher SOX17 expression as compared with using dissociated cells, with SOX17 expression subsequently decreasing along with increasing cell numbers, although lower initial cell-seeding density (2.5 × 10 4 cells cm −2 ) resulted in large variations in SOX17 expression. We also confirmed hepatocyte differentiation by monitoring the expression of -fetoprotein (AFP) and hepatocyte nuclear factor 4 (HNF4A differentiation marker mRNA on day 12 (Fig. 2G) . The use of cell aggregates with lower initial cell-seeding density (2.5 × 10 4 cells cm −2 ) did not result in AFP and HNF4A
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expression. Based on these results, we used 5 × 10 4 cells cm −2 for further differentiation protocols consequent to our observation of higher purity and improved robustness at higher initial cell densities.
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Under the optimized conditions, we tested DE differentiation from 253G1 hiPSCs 42 and differentiation to highly pure CXCR4-positive DE cells, and found that this condition gave similar results as H9 hESCs, even with 253G1 hiPSCs (Supplementary Fig. S1 ).
33
Optimization to induce anterior definitive-endoderm (ADE) specification and hepatocyte
commitment (HC)
We established a robust and efficient procedure for ADE specification from DE cells, as well as HCs, to generate hepatic progenitor cells (Fig. 3 and Supplementary Table S1 ). For ADE specification as shown in Fig. 1 and Experimental, it was necessary to maintain activin A levels but remove bFGF to reduce active cell-signaling pathways and maintain pluripotency. 43 Alternatively, HC requires BMP4 and FGF10
commitment (HC)
signaling. 44 ADE specification and HC require several days to achieve treatment-induced changes in phenotype. Therefore, we treated cells with activin A and a combination of BMP4 and FGF10 in RPMI medium supplemented with B27 supplement for ADE and HC, respectively, and varied these treatment periods to identify the optimal timing (~1-3 days for ADE and ~1-4 days for HC) for hepatic progenitor cell differentiation (Fig. 3A) by observing the expression levels of genes associated with each differentiation phenotype following treatment as determined by quantitative RT-PCR ( Fig. 3B and 
Supplementary Table S3).
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Supplementary Table S3).
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
Supplementary Table S3).
The copyright holder for this preprint .
Supplementary Table S3).
Following 1 day of treatment for ADE specification, cells continued to express most of the pluripotency associated genes including NANOG, SOX2, and OCT4 (or POU5F1) (Fig. 3B, "Pluripotency"). However, when the treatment for ADE specification was extended to 3 days, the expression levels of pluripotency associated genes was decreased, whereas that of genes associated with extraembryonic endoderm (SOX7, LAMB1, and HNF1A), mesoendoderm (GSC and NODAL), endoderm (FOXA2, SOX17, CXCR4, HNF1B, and HNF4A), and early hepatic cells (AFP, CYP3A7, DLK1, PROX1, and TBX3) was increased. Although 1-day treatment for ADE specification did not induce expression of genes associated with hepatic nuclear receptors and/or transporters, expression of these genes was observed on days ~2 and ~3. These results indicated that 3 and 4 days of treatment for ADE and HC, respectively, were optimal to obtain hepatic progenitor cells. (1, 2) represents 1-day treatment of ADE specification and 2-day treatment for hepatocyte commitment. Center lines show the medians; box limits indicate 25% and 75%; whiskers extend 1.5 times the interquartile range from 25% and 75%. These experiments were carried out at least three times independently.
Supplementary Table S3).
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Supplementary Table S3).
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
Supplementary Table S3).
The copyright holder for this preprint . http://dx.doi.org/10.1101/232215 doi: bioRxiv preprint first posted online Dec. 13, 2017;
Evaluation of hepatocyte differentiation status by observing gene-expression patterns
To confirm the gene expression associated with hepatic differentiation, we conducted multiplex quantitative RT-PCR, followed by clustering analysis (Fig. 4, Supplementary Fig. S2 and
Supplementary Table S3). Clustering analysis based on gene-expression patterns resulted in six clusters.
Class 1 genes in the undifferentiated cells exhibited the highest expression levels, which decreased after the initiation of differentiation. Most Class1 genes, including NANOG, SOX2, and POU5F1, were associated with pluripotency. Class 2 included endoderm genes, such as CXCR4, GATA6, and SOX17, which exhibited peak expression at day 5. Class 3 included early hepatic genes, such as DLK1 and TBX3, which exhibited peak expression at day 12, as well as some genes associated with the cytochrome P450 (CYP) family (CYP1A2, CYP2D6, and CYP2E1). Class 4 included many hepatic genes, such as AHSG, APOA4, and HNF1A, as well as hepatic transporter genes and solute-carrier-family genes, such as SLC10A1, SLC22A1, and SLC22A2, and exhibited the highest levels of expression at days 20 or 24. Class 5 genes, including AQP1, B2M, CD9, CYP2C8, NR3C1, SLC22A2, and SOX7, showed the highest expression at day 30, but did not appear to exhibit functional enrichment. Class 6 showed unique gene-expression patterns, with genes initiating expression on day 20 and peaking at day 35. These includes hepatic genes, such as ALB, SERPINA, and TAT, as well as other early hepatic genes. These gene-expression data suggested that our hepatic differentiation method resulted in a natural developmental process. 
Differences in gene expression between hPSC-derived hepatocyte-like cells and liver lysate
To investigate similarities between mature liver and cultured cells, we compared gene-expression patterns between hPSC-derived hepatocyte-like cells and mature liver lysate ( Fig. 5 and Supplementary Table   S3 and S4) using hPSC-derived hepatocyte-like cells at days 0, 24, and 35 of differentiation and liver lysate from a healthy donor. Hepatic marker genes, such as ALB and HNF4A, exhibited significant increases during the culture process, indicating progression of differentiation and increasing similarity between hPSC-derived hepatocyte-like cells and mature liver lysate. Additionally, genes including AFP, APOA4, KRT7, KRT8, KRT18, KRT19, DLK1, GATA6, and HNF1B exhibited increased expression between days 0 and 24, which was higher than the expression of these genes observed in the liver lysate.
Differences in gene expression between hPSC-derived hepatocyte-like cells and liver lysate
AFP is a marker of hepatoblasts and fetal livers, and APOA4 exhibits high expression levels in fetal liver.
KRT7, KRT8, KRT18
, and KRT19 are expressed in cholangiocytes, DLK1 is a hepatic progenitor-cell-surface marker, and GATA6 and HNF1B are transcription factors. Specifically, both AFP and DLK1 were strongly expressed in the hPSC-derived cells to a greater degree than in liver lysate, indicating that these cells did not reach the status mature adult liver. However, genes from the CYP family in hPSC-derived cells exhibited lower expression levels relative to those observed in liver lysate, suggesting that our method requires improvement in terms of CYP-family expression. All rights reserved. No reuse allowed without permission.
KRT7, KRT8, KRT18
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
KRT7, KRT8, KRT18
The copyright holder for this preprint . http://dx.doi.org/10.1101/232215 doi: bioRxiv preprint first posted online Dec. 13, 2017;
Matured hPSC-derived hepatocyte-like cells in the Liver-on-a-Chip
To facilitate maturation levels for establishment of the Liver-on-a-Chip platform, we developed a simple 3D-culture method using a microfluidic device. We hypothesized that 3D-culture conditions would provide a more suitable environment for hepatocytes as compared with conventional 2D-culture methods 27 toward mimicking 3D micro liver tissues (Fig. 6A) . Previously, we reported a microfluidic device enabling 3D culture of hPSCs 29, 30 (Fig. 6B) , which was utilized in the present study to perform 3D culturing of hepatocyte-like cells from day 12 to enhance the maturation process. The microfluidic device was made of polydimethylsiloxane (PDMS), which exhibits good biocompatibility and gas permeability, 47 and contained two microfluidic 3D cell-culture chambers [10 mm (L) × 1.5 mm (W) × 150 µm (H)] with a cell inlet (0.75-mm diameter) and a medium reservoir (3-mm diameter). To confirm the maturity of hPSC-derived hepatocyte-like cells at the protein level, we conducted immunocytochemistry for one of the maturation markers, 1 anti-trypsin (A1AT). Our results indicated that the A1AT hepatocyte-maturation marker was more strongly expressed in 3D-cultured hepatocyte-like cells derived from H9 hESCs over 33 days using our method as compared with levels observed in 2D-cultured cells ( Fig. 6C and Supplementary Fig. S3 ). Although undifferentiated H9 hESCs did not express hepatic genes, we also confirmed increased mRNA expression of other hepatocyte-marker genes, such as UGT1A1, CYP3A4, CYP2A9, CYC2C19, and CYP2D6, in the 3D-cultured H9 hESC-derived hepatocyte-like cells relative to levels observed in 2D-cultured cells ( Fig. 6D and Supplementary Table   S5 ). In contrast, HepG2 cells did not express UGT1A1, TDO1, MDR/TAP, CYP3A4, CYP2A9, CYP2C19, or CYP2D6. As expected, the liver extract from the healthy donor strongly expressed all tested genes at levels exceeding those of hepatocyte-like cells derived from H9 hESCs. These results indicated that the hepatocyte-like cells derived from H9 hESCs exhibited improved gene expression relative to a HepG2 cell line and 2D-cultured hepatocyte-like cells, but were unable to match levels observed in liver extracts from a healthy donor.
Matured hPSC-derived hepatocyte-like cells in the Liver-on-a-Chip
To investigate differences in functionality, an uptake/excretion assay for indocyanine green (ICG), which is a non-invasive marker of drug uptake/excretion in the liver, 45, 46 was performed on cells from both 3D-and 2D cultures ( Fig. 6E and Supplementary Fig. S4) . (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
Matured hPSC-derived hepatocyte-like cells in the Liver-on-a-Chip
The copyright holder for this preprint . http://dx.doi.org/10.1101/232215 doi: bioRxiv preprint first posted online Dec. 13, 2017; hiPSCs were provided by RIKEN BioResource Research Center (Ibaraki, Japan). H9 hESCs and 253G1 To induce endoderm differentiation, cultured hPSCs were washed with PBS and treated with TryPLE Express at 37°C for 5 min, followed by the addition of basal medium and transfer of the cell suspension into a 15-mL tube. Cells were centrifuged at 200 × g for 3 min and the supernatant was removed. Cells were resuspended in mTeSR-1 medium supplemented with 10 µM Y27632 and 100 ng mL −1 activin A, plated on a serum-coated culture dish, and cultured in a humidified incubator at 37°C with 5% CO 2 for 24 h. At the end of day 1, the medium was replaced with fresh mTeSR-1 medium supplemented with 10 µM Y27632 and 100 ng mL −1 activin A and cultured for another 24 h. On day 2, the medium was replaced
Matured hPSC-derived hepatocyte-like cells in the Liver-on-a-Chip
All rights reserved. No reuse allowed without permission.
Matured hPSC-derived hepatocyte-like cells in the Liver-on-a-Chip
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
Matured hPSC-derived hepatocyte-like cells in the Liver-on-a-Chip
The copyright holder for this preprint . http://dx.doi.org/10.1101/232215 doi: bioRxiv preprint first posted online Dec. 13, 2017; with mTeSR-1 medium supplemented with 10 µM Y27632, 100 ng mL −1 activin A, 10 ng mL −1 BMP-4, replaced with mTeSR-1 medium supplemented with 10 µM Y27632, 100 ng mL −1 activin A, 10 ng mL −1 BMP-4, and 10 µM LY294002, and cells were incubated for 24 h. On day 4, medium was replaced with Roswell Park Memorial Institute (RPMI) medium supplemented with B-27 (Life Technologies), 100 ng mL −1 activin A, and 100 ng mL −1 bFGF, and cells were incubated for 24 h.
Matured hPSC-derived hepatocyte-like cells in the Liver-on-a-Chip
To induce ADE specification, cells were treated with RPMI medium supplemented with 50 ng mL penicillin/streptomycin, and 1 mM nonessential amino acids, and the medium was changed every 2 to 3 days. The cells were passaged with trypsin-EDTA solutions at a 1:10 to 1:20 subculture ratio.
Matured hPSC-derived hepatocyte-like cells in the Liver-on-a-Chip
Flow cytometry. Cells were harvested with TrypLE Express and rinsed with PBS twice prior to cell counting. For staining with antibodies, cells were diluted to a final concentration of 1 × 10 7 cells mL −1 in simple and offer easy handling without requiring any special instruments. They can be applicable for increased throughput by means of straightforward device format design, such as the use of microplates, following the recommendations of the recommendation of the Society for Biomolecular Screening (SBS). Accordingly, we consider that our Liver-on-a-Chip platform will fulfill current requirements and serve as a useful tool in the field of drug discovery.
Conflicts of interest
Kyoto University ( All rights reserved. No reuse allowed without permission.
Conflicts of interest
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
Conflicts of interest
The copyright holder for this preprint . http://dx.doi.org/10.1101/232215 doi: bioRxiv preprint first posted online Dec. 13, 2017; 

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