A simple and complete system to generate mature hepatocytes from various iPS cell sources
- Use a standardized protocol with ready-to-use media and supplements to create panels of iPSC-derived hepatocytes
- Generate >90% pure hepatocytes that express mature hepatic markers and show relevant CYP activities
- Start with any patient-specific or disease-relevant iPS cell line
Human hepatocytes are highly attractive candidates for cell-based therapy for chronic liver disease, and are essential to many studies in areas such as disease modeling, hepatitis research, and alcoholic fatty liver disease. However, the use of human hepatocytes is limited by their lack of availability and large inter-individual variations (1). These challenges also affect drug development, where there is a lack of robust and predictive cellular models during preclinical development and safety and toxicology evaluation. This in turn results in an enormous failure rate for newly developed drugs and substantial loss of both time and money for the pharmaceutical industry.
One way to circumvent the dearth of primary human hepatocytes is to generate them from induced pluripotent stem (iPS) cells. This method offers the unique possibility of an unlimited source of hepatocytes from various human donors. However, current hepatocyte differentiation protocols exhibit varying differentiation efficiencies across diverse iPS cell lines derived from multiple donor sources (2). Moreover, in order to be useful, iPS-derived hepatocytes should recapitulate the diversity of metabolic phenotypes observed in human populations (3), express drug metabolizing enzymes at equivalent levels to primary human hepatocytes, and maintain stable functionality in vitro (4). The Cellartis iPS to Hepatocyte Differentiation System addresses all these challenges and allows for the simple generation of mature hepatocytes in vitro.
A simple and universal system for differentiation of human iPS cells into hepatocytes
The Cellartis iPS to Hepatocyte Differentiation System is a complete system that includes all media, supplements, and a protocol to differentiate any iPS cell line into hepatocytes (5). The protocol mimics liver development in vivo: iPS cells are first differentiated into definitive endoderm (DE) cells and are then further differentiated into mature hepatocytes. Differentiated mature hepatocytes are ready for your research applications at differentiation day 21, and have an experimental time window of at least 11 days.
iPS Cell to Hepatocyte Differentiation System Overview. The complete system includes media, supplements, and a protocol to differentiate any iPS cell line into hepatocytes.
A homogeneous culture of highly pluripotent iPS cells is an essential starting point for efficient and reproducible hepatocyte differentiation. We have developed an optimized iPS cell culture system, the Cellartis DEF-CS Culture System, which is included in the Cellartis iPS to Hepatocyte Differentiation System. DEF-CS enables monolayer culture of iPS cells that are highly pluripotent and exhibit low spontaneous differentiation and stable karyotypes, thereby providing iPS cells that are highly amenable to downstream differentiation (5).
Culture of undifferentiated iPS cells in the Cellartis DEF-CS Culture System. Panel A. DEF-CS facilitates the expansion of iPS cell line ChiPSC4 in a non-colony, 2D monolayer. Panel B. In iPS cell line ChiPSC18, the karyotype remains stable through 20 passages when cultured in DEF-CS. Panel C. iPS cells cultured in DEF-CS highly express pluripotency markers OCT4 (green, top panel) and SSEA-4 (green, bottom panel). Nuclei are stained with DAPI (blue). Panel D. Multiple iPS cell lines exhibit robust cell growth and a uniform doubling time in DEF-CS. Data adapted from Asplund et al. (5).
Using a protocol that combines media, supplements, and coating reagents optimized for 2D monolayer culture, the pluripotent stem cells are then differentiated into definitive endoderm, which is an essential embryonic mid-stage for all endodermal cell types (6). This process takes 7 days, during which the pluripotency transcription factor OCT4 is downregulated and several markers of DE cells (SOX17, FOXA2, CXCR4, and HHEX) are upregulated (5). DE cells are then passaged, re-plated, and prompted to become hepatocytes. Functional hepatocytes are available for experimentation within three weeks.
Derivation of homogenous definitive endoderm cells from hiPS cell lines. Panel A. RT-qPCR analysis indicates down-regulation of the expression of stem cell markers OCT4 (A1) and NANOG (A2) in DE cells. In contrast, mRNA expression of DE markers CXCR4 (A3) and SOX17 (A4) is strongly up-regulated in DE cells compared to undifferentiated hiPS cells. Low expression of the extraembryonic marker SOX7 (A5) indicates minimal occurrence of extraembryonic endoderm cells. Panel B. Immunocytochemical staining for the DE marker SOX17 (B2, B5) and the stem cell marker OCT4 (B3, B6) reveal minimal presence of OCT4 immuno-positive nuclei and a majority of SOX17 immuno-positive nuclei in DE cells derived from hiPS cell lines ChiPSC6b and P11025. Nuclei are stained with DAPI (B1, B4).
To evaluate the robustness and applicability of our protocol, we tested hepatic differentiation from 20 different iPS cell lines. Hepatocytes generated using this system morphologically resemble primary hepatocytes in that they are polygonal cells, bi-nucleated, and have distinct cell borders. We also tested for expression of mature hepatocyte markers and loss of the OCT4 transcription factor. Hepatocytes derived from all 20 iPS cell sources were immunopositive for the transcription factor HNFα, which is a master transcription factor responsible for hepatocyte fate specification. The vast majority, 93.8% ± 0.7 (mean ± SEM), of DAPI-stained hepatocyte nuclei also stained positive for HNFα, and no OCT4-positive cells were observed, indicating the absence of any residual pluripotent cells (5).
Hepatocytes derived from multiple iPS cell donors exhibit expected morphology. Panel A. 20 different iPS cell lines representing 19 different donors are successfully differentiated using the iPS Cell to Hepatocyte Differentiation System. Representative images of 16 lines, taken 21–25 days after the start of differentiation, show that the iPS cell-derived hepatocytes morphologically resemble primary hepatocytes and exhibit consistent differentiation patterns. Panel B. At differentiation day 28, iPS cell-derived hepatocytes are immunostained with HNF4α and nuclei are stained with DAPI. On average, 93.8% of cells are HNF4α-positive, as indicated by the horizontal line.
We also tested for expression of fetal and mature hepatocyte markers during differentiation into hepatocytes using RT-qPCR. Interestingly, the levels for the fetal hepatic marker AFP are highest at day 21 and gradually decrease as the hepatocytes mature. The opposite is true for the adult hepatocyte markers albumin, PXR, CYP1A2, CYP2C9 and CYP3A4, which are gradually upregulated in the maturing hepatocytes. CYP1A2, 2C9 and 3A4 are important drug metabolizing enzymes detected in adult liver tissue but not in fetal liver tissue. Taken together, these data indicate a homogeneous population of mature hepatocytes derived from multiple iPS cell lines.
iPS cell-derived hepatocytes reflect maturing hepatocyte mRNA expression profiles. mRNA expression of several hepatocyte markers was analyzed between day 11 and 42 after the start of differentiation using RT-qPCR. mRNA levels were normalized to the reference gene CREBBP. The levels for the fetal hepatocyte marker AFP are highest at day 21 and gradually decrease as the hepatocytes mature, while the opposite is true for the adult hepatocyte markers albumin, PXR, CYP1A2, CYP2C9 and CYP3A4, which are gradually upregulated in the maturing hepatocytes.
Drug metabolism is a central hepatocyte function. A critical metric for terminal hepatocyte differentiation is the expression and activity of drug metabolizing enzymes in the cytochrome P450 (CYP) family. Therefore, in addition to RT-qPCR analysis of the enzymes CYP1A2, CYP2C9 and CYP3A4 shown above, we measured the activities of key CYP enzymes by LC/MS. We detected CYP1A, CYP3A, CYP2C9 and CYP2C19 activity at levels comparable to human primary hepatocytes cultured for in total 20 hr post-thawing (cryo hpep data, below). Interestingly, we observed variation in CYP activities (Phase I metabolism) between hepatocytes derived from different iPS cell lines or from different primary hepatocytes. We then determined if the foregoing variation was due to inter-experimental variation or differences in the donor source. Hepatocytes were repeatedly derived from ChiPSC18 (n=4), and CYP activity assays were performed on the different batches. We observed very small batch-to-batch variation in CYP activity (ChiPSC18 data, below). These data strongly indicate that the variability in CYP activity between cell lines was not due to an artifact of the differentiation protocol, but instead are representative of genuine phenotypic variations in iPS cell-derived hepatocytes from various human sources.
CYP activity of human iPS cell-derived hepatocytes recapitulates the inter-individual variation of the human population. CYP activity was measured by LC/MS and normalized to the protein content per well in iPS cell-derived hepatocytes (29 days after the start of differentiation). Activities were comparable with cryopreserved human hepatocytes (cryo hphep) from four different donors. Hepatocytes derived from five different hiPS cell lines show diverse CYP activity profiles, reflecting the metabolic diversity found in human primary hepatocytes from different donors. For example, CYP2C19 activity is low in ChiPSC18, but high in ChiPSC6b, reflecting naturally occurring inter-individual variation.
The robust and reproducible generation of hepatocytes from iPS cells has enormous implications for basic and clinical research, drug development, and cell-based therapy. The Cellartis iPS to Hepatocyte Differentiation System is a complete system with a universal differentiation protocol to make hepatocytes from various iPS cell lines. It includes the DEF-CS media for maintenance of iPS cells in monolayer culture in order to generate optimal starting material with low spontaneous differentiation and high pluripotency for subsequent differentiation into DE cells and hepatocytes. Our system allows for directed differentiation of 20 different iPS cells resulting in cells with a clear hepatocyte morphology with no residual pluripotent cells. The iPS-derived hepatocytes express markers typical of mature hepatocytes such as HNFα, albumin, PXR, and Phase I metabolizing enzymes. Finally, the iPS cell-derived hepatocytes faithfully recapitulate the metabolic diversity found in the human population.
The Cellartis iPS to Hepatocyte Differentiation System has powerful utility for a breadth of applications from basic liver biology research, disease modeling, hepatitis studies, and pre-clinical work to cell-based therapy for chronic liver diseases. Importantly, hepatocytes made using this system reflect the metabolic diversity inherent to human cells. Therefore, the Cellartis iPS to Hepatocyte Differentiation System will allow a researcher to make panels of iPS cell-derived human hepatocytes to interrogate potential patient-specific phenotypes. Safety and toxicology labs interested in developing in-house hepatocyte generation methods would also be interested in such cellular panels. In all, the Cellartis iPS to Hepatocyte Differentiation System greatly simplifies the generation of hepatocytes from various iPS cells, providing any customer a complete solution for their research.
Differentiation of iPS cells into hepatocytes
The differentiation protocol is as published by Asplund et al., with some modification to the maturation stage (5). Briefly, iPS cells are cultured in DEF-CS at a defined cell density in day 0 cell culture medium. Medium changes over the next seven days result in homogenous DE cells that are then enzymatically dissociated and re-plated for further hepatocyte differentiation. The hepatocyte differentiation media guides the DE cells through the same developmental stages that occur during liver development in vivo: ventral foregut, hepatoblast, fetal-like hepatocyte and mature hepatocyte. This process takes another two weeks. At the end of this process, hepatocyte maintenance media provided allows for maintenance of function for at least another 11 days.
Cells were stained with primary anti-HNF4α antibody (Santa Cruz Biotechnology) followed by donkey anti-rabbit secondary antibody and counter stained with DAPI (5). Representative HNFα pictures were merged with DAPI, and the number of HNFα-DAPI double positive nuclei was evaluated.
Analyses were performed using OCT4 (Hs01654807_s1), NANOG-1 (Hs02387400_g1), SOX17(Hs00751752_s1), CXCR4(Hs00237052_m1), SOX7(Hs00846731_s1), CREBBP (Hs00231733_m1), AFP (Hs00173490_m1), Albumin (Hs00910225_m1), CYP1A2 (Hs01070374_m1), CYP2C9 (Hs004260376_m1), and CYP3A4 (Hs00604506_m1) TaqMan probes (Applied Biosystems). Expression levels were calculated using the Î”Î”Ct method, normalized to CREBBP expression.
CYP Activity Assay
Cells were washed twice with warm WME without phenol red (Thermo Fisher). 26 µM phenacetin (CYP1A), 50 µM mephenytoin (CYP2C19), 9 µM diclofenac (CYP2C9) and 3 µM midazolam (CYP3A) were added to the plates in warm WME supplemented with 0.1% PEST, 25 mM HEPES, and 2 mM L-glutamine. After 2h, 100 µl of supernatant was collected and stored at -80C until LC/MS analysis of paracetamol, hydroxy-mephenytoin, hydroxy-diclofenac, and hydroxy-midazolam was performed. Protein amount per well was quantified by the Pierce BCA Protein Assay Kit (Thermo Scientific). Metabolite concentrations were normalized to protein amount per well and assay duration.
- Behbahan et al. (2011). New approaches in the differentiation of human embryonic stem cells and induced pluripotent stem cells toward hepatocytes. Stem Cell Rev 7(3): 748–759.
- Kajiwara et al. (2012). Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells. PNAS 109(31): 12538–12543.
- Li A. (2008). Human hepatocytes as an effective alternative experimental system for the evaluation of human drug properties: general concepts and assay procedures. Altex 25(1): 33–42.
- Sartipy et al. (2011). Concise review: Human pluripotent stem cell-based models for cardiac and hepatic toxicity assessment. Stem Cells 29(5): 744–748.
- Laydon, D.J., Bangham, C.R. & Asquith, B. (2015) Estimating T-cell repertoire diversity: limitations of classical estimators and a new approach. Phil. Trans. R. Soc. B. 370(1675):20140291.
- Asplund et al. (2015). One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells. Stem Cell Rev and Reports. 12(1): 90–104
- Zaret et al. (2008) Generation and Regeneration of Cells of the Liver and Pancreas. Science. 322(5907): 1490–1494.