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First published online February 21, 2008
Stem Cells Vol. 26 No. 5 May 2008, pp. 1117 -1127
doi:10.1634/stemcells.2007-1102; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS

Efficient Differentiation of Functional Hepatocytes from Human Embryonic Stem Cells

Sadhana Agarwal, Katherine L. Holton, Robert Lanza

Advanced Cell Technology, Worcester, Massachusetts, USA

Key Words. Human embryonic stem cells • Differentiation • Hepatic • Liver

Correspondence: Correspondence: Robert Lanza, M.D., Advanced Cell Technology, 381 Plantation Street, Worcester, Massachusetts 01605, USA. Telephone: 508-756-1212, ext. 655; Fax: 508-756-4468; e-mail: rlanza{at}advancedcell.com

Received on December 31, 2007; accepted for publication on February 15, 2008.

First published online in STEM CELLS EXPRESS  February 21, 2008.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Differentiation of human embryonic stem cells (hESCs) to specific functional cell types can be achieved using methods that mimic in vivo embryonic developmental programs. Current protocols for generating hepatocytes from hESCs are hampered by inefficient differentiation procedures that lead to low yields and large cellular heterogeneity. We report here a robust and highly efficient process for the generation of high-purity (70%) hepatocyte cultures from hESCs that parallels sequential hepatic development in vivo. Highly enriched populations of definitive endoderm were generated from hESCs and then induced to differentiate along the hepatic lineage by the sequential addition of inducing factors implicated in physiological hepatogenesis. The differentiation process was largely uniform, with cell cultures progressively expressing increasing numbers of hepatic lineage markers, including GATA4, HNF4{alpha}, {alpha}-fetoprotein, CD26, albumin, {alpha}-1-antitrypsin, Cyp7A1, and Cyp3A4. The hepatocytes exhibited functional hepatic characteristics, such as glycogen storage, indocyanine green uptake and release, and albumin secretion. In a mouse model of acute liver injury, the hESC-derived definitive endoderm differentiated into hepatocytes and repopulated the damaged liver. The methodology described here represents a significant step toward the efficient generation of hepatocytes for use in regenerative medicine and drug discovery.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Two hallmarks of human embryonic stem cells (hESCs)—their pluripotency and capacity for unlimited self renewal—suggest that they could serve as a potentially inexhaustible source of cells for replacement therapy [1, 2]. As with other tissues, there is a scarcity of donor livers and hepatocytes, which is compounded by the low recovery and proliferative capacity of adult primary hepatocytes [3–5]. In addition to their potential use for the treatment of liver disease, hESC-derived hepatocytes could be used to study the developmental biology of hepatogenesis [1, 6–8]. Large-scale differentiation of hESCs to hepatocytes could also provide a valuable model system for novel pharmaceutical drug discovery assays, as well as new drug metabolism and cytotoxicity screens, particularly since the liver is a major site for detoxification [9, 10].

Definitive endoderm (DE) is the embryonic germ layer that develops into the liver, lung, pancreas, thyroid, and intestines [11, 12]. During early development, the inner cell mass of the preimplantation blastocyst differentiates into the epiblast (which is the origin of the three germ layers: ectoderm, mesoderm and definitive endoderm) and the primitive endoderm (which forms the extraembryonic tissues, visceral endoderm and parietal endoderm, which do not contribute directly to the developing embryo proper [2, 13]). Definitive endoderm is formed during gastrulation when pluripotent epiblast cells migrate through the primitive streak of the embryo to form either mesoderm or DE, which then displaces the pre-existing primitive (visceral) endoderm [11, 12, 14]. Although not completely understood yet, a large body of research has led to the description of various aspects of the distinct sequential stages of hepatocyte development [11, 14–18]. This includes accounts of the extensive morphogenetic changes involved, as well as identification of some of the underlying inducing factors, signaling pathways, key transcription factors, and alterations in gene expression. At the earliest stages, studies establish the requirement for high levels of the transforming growth factor (TGF)-β family member Nodal in the specification of DE during gastrulation [15, 19, 20]. Postgastrulation, the embryo undergoes substantial invaginations and morphogenetic movements, and the DE is transformed from a sheet of cells to the primitive gut tube [11, 12]. The ventral domain of the foregut commits to a liver-cell fate, induced, in part, by fibroblast growth factor (FGF) signaling originating from the adjacent cardiac mesoderm and enhanced by bone morphogenetic protein (BMP) signaling from mesenchymal cells of the neighboring septum transversum [14, 17, 21–23]. The hepatic endoderm then proliferates and buds into the collagen-rich environment of the septum transversum, where hepatocyte growth factor (HGF) promotes hepatic growth [14, 24]. As hematopoietic cells migrate into the fetal liver bud, they produce additional factors, such as the interleukin-6 family cytokine oncostatin M (OSM), that, along with the glucocorticoid dexamethasone (Dex), have been implicated in the maturation of the hepatocytes [17, 25, 26]. Thus, the specification and subsequent maturation of hepatocytes from DE is governed by a series of interactions with other juxtaposed cell types in the developing embryo. Potentially, elucidation of these interactions can be used to develop culture conditions that would allow the derivation of hepatocytes from hESCs in vitro in a manner that mimics differentiation in vivo.

Several studies have examined the differentiation of mouse embryonic stem cells (mESCs) and hESCs to hepatic cells and have obtained various extents of hepatic induction ([27–29]; reviewed in [30]). The formation of DE from mESCs [31–34] and hESCs [35, 36] has been described, and the derived DE can be induced to produce hepatic cells [29, 34]. These various studies differ in the form of ESCs they start with, differentiation matrices, and induction schemes. For hESCs, the strategies range from starting with embryoid bodies (EBs) that are later plated on diverse matrices [27, 37–39], to differentiation on mouse embryonic fibroblast (MEF) feeder layers [29, 40], to differentiation of adherent feeder-free hESC cultures [28]. Several inducing factors, some associated with hepatic development and others used in the culture of primary hepatocytes, have been investigated [6, 30]. For hESCs, these include the nonspecific agents sodium butyrate [37] and dimethyl sulfoxide (DMSO) [28] and specific factors, some linked to hepatic development [27–29, 39]. These studies establish the feasibility of hepatic differentiation of hESCs and suggest induction strategies. However, current methods are inadequate for large-scale applications because of several impediments, including inefficient differentiation resulting in heterogeneous cultures containing many cell types and low proportions of hepatocytes [27–29, 37–40].

On the basis of mammalian hepatic development, we have established a well-defined, efficient method for generating hepatocytes from hESCs in vitro. Previous procedures [35] were adapted to facilitate the production of sizeable percentages of healthy DE cells. DE was then induced to differentiate using factors involved in early hepatic development, on a collagen I matrix in serum-free media. The differentiating cultures exhibited a sequential pattern of expression of hepatic genes, generating large proportions of hepatocytes that expressed late stage liver cell genes and exhibited functional hepatic characteristics. In a mouse transplantation model of liver injury, DE was shown to differentiate and repopulate the liver in vivo.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture
Experiments were carried out with the hESC lines WA09 (H9) and WA01 (H1)-green fluorescent protein (GFP) (a stable derivative of WA01 carrying the transgene for GFP). hESCs were cultured on mitomycin C-treated MEF feeder layers in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Mediatech, Manassas, VA, http://www.cellgro.com) containing 20% KnockOut Serum Replacement (KOSR; Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 4 ng/ml basic fibroblast growth factor (Invitrogen), 1 mM nonessential amino acids (Invitrogen), 2 mM GlutaMAX (Invitrogen), penicillin/streptomycin (Invitrogen), and 0.55 mM 2-mercaptoethanol (Invitrogen) [35]. The cells were passaged using 0.05% trypsin-0.53 mM EDTA (Invitrogen) at a ratio of 1:3 to 1:6 every 3–4 days [41]. HepG2 cells (American Type Culture Collection, Manassas, VA, http://www.atcc.org) were cultured per the manufacturer's instructions.

Differentiation of hESCs to Definitive Endoderm
Eighty percent confluent cultures were washed with phosphate buffered saline (PBS) with calcium and magnesium (Mediatech) and placed in RPMI medium (Mediatech supplemented with GlutaMAX and penicillin/streptomycin and containing 0.5% defined fetal bovine serum (FBS; HyClone) and 100 ng/ml Activin A (R&D Systems Inc., Minneapolis, http://www.rndsystems.com). Three days postinduction, the medium was refreshed using the same RPMI-based medium with 100 ng/ml Activin A but altered FBS concentration. We have used several alternatives (including 0.5% FBS, 2% FBS, and substitution with 2% KOSR) with comparable results. The latter is preferred to reduce serum use. Differentiation was continued for another 2 days.

Differentiation of Definitive Endoderm to Hepatocytes
Definitive endoderm cultures were passaged with 0.05% trypsin-0.53 mM EDTA and plated at a ratio of 1:1 on collagen I (Inamed, Fremont, CA, http://www.inamedbiomaterials.com)-coated dishes (5 µg/cm2), in RPMI medium supplemented with GlutaMAX and penicillin/streptomycin and containing 2% KOSR, 10 ng/ml FGF-4 (R&D Systems), and 10 ng/ml HGF (R&D Systems). Three days later the cells were switched to minimal MDBK-MM medium (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) supplemented with GlutaMAX and penicillin/streptomycin and containing 0.5 mg/ml bovine serum albumin (BSA) (Sigma-Aldrich), 10 ng/ml FGF-4, and 10 ng/ml HGF. After another 3 days, the cells were switched to complete hepatocyte culture medium (HCM) supplemented with SingleQuots (Lonza, Walkersville, MD, http://www.lonza.com) and containing 10 ng/ml FGF-4, 10 ng/ml HGF, 10 ng/ml oncostatin M (R&D Systems), and 10–7 M dexamethasone (Sigma-Aldrich). Differentiation was continued for another 9 days. At each stage, the medium was refreshed every 2 days.

Immunofluorescence
Cells were fixed with 4% paraformaldehyde (PFA) and blocked/permeabilized in PBST (with 0.2% Triton X-100 [Sigma-Aldrich]) containing 10% normal donkey serum (NDS; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) before primary antibody incubations in PBST/NDS overnight at 4°C. After washing in PBS, cells were incubated with biotinylated secondary antibodies (Jackson Immunoresearch Laboratories) for 45 minutes at room temperature. After further washing, Alexa-594-conjugated streptavidin (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) was added for 15 minutes at room temperature before extensive washing in PBS and mounting in Prolong Gold with 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes). The antibodies used were as follows: anti-Sox17, anti-FoxA2 (R&D Systems), anti-Oct4, anti-GATA4, anti-Sox7, anti-HNF4{alpha} (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), anti-{alpha}-fetoprotein (anti-AFP; Dako, Glostrup, Denmark, http://www.dako.com), anti-CD26 (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml), anti-albumin (Sigma-Aldrich), and anti-{alpha}-1-antitrypsin (anti-AAT; GeneTex, San Antonio, TX, http://www.genetex.com).

Live-Cell Suspension Immunostaining
Cells were dissociated using trypsin-EDTA. Cell pellets were resuspended in PBS containing 0.18% glucose/0.2% BSA and incubated with an anti-CXCR4 antibody (R&D Systems) for 1 hour at room temperature while rocking. After PBS washes, cells were fixed with 4% PFA, washed, and incubated with biotinylated secondary antibodies (in blocking buffer: 5.0% normal goat serum [Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com], 1% fraction V BSA [Sigma-Aldrich], and 0.2% Triton-X-100 in PBS) for 45 minutes at room temperature. After two additional washes, Alexa-594-conjugated streptavidin (in blocking buffer) was added for 15 minutes at room temperature before two final washes. Cell pellets were resuspended in ~50 µl of PBS and transferred to a slide, dried, and mounted in Prolong Gold with DAPI.

Immunoblotting
Trypsin-EDTA-dislodged cells were lysed in suspension with Protease Inhibitor Cocktail (Sigma-Aldrich)-supplemented RIPA buffer (Boston Bioproducts, Worcester, MA, http://www.bostonbioproducts.com). Protein concentrations were determined (BCA kit; Pierce, Rockford, IL, http://www.piercenet.com), and equal amounts of total protein were resolved by SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, http://www.bio-rad.com), transferred to nitrocellulose, probed with primary antibodies (same as immunofluorescence, except anti-Sox17 from Santa Cruz Biotechnology), and detected by chemiluminescence (Pierce).

Fluorescence-Activated Cell Sorting Analysis
Cells were processed as described [35] and analyzed by flow cytometry using ModFit software.

Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was isolated using RNeasy Mini kits (Qiagen, Hilden, Germany, http://www1.qiagen.com). Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the One Step RT-PCR kit (Qiagen). Primer sequences and annealing temperatures were as follows: AFP, 5'-TGCAGCCAAAGTGAAGAGGGAAGA-3' (forward) and 5'-CATAGCGAGCAGCCCAAAGAAGAA-3' (reverse), 60°C; Alb, 5'-TGCTTGAATGTGCTGATGACAGGG-3' (forward) and 5'-AAGGCAAGTCAGCAGGCATCT-CATC-3' (reverse), 60°C; AAT, 5'-CAGAGGAGGCACCCCTGAA-3' (forward) and 5'-AGTCCCTTTCTCGTCGATGGT-3' (reverse), 60°C; Cyp3a4, 5'-ATGAAAGAAAGTCGCCTCG-3' (forward) and 5'-TGGTGCCTTATTGGGTAA-3' (reverse), 55°C; Cyp7a1, 5'-GTGCCAATCCTCTTGAGTTCC-3' (forward) and 5'-ACTCGGTAGCAGAAAGAATACATC-3' (reverse), 55°C; and GAPDH, 5'-GTCCATGCCATCACTGCCA-3' (forward) and 5'-TTACTCCTTGGAGGCCAT-3' (reverse), 60°C.

Periodic Acid-Schiff Assay for Glycogen
Cells were fixed with 4% PFA and stained using a periodic acid-Schiff (PAS) staining system (Sigma-Aldrich). Cells were counterstained using Hematoxylin-QS (Vector Laboratories) and mounted with Vectamount AQ (Vector Laboratories).

Cellular Uptake and Release of Indocyanine Green
Indocyanine green (ICG) (Sigma-Aldrich) was suspended in DMSO (Sigma-Aldrich) for a stock at 5 mg/ml and freshly diluted in culture medium to 1 mg/ml. Cells were incubated in diluted ICG for 30 minutes at 37°C. After washing, cellular uptake of ICG was documented. Cells were returned to culture medium and incubated for 6 hours. Loss (release) of cellular ICG stain was examined.

Albumin Secretion Assay
Immuno dot blots for human-specific albumin present in cell-conditioned media were performed as described [27]. Briefly, conditioned medium (collected over 2 days from equivalent numbers of cells) was placed onto nitrocellulose membranes, along with increasing amounts of the standard, human serum albumin (Sigma-Aldrich), and unconditioned medium controls. After blocking, the membrane was probed with a human-specific primary antibody against albumin (Sigma-Aldrich), developed with chemiluminescence (Pierce), and analyzed using a Kodak Imaging Station (Kodak, Rochester, NY, http://www.kodak.com).

Mouse Model for Hepatic Repopulation
Ten-week-old male NOD-SCID mice (Jackson Immunoresearch Laboratories) were injected intraperitoneally with retrorsine (60 mg/kg of body weight; Sigma-Aldrich). Two weeks later, acute liver injury was caused by intraperitoneal injection of carbon tetrachloride (CCl4 [Sigma-Aldrich]; 0.5 ml/kg of a 1:10 dilution in olive oil). One day later, trypsin-EDTA-dislodged 1 x 106 DE cells in 0.1 ml of PBS (or PBS alone) were injected into the portal vein using a 32-gauge needle. Four weeks later, livers were harvested, (and snap frozen) embedded in Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA, http://www.sakura.com), until cryosectioning. Sections were fixed with 4% PFA and analyzed by immunohistochemistry using Vectastain Elite ABC horseradish peroxidase staining kits (with Nickel, Vector Laboratories) and an Avidin/Biotin blocking kit (with Nickel, Vector Laboratories) before development with a DAB substrate kit (Vector Laboratories), counterstaining with Hematoxylin-QS, and mounting with Vectamount AQ. Primary antibodies were as follows: anti-GFP (Santa Cruz Biotechnology), anti-human mitochondria (Chemicon, Temecula, CA, http://www.chemicon.com), human-specific anti-CD26 (BD Pharmingen), and human-specific anti-AAT (GeneTex).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Derivation and Characterization of hESC-Derived Definitive Endoderm
Based on the prerequisite for high levels of Nodal in the induction of endoderm during gastrulation, DE was derived from hESCs in low serum and high concentrations of Activin A (a more readily available TGF-β family member that binds the Nodal receptor and stimulates similar signaling pathways [31–33, 35]). Previous procedures [35] were modified to enhance the generation of DE from hESCs. Manual passaging of hESCs was replaced with using trypsin-EDTA, which greatly simplified the process and allowed for scaleable production of DE. Preliminary experiments indicated that efficient derivation from trypsin-passaged hESCs required cultures to be 80% confluent before differentiation. Differentiation was initiated in low serum (0.5% FBS) and high Activin A (100 ng/ml) in RPMI medium. After the 5-day differentiation process, the resultant cells were found to be very fragile and survived downstream handling poorly. Therefore, 3 days after induction, the medium was refreshed while maintaining the Activin A concentration. Various media were used for this step (including RPMI containing 0.5% FBS, 2% FBS, or, preferably, 2% KOSR, to reduce the use of serum) with similar outcomes. Five days postinduction, healthy DE was obtained.

Figures 1 and 2 depict the characterization of the DE generated. Morphologically, the cells gradually transformed from typical, defined, tight hESC colonies (at time 0 [T0]) into less dense, flatter cells containing prominent nuclei (at a time of 5 days [T5]; Fig. 1A). Notably, a majority of the cells appeared to change synchronously, leading to a mostly uniform culture. This was accompanied by characteristic changes in protein expression as analyzed by immunofluorescence (Fig. 1B). No unique expression marker specific for DE has been described yet; rather, genes expressed in DE are expressed in primitive endoderm or other embryonic lineages, particularly mesoderm [1, 30, 35]. The cells were therefore examined for the distinguishing pattern of expression of a panel of markers, including those that are expected to be expressed in DE and those that are not. Comparing hESCs at the beginning of the differentiation process (T0) to 5 days post-Activin A treatment (T5), most of the cells lost their expression of the pluripotent marker Oct4 [42] while concomitantly gaining strong expression of DE transcription factor markers Sox17 (not expressed in mesoderm [43]) and FoxA2 [44] (Fig. 1B). Induction of transcription factor GATA4, which proceeds to have critical regulatory roles in hepatic development [14, 18, 45], was also detected in most cells. Importantly, the cells did not gain any detectable expression of the transcription factor Sox7 (expressed in primitive [parietal and visceral] endoderm but not DE [43]) or AFP, a marker for primitive (visceral) endoderm at this stage [46].


Figure 1
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  		Figure 1. Derivation of definitive endoderm from hESCs. hESCs at the start of the induction process (T0) or 5 days post-Activin A treatment (T5) were examined by phase contrast microscopy (A) and immunofluorescence for expression of a defining panel of proteins (B), as indicated. Magnification, x200. Abbreviations: AFP, {alpha}-fetoprotein; hES, human embryonic stem; T, time.

 


Figure 2
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  		Figure 2. Characterization and quantitation of hESC-derived definitive endoderm. (A): Immunoblotting analysis of protein extracts of untreated hESCs (T0) or 5-day Activin A-treated cells (T5) for several marker proteins, as indicated on the left. The positions of molecular weight standards (in kD) are indicated on the right. (B): Live-cell suspension immunostain for cell surface expression of CXCR4 on Activin A-treated cells. Shown are CXCR4 fluorescence, DAPI (nuclei), and merged views of the same field. Magnification, x200. (C): Quantitative depiction of flow cytometric analysis for CXCR4 expression on untreated hES or derived DE for the hES lines WA09 or WA01-GFP, as indicated. Abbreviations: AFP, {alpha}-fetoprotein; DAPI, 4,6-diamidino-2-phenylindole; DE, definitive endoderm; GFP, green fluorescent protein; hES, human embryonic stem cells; kD, kilodaltons; T, time.

 
Immunofluorescence observations were corroborated by immunoblotting analysis (Fig. 2A). Comparing protein expression levels in hESCs (T0) to those in induced cells (T5) showed a clear loss of expression of Oct4 and unambiguous gain of Sox17, FoxA2, and Gata4 proteins, but no detection of AFP protein, in the induced cells. Activin A-treated cultures were also examined for expression of the cell surface chemokine receptor CXCR4 (expressed in definitive but not primitive endoderm [47]). In a live-cell suspension immunostain procedure, most of the differentiated cells were found to exhibit a cell surface expression pattern for CXCR4 (Fig. 2B). The derivation process was quantified by fluorescence-activated cell sorting (FACS) analysis of CXCR4 expression [33, 35] (Fig. 2C). CXCR4-expressing cells were highly enriched following the derivation process (an average of 72% of cell populations expressed CXCR4 for the hESC-line WA09). Although most of our experiments were performed with WA09, similar results were obtained for WA01-GFP, as depicted here for CXCR4 expression analysis by FACS (Fig. 2C). In additional experiments, early transient expression of the primitive streak and mesoderm marker Brachyury [35, 48] and induction of Goosecoid (expressed in definitive but not primitive endoderm [49]; data not shown) further confirmed the derived cells to be definitive endoderm. These data clearly establish the robust induction of DE from hESCs under the conditions described here.

Directed Differentiation of Definitive Endoderm to Hepatocytes
Following known facets of hepatocyte developmental biology, we investigated the sequential addition of inducing factors to hESC-derived DE, including Activin A, FGFs, BMPs, HGF, OSM, and Dex, using a collagen I extracellular matrix. To increase the efficiency and reduce the heterogeneity in the process of hepatocyte induction, various serum-free media were tested to maximize the proportion of hepatocyte induction obtained while minimizing the production of other cell types. Differentiation was initially evaluated by immunofluorescence analysis for the expression of AFP, a marker of earliest hepatic specification, and albumin (Alb), the activation of which correlates with further hepatic differentiation [34, 50–52]. Accordingly, we developed a novel, multistep protocol for generating hepatocytes, as illustrated in Figure 3A. Briefly, differentiation was commenced (T0) by treating hESCs with Activin A in 0.5% FBS containing RPMI medium. The medium was changed at T3 to Activin A in 2% KOSR containing RPMI medium. Confluent DE cultures obtained at T5 were dislodged with trypsin-EDTA and plated on collagen I-coated dishes in 2% KOSR/RPMI medium supplemented with the early inducing factors FGF-4 and HGF. Three days later (T8), the medium was changed to a minimal medium (MDBK-MM) supplemented with BSA, while FGF-4 and HGF concentrations were maintained (replacing the medium at this step greatly aided the maintenance of the homogeneity of the culture). After an additional 3 days of culture (T11), the cells were switched to complete HCM supplemented with oncostatin M and dexamethasone in addition to FGF-4 and HGF. The cells were allowed to mature in this cocktail until T20.


Figure 3
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  		Figure 3. Differentiation of hESCs to hepatocytes in vitro. (A): Schematic representation of the developed multistep differentiation procedure. (B): Phase contrast images of differentiating cells at 8 (T8), 11 (T11), 15 (T15), and 20 (T20) days post-initial Activin A treatment. Arrows indicate cytoplasmic vacuole formation. Magnification, x200. Abbreviations: BSA, bovine serum albumin; Dex, dexamethasone; FBS, fetal bovine serum; FGF, fibroblast growth factor; HCM, hepatocyte culture medium; hES, human embryonic stem; HGF, hepatocyte growth factor; KOSR, KnockOut Serum Replacement; MM, MDBK-MM; OSM, oncostatin M; T, time.

 
The progression of the cells was examined throughout the differentiation process, including at T8, T11, T15, and T20 (days post-initial Activin A treatment). After replating on collagen I, the DE reattached, growing into a confluent monolayer by 8 days postdifferentiation (T8; Fig. 3B). Morphologically, the cultures remained mostly homogeneous, aside from some cells appearing to be smaller and closer together (T11 and T15). As the cells matured (from T15 to T20), they developed cytoplasmic vacuoles, as previously reported for ESC-derived hepatocytes [28, 34] and resembling confluent cultures of polygonal, vacuolated primary human hepatocytes [28, 53].

Progression of Differentiating Cells Through Developmentally Regulated Hepatic Gene Induction
Immunofluorescence analysis showed that the differentiating DE exhibited a time-dependent, ordered pattern of hepatic and stage-specific gene expression (Fig. 4). GATA4 was induced in DE (T5; Fig. 1B) and continued to be expressed through the time course of hepatic differentiation, appearing more intense in the clusters of smaller, closer cells. HNF4{alpha}, a key transcription factor that regulates a cascade of liver-specific transcription [14, 18, 54], also displayed clear nuclear expression in all cells at the time points tested (T8–T20). Protein expression of AFP, an early marker for hepatic commitment [50] was, however, first detected 11 days postinduction (T11) and only in a subset of the cells. The levels of expression and the percentage of AFP-positive cells increased progressively to include most of the cell population by T20. Similarly, increasingly later-stage hepatic proteins, albumin [34, 50–52], the membrane-bound CD26 (Dipeptidylpeptidase IV) [55, 56] and the later-stage, primarily liver-specific enzyme AAT [57] were first detected in a fraction of the cell population at the T15 time point. The intensity and extent of their expression enhanced significantly to encompass a majority of the cells by T20. Quantitation of positive-scoring cells (as a percentage of total number of DAPI-positive nuclei in multiple fields at T20) showed that 82.7% ± 3.8% of the cells scored positive for AFP, 67.4% ± 0.35% of cells expressed Alb, and 84.7% ± 4.6% were AAT-positive at the end of the differentiation process. These observations indicate that this strategy promotes the induction of high proportions of late-stage marker-expressing hepatocytes from DE in a manner that is analogous to the sequence of physiological hepatic lineage commitment.


Figure 4
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  		Figure 4. Progression of the differentiating cells through a cascade of developmentally regulated hepatic gene induction in vitro. Immunofluorescence analysis of the expression of a panel of indicated stage-specific hepatic genes in differentiating cells at 8 (T8), 11 (T11), 15 (T15), and 20 (T20) days post-initial Activin A treatment. Magnification, x200. Abbreviations: AAT, {alpha}-1-antitrypsin; AFP, {alpha}-fetoprotein; Alb, albumin; T, time.

 
The expression profile of hepatic lineage genes through the time course of differentiation (T0–T20) was also analyzed at the RNA level by RT-PCR (Fig. 5). RNA from the hepatic cell line HepG2 was examined as a positive control. As expected, Afp transcripts were detectable at T11 and beyond. Using this more sensitive assay (compared with immunofluorescence), expression of Alb and AAT was also detected starting at T11 and maintained thereafter. The examination of their relative levels of expression at the time points post-T11 requires further, more quantitative analysis by real-time RT-PCR. Notably, expression of two members of the key cytochrome P450 superfamily of oxidative metabolism enzymes [58], Cyp7a1 [59] and Cyp3a4, was also found to be activated, but not until T20, when the generated hepatocytes were at their most mature—further demonstrating increasing hepatic maturation in the differentiation cultures with time.


Figure 5
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  		Figure 5. Time course of hepatic gene transcription in the differentiating cells. Reverse transcription-polymerase chain reaction (RT-PCR) expression analysis of total RNA prepared from cells at various time points through differentiation (T0–T20, in days post-Activin A treatment), using primers specific to various hepatic genes and to the ubiquitous housekeeping gene GAPDH, as indicated on the left (with the expected sizes of respective RT-PCR products). RNA from the hepatic cell line HepG2 was included as a positive control. The positions of molecular weight markers (in bp) are indicated on the right. Abbreviations: AAT, {alpha}-1-antitrypsin; AFP, {alpha}-fetoprotein; Alb, albumin; bp, base pairs; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; T, time.

 
hESC-Derived Hepatic Cells Exhibit Hepatocyte-Like Functions
To examine functionality, we tested the ability of the differentiating cell populations to store glycogen, a characteristic of functional hepatocytes [60]. Cells at T8, T11, T15, and T20 were stained for cytoplasmic glycogen using the PAS staining procedure (Fig. 6A). Nuclei were counterstained using hematoxylin (blue-violet). Cells at T8 showed virtually no glycogen staining, whereas T11 cells exhibited few pockets of staining (pink to dark red-purple cytoplasm). At T15, a dramatic increase in the cytoplasmic glycogen stain was observed in clusters of smaller, closer differentiating cells, and by T20, cytoplasms of the majority (~80%) of the cells stained pink to dark red-purple, indicating that they had the capacity to store glycogen, as do functional hepatocytes. Hence, consistent with the gain of expression of mature hepatic markers, the differentiating cells also exhibit a progressive gain of hepatic functionality.


Figure 6
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  		Figure 6. Human embryonic stem cell (hESC)-derived hepatic cells exhibit hepatocyte-like functions. (A): Glycogen storage. Periodic acid-Schiff assay was performed on differentiating cells at 8 (T8), 11 (T11), 15 (T15), and 20 (T20) days post-initial Activin A treatment. Nuclei were counterstained with hematoxylin (blue-violet). Glycogen storage is indicated by pink or dark red-purple cytoplasms. (B): Indocyanine green (ICG) uptake and release. hESCs (T0), cells at the end of the differentiation process (T20), and HepG2 cells (positive control) were examined for their ability to take up ICG (left column) and release it 6 hours thereafter (right column). Magnification, x200. (C): Albumin production. hESCs (T0) and cells at the end of the differentiation process (T20) were examined for their ability to secrete albumin, using a human-specific anti-albumin antibody and Immuno dot blot analysis. Abbreviations: hAlb, human albumin; T, time.

 
The vital liver cell function of excretion of diverse compounds from the circulation involves hepatocellular uptake, conjugation, and subsequent release of the compounds [61]. ICG is a nontoxic organic anion that is eliminated exclusively by mature hepatocytes and is used clinically to test hepatic function [62]. Uptake and release of ICG can be used to identify hepatocytes in ESC differentiation models [63]. To investigate this function in our induced cells, we compared the capacity of hESCs (T0) with that of cells at the end of the differentiation process (T20) to take up and release ICG. The function of HepG2 cells was assessed as a positive control. As evident in Figure 6B, hESCs (T0) did not take up any ICG. In contrast, the differentiated cells (T20), displayed a pronounced capacity to take up ICG and release it 6 hours later. Approximately 50% of the HepG2 cells also took up ICG and eliminated it similarly. Thus, hepatic cells generated from hESCs using our induction procedures demonstrate the capacity of mature hepatocytes for uptake and excretion of ICG-like compounds.

We also examined the capacity of the hESC-derived hepatic cells to secrete albumin, a critical hepatocyte function. The levels of human-specific albumin, secreted into the culture medium by hESCs (T0) or by cells at the end of the differentiation process (T20), were examined using an immuno dot blot assay. As shown in Figure 6C, significant levels of human albumin were detected in media conditioned with the differentiated cells (T20). These results are consistent with the expression of albumin at the RNA (Fig. 5) and protein (Fig. 4) levels.

Differentiation of hESC-Derived Definitive Endoderm to Hepatocytes In Vivo
To determine whether hESC-derived DE were capable of giving rise to hepatocytes in vivo, we used a mouse transplantation model for hepatic repopulation following carbon tetrachloride (CCl4)-induced liver injury. Studies on liver repopulation with cell transplants have established that reconstitution requires the liver to be in a state of injury and the transplanted cells to have a selective advantage over host hepatocytes [64, 65]. Immunosuppressed NOD-SCID mice were treated with retrorsine to inhibit endogenous hepatocyte proliferation [66] and subjected to acute liver injury with CCl4 [67]. One day post-CCl4 administration, DE cells derived from GFP-positive hESCs (WA01-GFP) were injected into the portal vein. Control mice were injected with buffer. Four weeks later, livers were harvested, and sections were examined by immunohistochemistry with antibodies to the following donor cell-specific markers: GFP, human-mitochondrial antigen, human-specific liver enzyme CD26, and human-specific liver enzyme AAT (Fig. 7). In contrast to livers of sham-injected mice, livers of DE-injected mice showed the presence of cells that expressed GFP, human-specific mitochondrial antigen, and human liver enzymes CD26 and AAT. The occurrence of positive signals was sporadic and low, but the presence of human cells was clearly detected in the livers of all the mice that received DE injections. These data indicate that hESC-derived DE cells were able to integrate into the adult liver and differentiate into liver cells in vivo, just as they do in vitro.


Figure 7
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  		Figure 7. Differentiation and integration of human ESC-derived definitive endoderm cells in a mouse model for hepatic repopulation. Immunohistochemistry analysis of sections of livers of sham-injected (left column) or WA01-GFP-derived DE-injected (right column) mice for the presence of donor cell-specific markers GFP, huMito, huCD26, and huAAT. Positive signals were detected with a DAB-substrate kit (which stains black/brown). Tissue sections were counterstained with hematoxylin (which stains blue-violet). Magnification, x400. Abbreviations: DE, definitive endoderm; GFP, green fluorescent protein; huAAT, human-specific {alpha}-1-antitrypsin; huCD26, human-specific CD26; huMito, human mitochondria.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
We describe a multistep protocol for the efficient derivation of hepatocytes from hESCs, through a process that resembles the progressive specification of definitive endoderm and hepatocytes during mammalian development. The use of this method yields largely synchronous populations of hepatocytes that are generated in clinically preferred conditions, with minimum use of serum and cell feeder layers.

In a recapitulation of the initial differentiation of pluripotent epiblast cells to DE, pluripotent hESCs were first induced to generate definitive endoderm [35]. Since they share an overlapping pattern of gene expression, it was imperative to discriminate between the formation of primitive versus definitive endoderm [1, 30]. The cells we obtained were Sox17+, FoxA2+, Gata4+, CXCR4+, Gsc+, AFP–, and Sox7– (Figs. 1, 2). The lack of expression of Sox7 and AFP (expressed in primitive but not definitive endoderm), in addition to the induction of CXCR4, goosecoid, and, transiently, brachyury (expressed in definitive but not primitive endoderm), ruled out the possibility of generation of primitive endoderm in our conditions. After the DE cells were dissociated and replated on a collagen I matrix, the cells maintained their remarkable uniformity (Fig. 3). The sequential application of suitable combinations of hepatic development-based inducers, in appropriate medium formulations and stages, resulted in the visibly coordinated differentiation of the cells that exhibited a temporal regulation of hepatic stage-specific gene expression (Figs. 4, 5) and acquired increasing hepatic characteristics over time (Figs. 4Go–6). The majority (~70%) of the cells at end of the differentiation protocol remained substantially uniform with little cellular heterogeneity, resembled hepatocytes morphologically, expressed a repertoire of genes expressed in mature hepatocytes (GATA4, HNF4{alpha}, AFP, CD26, Alb, AAT, Cyp3A4, and Cyp7A1), and displayed hepatocyte-like functions. The expression of the cytochrome P450 enzymes was particularly significant since Cyp7A1 has been shown to be adult liver-specific and not expressed in other organs or extraembryonic tissues [59]. Furthermore, Cyp3A4 is the most abundant CYP protein in the human liver and is involved in the metabolism of a large percentage of current pharmaceutical drugs [68]. The prevalence of drug-metabolizing enzymes in hESC-derived hepatocytes is critical to their application both in cell replacement and in drug metabolism and hepatotoxicity screens in vitro, as previously reported for glutathione transferases [40] and the cytochrome P450 enzymes [27–29, 37, 69].

Although the hESC-derived hepatocytes described here exhibit characteristics of mature hepatocytes (gene expression/protein profile and hepatic functions), it should be noted that they also appear to retain some immature characteristics, such as relatively low levels of expression of the cytochrome P450 transcripts and continued expression of AFP, a marker of fetal rather than adult hepatocytes. Interestingly, previous ESC studies report the production of hepatocytes with similar features, in particular with continued expression of AFP (for example in mESC-derived hepatocytes [34] and hESC differentiation [27–29]). Further investigation is required to examine whether hepatocytes derived in vitro from hESCs can be matured further in culture (with additional key inducing factors and/or more time) or whether the final maturing steps require an in vivo environment.

The notable persistence of uniformity in our cultures suggests the coordinated stimulation of signaling pathways in the responding cells, as they transitioned through successive differentiation stages. This can, at least in part, be attributed to the uniform exposure of all cells in monolayer cultures to inducing factors and the minimum use of serum (which can be variable and contains unidentified factors). Importantly, when synchronized definitive endoderm cell cultures were replated on collagen I, concordant differentiation was sustained. The choice of serum-free medium used at the different stages proved to be critical for this outcome. In particular, a shift to the minimalist MDBK-MM medium, at an early stage of hepatic specification, helped suppress the production of other cell types (unpublished observations). Subsequently, the shift to the hepatocyte-growth supporting HCM was important for the maintenance of healthy cells through the maturation steps.

The selection of inducing factors used was principally directed by their known roles in hepatic development. Empirically, however, it was more effective to sometimes diverge from expected sequences. This could reflect the dissimilarities between the environment in vivo versus in vitro. One noticeable example was the lack of requirement for BMP4 addition, which only served to increase the heterogeneity in the cultures (unpublished observations). BMP4 was reported to be essential for the generation of hepatocytes from mESCs [34] and the disparity with our observations could reflect species variations, the substantial technical differences in the EB-based approach of that report, or the compensation for added BMP4 in our system by other included factors, such as HGF. Interestingly, addition of HGF restored growth in mutant mouse liver explants (impaired in the BMP4 superfamily, TGF-β signaling), suggesting that HGF may provide parallel signals to TGF-β-induced pathways [70]. The system of hepatocyte differentiation established here, with its progression through distinct stages of lineage commitment, could provide a powerful tool to dissect such molecular pathways involved at each stage.

When examined in a transplantation model for hepatic repopulation, the hESC derived DE cells were found to differentiate to hepatocytes both in vivo and in vitro. Human liver cells were clearly detectable in the recipient animals (Fig. 7), indicating successful, albeit low levels (<1%) of integration. However, the low efficiency observed may reflect the fact that the DE cells were not transplanted at the optimal developmental stage. Future studies will be necessary to determine the most suitable stage(s) in the differentiation procedure for the successful transplantation and integration of the cells into the livers.


   SUMMARY
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
Disclosure of Potential...
 Acknowledgments
 References
 
We have developed a high-yielding procedure for the generation of hepatocytes from hES cells in vitro, which proceeds in a stepwise fashion and mirrors events of hepatogenesis in vivo. Large-scale production of hepatocytes using this method should greatly bolster their applications in basic research, clinical medicine, and preclinical drug discovery.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
Acknowledgments
 References
 
R.L., S.A., and K.L.H. own stock in Advanced Cell Technology. R.L. has served as an officer or member of the Board for Advanced Cell Technology.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Lucy Vilner and Jennifer Shepard for cell culture, Tong Lin and Tonya Russell for help with the animal experiments, and Sandy Becker and Irina Klimanskaya for valuable advice.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 

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