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Hepatic differentiation of porcine induced pluripotent stem cells in vitro
The Veterinary Journal 194 (2012) 369–374
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Hepatic differentiation of porcine induced pluripotent stem cells in vitro Rajagopal N. Aravalli a,⇑, Erik N.K. Cressman a, Clifford J. Steer b,c a
Department of Radiology, University of Minnesota Medical School, 420 Delaware Street SE, Minneapolis, MN 55455, USA Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455, USA c Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA b
a r t i c l e
i n f o
Article history: Accepted 19 May 2012
Keywords: Hepatocyte iPS Differentiation Porcine Stem cell Liver
a b s t r a c t Porcine hepatocytes are potentially important in liver regeneration and in the treatment of humans with acute and chronic liver diseases. Induced pluripotent stem (iPS) cells are a valuable source of hepatocytes for these applications as they have unlimited potential to propagate in vitro. An efficient and robust differentiation of iPS cells generated from porcine fetal fibroblasts into functional hepatocyte-like cells in vitro is reported. The methodology followed a three-step differentiation protocol using several growth factors, namely, activin A, basic fibroblast growth factor, bone morphogenetic protein-4, and oncostatin M. Porcine iPS cell-derived hepatocyte-like (piPS-Hep) cells were characterized by morphological analysis and were tested for the expression of hepatocyte-specific genes using RT-PCR. Functional analyses for albumin production and glycogen storage were also carried out. These differentiated hepatocyte-like cells could represent a valuable source for studies of drug metabolism and for cell transplantation therapy for a variety of liver disorders. Ó 2012 Elsevier Ltd. All rights reserved.
Introduction Liver failure is associated with high morbidity and mortality in humans and liver transplantation is the only viable treatment option for hepatic failure (Lee, 1993). At present, because of the scarcity of donor livers for transplantation, efforts are being made to develop alternate strategies, such as artificial liver support systems (Sen and Williams, 2003). Hepatocyte transplantation is also currently being studied to bridge the gap to transplantation until an organ becomes available (Fitzpatrick et al., 2009; Zhang et al., 2010). However, xenotransplantation from pigs could eliminate the shortage of livers. In addition, studies are being conducted on drug metabolism in hepatic cells in order to develop novel therapeutics for liver diseases, and more generally in predicting pharmacokinetics of new drug candidates (Langsch et al., 2009). The pig is the animal model for these efforts, since it is close to humans in terms of anatomy, physiology and size of the liver (Nieuwoudt et al., 2006; Hara et al., 2010; Caperna et al., 2011; Fondevila et al., 2011). Liver therapy using porcine hepatocytes is gaining significant attention because hepatocytes can be isolated in large numbers and can be propagated in cell culture and bioreactors (De Bartolo and Bader, 2001; Maringka et al., 2010). However, due to high costs and concerns about health and safety issues, attempts are being made to obtain functional hepatocytes from other sources such as stem cells (Zhang et al., 2010). Stem cells are pluripotent in nature ⇑ Corresponding author. Tel.: +1 612 626 5550. E-mail address: [email protected] (R.N. Aravalli). 1090-0233/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2012.05.013
and can be differentiated into hepatocyte-like cells and other liver cell types. Nevertheless, a number of hepatic stem/progenitor cell types isolated from different animal species have proven to be difficult not only to isolate in large numbers, but also to maintain in culture for prolonged periods of time (Aravalli et al., 2010). The discovery of induced pluripotent stem (iPS) cells (Takahashi and Yamanaka, 2006) and their subsequent generation using many mammalian somatic cell types have made iPS cells an attractive source of hepatocytes for liver transplantation and for metabolic studies (Asgari et al., 2010). To date, four reports have been published on the generation of porcine iPS cells by expressing reprogramming factors in porcine fibroblasts using retroviral vectors (Esteban et al., 2009), lentiviral vectors (Ezashi et al., 2009; Wu et al., 2009), and a polycistronic plasmid (Montserrat et al., 2011). However, none of these iPS cells have been differentiated into hepatic cells. In this study, we generated porcine iPS cells using fetal fibroblasts by expressing three human reprogramming factors, namely, sex determining region Y-box 2 (Sox2), Krüppellike factor 4 (Klf4) and octamer-binding transcription factor 4 (Oct4), from a non-integrating episomal vector. Here, we report, for the first time, the differentiation of porcine iPS cells into functional hepatocyte-like cells in vitro. Materials and methods Fibroblast cultures Porcine fetal fibroblasts (PFFs) isolated from fetuses of Yucatan pregnant sows at 25–40 days post-fertilization were kindly provided by Dr. Scott Fahrenkrug (University of Minnesota). Fetal viscera and heads were removed prior to mincing
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of tissue with scalpel and mechanical disruption. Minced tissue was then added to a 50 mL tube containing 5 volumes of growth medium to tissue mass (DMEM enriched with 10% fetal bovine serum [FBS] and 2 antibiotic/antimycotic solution). Type I Collagenase (Worthington Biochemical) reconstituted at 2000 U/mL in phosphate-buffered saline (PBS) was added at a final concentration of 200 U/mL prior to 2–4 h of incubation at 37 °C with periodic mechanical disruption. Digested material was allowed to settle for 5 min and cells were pelleted by centrifugation. Cells were then re-suspended in growth medium and plated onto several plates at densities to achieve 100% confluence in 1–2 days. PFFs thus obtained were cultured continuously in DMEM medium (Invitrogen) with 10% FBS and penicillin/streptomycin. Generation of iPS cells PFFs were seeded at a density of 2 105/cm2 in 6-well plates and were transfected with 3 lg of episomal vector pEP4EO2SET2K (Yu et al., 2009) purchased from Addgene using the Fugene 6 transfection reagent (Roche). This vector contains open reading frames for three human reprogramming factors (Oct4, Sox2 and Klf4) under the control of a strong elongation factor alpha promoter. Following transfection, cells were grown in mouse embryonic stem cell medium (DMEM, 15% FBS, 1 mM glutamine, 0.1 mM minimal non-essential amino acids, penicillin–streptomycin, 55 lM 2-mercaptoethanol, and no leukemia inhibitory factor). The medium was changed every 2 days. Subsequently, iPS cells were maintained in Knockout-DMEM medium (Invitrogen) with 20% knockout serum (Invitrogen) and ampicillin/streptomycin in gelatin-coated plates, and on feeder layers of Gibco mouse embryonic fibroblasts (MEFs) that were inactivated by gamma irradiation (Invitrogen). Alkaline phosphatase staining Alkaline phosphatase (AP) specifically stains undifferentiated iPS cells and is commonly used to visualize these cells in culture. In contrast, the feeder layer of differentiated cells does not stain. StemTAG AP kit (Cell Biolabs) was used for staining according to the manufacturer’s recommendations. Differentiation of porcine iPS cells Following the disassociation with trypsin and collagenase IV, porcine iPS cells (piPSCs) were placed in suspension culture in 12-well HydroCell plates (Thermo Fisher Scientific) at the density of 1 106 per well in mouse ES medium. They were then aggregated into embryoid bodies and were plated on gelatin-coated plates in RPMI medium (Invitrogen) with B27 supplements (Sigma) on an MEF STO cell line (kindly provided by Dr. Neil Talbot, USDA) that was mitotically inactivated either by gamma irradiation or by mitomycin C (10 lg/mL). For hepatic differentiation, the human iPS differentiation method was used with few modifications (Si-Tayeb et al., 2010). Briefly, cells were grown as embryoid bodies. Two days after plating on STO feeders, growth medium was replaced with RPMI medium containing B27 supplements and activin A (100 ng/mL) to induce the formation of endoderm. Cells were grown for 5 days at 37 °C with 5% CO2, and the medium was replaced with RPMI/B27 containing 10 ng/mL fibroblast growth factor-2 (FGF-2), 10 ng/mL bone morphogenetic protein-4 (BMP-4) and 20 ng/mL hepatocyte growth factor (HGF). Cultures were grown for 5 days and the medium was replaced with RPMI/B27 containing oncostatin M. After 5 days in this medium, hepatocyte-like cells were clearly visible in plates. They were collected with trypsin and were subjected to biochemical and morphological analyses. In subsequent experiments, piPSC-derived hepatocyte-like cells (piPSC-Hep) were cultured in plates coated with matrigel (BD Biosciences) under feeder-free conditions and propagated in STO-conditioned medium. RT-PCR analysis Total RNA was isolated from cells using the Trizol reagent (Invitrogen) following the manufacturer’s recommendations. Reverse transcription was carried out using 2 lg of total RNA with Superscript III Reverse Transcription Kit (Invitrogen) in a total volume of 20 lL. Resulting cDNA was diluted 10 times in RNAse- and DNAse-free water, and 1 lL of it was used per reaction to perform PCR using porcine-specific primers as described previously (Caperna et al., 2011). Primer sequences for b-actin were: forward: 50 -CCGTGAGAAGATGACCCAGATCATGT-30 ; reverse: 50 CGTGATCTCCTTCTGCATCCTGTC-30 ; and GAPDH sequences were: forward: 50 -ACTCACGGCAAATTCAACGGC-30 ; reverse: 50 -ATCACAAACATGGGGGCATCG-30 . PCR was performed as follows: initial denaturation at 94 °C for 3 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing for 1 min at 60 °C, and elongation for 1.5 min at 72 °C. Immunohistochemistry piPSC-Hep cells were tested for production of albumin by immunohistochemistry using porcine-specific anti-albumin antibody (Abcam; ab79960). Cells were fixed in 4% paraformaldehyde for 10 min on cover plates. They were permeabilized with 0.1% Triton X-100, washed three times with PBS and stained overnight with
albumin antibody (dilution, 1:1000). Cells were washed again with PBS and albumin was detected with Dylight 549 secondary antibody (Jackson ImmunoResearch Laboratories). Cells were visualized using a Nikon DXM1200C microscope. Periodic acid Schiff assay Glycogen storage by differentiated piPSC-Heps was evaluated using the periodic acid Schiff (PAS) assay. Cells were fixed on cover slips with 4% paraformaldehyde for 10 min and were stained with PAS assay reagent (Sigma–Aldrich) according to the manufacturer’s recommendations. Cytochrome P450 ethoxyresorufin-O-deethylase (EROD) activity assay piPSC-Hep cultures were cultured in 96-well plates and were incubated with 5 lM 3-methylcholanthrene (3-MC, Sigma–Aldrich) in DMEM medium for 48 h to induce CYP1A1 activity. Cells were then exposed to 8 lM 7-ethoxyresorufin (Santa Cruz Biotechnology) for 30 min, as described previously (Donato et al., 1993). The medium was harvested and the concentration of the fluorescent product, resorufin, was assayed in the presence and absence of b-glucuronidase/arylsulfatase (Sigma– Aldrich) to determine the extent of possible conjugation reactions. Activity is presented as pmol resorufin product formed per 30 min/mg cell protein in cultures treated with and without 3-MC.
Results Generation of induced pluripotent stem cells To generate iPS cells, 1 106 PFF cells (passage 1) were transfected with the episomal vector using the Fugene 6 reagent, and plated in 6-well plates with mouse ES medium (Fig. 1A). The transfection efficiency was determined with a dsRed expression vector (Fig. 1B) and was found to be in the range of 10–12%. After propagating cells for 7–10 days, small iPS cell colonies developed (Fig. 1C), which displayed morphological characteristics of piPSC colonies consistent with previous reports (Esteban et al., 2009; Ezashi et al., 2009; Wu et al., 2009). In total, we generated six piPSC colonies. These colonies were isolated by mechanical disruption and were transferred onto mitomycin-treated MEF feeder layers (Fig. 1B). After 7 days of growth on feeder cells, secondary colonies were formed. These colonies stained positively for AP (Fig. 1D), thereby, demonstrating that piPSCs were successfully generated from PFF cells. Characterization of porcine iPS cells RT-PCR was used to determine whether the piPSCs express the reprogramming factors. PCR was performed with human primers for three reprogramming genes as well as with porcine-specific primers for other known reprogramming factors (Nanog and Lin28). As shown in Fig. 1E, piPSCs expressed all these factors demonstrating that they were indeed stem-like cells. Differentiation of iPS cells into hepatocyte-like cells To determine whether the porcine iPSCs are capable of differentiating into hepatocyte-like cells, we subjected them to a differentiating protocol based on the human iPSC differentiation method with some modifications (Si-Tayeb et al., 2010). A three-step protocol was used in this study to induce hepatic differentiation of piPSCs (Fig. 2). It involved the use of activin A to induce the formation of definitive endoderm, followed by the propagation of cells in the medium with FGF-2, BMP-4 and HGF to differentiate cells into hepatic lineage. In the final step, cells were grown in the presence of oncostatin M to induce maturation of the immature hepatocytelike cells. In this protocol, piPSCs were grown on STO feeder layers and were successfully differentiated into hepatocyte-like cells. In the first step, they slowly began to migrate and differentiate in RPMI with activin A (Fig. 2A).
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A
B
C
D
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Fig. 1. Generation of porcine inducible pluripotent stem (iPS) cells. Pig fibroblasts (A) were transfected with pEP4EO2SET2K, which contains the open reading frames of three human reprogramming factors (Klf4, Oct4 and Sox2) using the Fugene 6 reagent. pCAGGS-DsRed construct expressing the Ds-Red ORF was used as a control to determine transfection efficiency (B). About 5–7 days after the initial transfection, small iPS colonies began to develop (C). These colonies tested positive for alkaline phosphatase (D). After growing the colonies for 3 more days, they were mechanically dispersed and total RNA was isolated for RT-PCR using primers specific for all three human reprogramming factors, as well as for two porcine genes, Nanog and Lin28 (E). Porcine GAPDH was used as a positive control. As shown here, all these genes were expressed by iPS cells but not by the porcine fetal fibroblast (PFF) cells.
the presence of BMP-4, FGF-2 and HGF, and matured in medium with oncostatin M by displaying typical hepatocyte-like morphology (Fig. 2D and E). DAPI staining of these porcine iPSC-derived cells (piPSC-Hep) revealed that a large number of the cells were binucleated (Fig. 3B), a distinct feature of mature hepatocytes that strongly supported successful differentiation of the piPSCs. Functional characterization of iPS cell-derived hepatocytes
Fig. 2. Schematic representation of the porcine induced pluripotent stem cells (piPSC) differentiation protocol. piPSC colonies that developed on MEF feeders were transferred to matrigel-coated plates and cultured in RPMI medium until they grew between 50 and 100 lm in diameter. They were then grown in the presence of activin A to initiate differentiation of the definitive endoderm. After 5 days in this medium, cells were grown for an additional 5 days in medium containing BMP-4, FGF2, and HGF. This step allows for the proliferation of cells that have committed to the hepatic lineage. Finally, they were grown for 5 days in the presence of oncostatin M to induce their maturation into hepatocyte-like cells (piPSC-Hep). Binucleated cells are indicated by arrows (C).
After 4–5 days in culture, they transformed into a large vacuolelike structure (Fig. 2B). The cells became binucleated (Fig. 2C) in
To determine functionality, the hepatocyte-like cells were evaluated for expression of albumin by immunocytochemistry. Albumin production is another unique feature of hepatocytes. By using a porcine albumin-specific antibody, we showed that our piPSC-Heps produced albumin (Fig. 3C). RT-PCR was also performed to study the expression of hepatocyte-specific markers with porcine primers for albumin, a-fetoprotein (AFP), c-Met, hepatocyte nuclear factors 1A and 4 (HNF1A and HNF4), cytokeratins 8 and 18 (CK8 and CK18), transferrin, and transthyretin (TTR). As shown in Fig. 4 (left panel), piPSC-Heps expressed all these hepatocyte markers. A PAS assay was carried out to further study the hepatic function of differentiated iPSC-Heps, since mature hepatocytes synthesize and store glycogen. Most of the differentiated cells tested positive for glycogen storage (Fig. 4, right panel). piPSC-Heps were then assayed for inducible cytochrome P450IA1 (CYPIA1) activity following 3-MC treatment by measuring EROD activity, i.e., conversion of 7-ethoxyresorufin (7-ER) to the fluorescent product resorufin. piPSC-Heps converted 7-ER to resorufin at rates of nearly 250 pmol/mg cell protein per 30 min, suggesting that piPSC-Heps express functional CYPIA1 protein (Fig. 5). Most notably, EROD activity was negligible in the piPSC-Heps that were not induced by exposure to 3-MC (control). The human liver cancer cell line HuH-7 was used as a positive control for this study. Collectively, these findings demonstrated that our piPSC-Heps possess hepatocyte-specific functions. Discussion The discovery that somatic cells can be converted into pluripotent stem cells by expressing four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) has revolutionized biomedical research (Takahashi and Yamanaka, 2006). Since then, other groups have successfully generated iPS cells using these and other reprogramming
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Resorufin (pmol/mg protein/30 min)
Fig. 3. Albumin production in porcine induced pluripotent stem hepatocyte-like cells (piPSC-Heps). Expression of albumin is a hallmark of hepatocytes. To test whether piPSC-Heps express albumin, they were grown on gelatin-coated plates (A) and were fixed with 4% paraformaldehyde. Many cells in culture were binucleated, as seen with DAPI staining (B and inset). They were then stained with porcine antibody against albumin and were visualized using a Dylight 549 fluorescent secondary antibody (C). Differentiated piPSC-Heps expressed albumin and stained positively. Merged image (D). Scale bar, 50 lm.
300 250 200 150 100 50 0 Control
Fig. 4. Functional characterization of porcine induced pluripotent stem hepatocytelike cells (piPSC-Heps). RT-PCR (left panel). piPSC-Heps were collected on day 15 following the differentiation procedure, and total RNA was isolated from them (Lane 2). RT-PCR was performed using primers specific for several hepatocyte markers: albumin, alpha fetoprotein (AFP), transthyretin (TTR), hepatocyte nuclear factors 1A and 4 (HNF1A, HNF4A), cytokeratins 8 and 18 (CK8, CK18), c-Met, and transferrin; house-keeping gene, b-actin. piPSC-Heps expressed all these markers in contrast to porcine induced pluripotent stem cells (piPSCs). Total RNA from piPSCs was used as negative control (Lane 1), while total RNA from primary porcine hepatocytes was used as positive control (Lane 3). Glycogen storage in piPSC-Heps (right panel). piPSC-Heps (A) were tested for their ability to store glycogen using a periodic acid Schiff (PAS) assay. piPSC-Heps stained positive for glycogen (B and inset). Original magnification, 10; inset, 60; scale bar, 50 lm.
factors (Wu et al., 2011). These findings have opened a new avenue to obtain hepatocytes for cell transplantation and for studying liver diseases.
piPSC-Hep
HuH-7
Fig. 5. Cytochrome P450IA1 ethoxyresorufin-O-deethylase (EROD) activity in porcine induced pluripotent stem hepatocyte-like cells (piPSC-Heps). Resorufin production was measured after exposing piPSC-Heps to 5 lM 3-methylchloranthrine (3-MC) for 48 h. Control, porcine induced pluripotent stem cells (piPSCs) without addition of 3MC and b-glucoridinase/arylsulfatase; piPSC-Hep (negative control); piPSC cells with 3-MC and b-glucoridinase/arylsulfatase; HuH-7, human liver cancer cell line with 3-MC and b-glucoridinase/arylsulfatase (positive control). Values represent the mean ± SEM (n = 6). P < 0.05 as determined by ANOVA.
The ability of iPS cells to differentiate into most mammalian cell types represents an attractive source of hepatocytes for the study and treatment of liver diseases. iPS cells can proliferate indefinitely under appropriate culture conditions in vitro and can be differentiated into hepatocytes for a prolonged period time. However, the major challenge remains the generation of iPS cells using methods that involve minimal modification to the genome of the
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primary somatic cells. To date, four reports have been published on the generation of porcine iPS cells by expressing various reprogramming factors with retroviral vectors (Esteban et al., 2009), lentiviral vectors (Ezashi et al., 2009; Wu et al., 2009), and a single plasmid (Montserrat et al., 2011). However, none of these iPS cells were differentiated into any liver-specific cell type(s). In all cases, integration of transgene sequences into the host chromosome did occur, making them undesirable for liver studies. In the present study, we used three ‘Yamanaka factors’ (Sox2, Klf4, and Oct4) to generate iPS cells with PFFs, but omitted the fourth factor c-Myc based on the report that it is not required to produce iPS cells (Nakagawa et al., 2008). Furthermore, c-Myc expression during the generation of iPS cells renders them tumorigenic (Okita et al., 2007). We used a single non-integrating episomal vector (Yu et al., 2009) to express the reprogramming factors so these iPS cells were free from vector and transgene sequences and are ideal candidates to generate hepatocyte-like cells to study liver-specific functions. To our knowledge, this is the first report to describe hepatic differentiation of piPSCs. We have demonstrated robust differentiation of piPSCs into hepatocyte-like cells. To achieve our objective, we adopted a three step differentiation protocol: (1) growth of iPS cells for 5 days in the presence of activin A to induce the differentiation of definitive endoderm; (2) growth in medium supplemented with BMP-4, FGF and HGF for an additional 5 days to obtain hepatocyte-like cells; and (3) growth in medium for 5 days containing oncostatin M. After 15 days, >90% of the cells in the culture medium displayed hepatocyte-specific features such as the production of albumin, glycogen storage, and a large number of bi-nucleated cells. Several laboratories have reported the differentiation of rodent and human iPSCs into hepatocyte-like cells using different methods by employing various cytokines (Duan et al., 2007; Song et al., 2009; Li et al., 2010; Gai et al., 2010; Si-Tayeb et al., 2010; Sullivan et al., 2010; Iwamuro et al., 2010; Sancho-Bru et al., 2011). However, these protocols are lengthy (15–20 days) and need to be fine-tuned to obtain hepatocyte-like cells in sufficient numbers for autologous cell therapy. In order to achieve optimal differentiation efficiency, it is necessary to use the appropriate cell type as a feeder layer. Feeder layers secrete growth factors and cytokines that will influence stem cells. Our efforts to differentiate piPSCs on an MEF feeder layer resulted in a very low level of hepatic differentiation, and it never reached more than 50% efficiency (data not shown). However, when MEFs were replaced with mouse STO fibroblast feeder cells (Martin and Evans, 1975), differentiation efficiency improved dramatically, and over 90% efficiency was achieved. Furthermore, piPSC-Heps grew rapidly when they were replenished with STO-conditioned medium. Earlier studies have reported that the STO feeder cells produce an extracellular matrix containing collagen, laminin, and proteoglycans (Horák and Fléchon, 1998; Talbot et al., 2002; Caperna et al., 2011). In addition to the three-dimensional extracellular matrix environment, STO cells provide interaction with cell-bound and soluble proteins that may support hepatocyte differentiation (Caperna et al., 2011). These cells have been shown to secrete a number of growth factors that promote hepatocyte morphogenesis and function, including insulin-like growth factor (IGF)-binding proteins (Caperna et al., 2011), stem cell factor (Pesce et al., 1997), HGF (Montesano et al., 1991), leukemia inhibitory factor (LIF) (Rathjen et al., 1990), and BMP-4 (Qi et al., 2004). Thus, it is likely that these cells play a critical role in the differentiation of piPSCs into hepatocyte-like cells. We have shown that piPSC-derived hepatocyte-like cells express albumin, AFP, c-Met, HNF-1a, HNF-4A, TTR, CK8, CK18, transferrin, and CYP1A1 and can store glycogen. Hengstler et al. (2005) have described specific characteristics that define a ‘hepatocyte-like cell’
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that include both quantitative analysis of enzymatic activities as well as qualitative analyses such as RT-PCR and immunohistochemistry. As shown here, piPSC-Heps displayed both qualitative and quantitative traits of hepatocytes. Furthermore, morphological analysis clearly demonstrated that a large number of differentiated cells were binucleated, a hallmark of hepatocytes, and formed structures both on STO feeders as well as under feeder-free conditions. Lack of porcine-specific antibodies and reagents currently preclude further studies such as those on phase II metabolism. However, the analysis conducted in this study clearly demonstrated that piPSC-Heps possess hepatocyte-specific functions. Conclusions piPSC-Heps displayed qualitative, morphologic and quantitative traits of hepatocytes and possessed hepatocyte-specific functions. The porcine hepatocyte-like cells generated from transgene-free piPSCs may be ideal candidates to study drug metabolism and may have potential for cell therapy of liver diseases. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgments We thank Dr. Scott Fahrenkrug for porcine fetal fibroblasts, Dr. Neil Talbot for STO-feeder cells and primary porcine hepatocytes, and Phillip Wong for assistance with the RT-PCR studies. This work was supported by the start-up funds from the Department of Radiology to R.N.A. References Aravalli, R.N., Sahin, M.B., Cressman, E.N., Steer, C.J., 2010. Establishment and characterization of a unique 1 lm diameter liver-derived progenitor cell line. Biochemical and Biophysical Research Communications 391, 56–62. Asgari, S., Pournasr, B., Salekdeh, G.H., Ghodsizadeh, A., Ott, M., Baharvand, H., 2010. Induced pluripotent stem cells: A new era for hepatology. Journal of Hepatology 53, 738–751. Caperna, T.J., Blomberg, L.A., Garrett, W.M., Talbot, N.C., 2011. Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media. In Vitro Cell and Developmental Biology Animal 47, 218–233. De Bartolo, L., Bader, A., 2001. Review of a flat membrane bioreactor as a bioartificial liver. Annals of Transplantation 6, 40–46. Donato, M.T., Gomez-Lechon, M.J., Castell, J.V., 1993. A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Analytical Biochemistry 213, 29–33. Duan, Y., Catana, A., Meng, Y., Yamamoto, N., He, S., Gupta, S., Gambhir, S.S., Zern, M.A., 2007. Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo. Stem Cells 25, 3058–3068. Esteban, M.A., Xu, J., Yang, J., Peng, M., Qin, D., Li, W., Jiang, Z., Chen, J., Deng, K., Zhong, M., Cai, J., Lai, L., Pei, D., 2009. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. Journal of Biological Chemistry 284, 17634–17640. Ezashi, T., Telugu, B.P., Alexenko, A.P., Sachdev, S., Sinha, S., Roberts, R.M., 2009. Derivation of induced pluripotent stem cells from pig somatic cells. Proceedings of the National Academy of Sciences of the USA 106, 10993–10998. Fondevila, C., Hessheimer, A.J., Flores, E., Vendrell, M., Muñoz, J., Escobar, B., Calatayud, D., Taurá, P., Fuster, J., García-Valdecasas, J.C., 2011. Step-by-step guide for a simplified model of porcine orthotopic liver transplant. Journal of Surgical Research 167, e39–e45. Fitzpatrick, E., Mitry, R.R., Dhawan, A., 2009. Human hepatocyte transplantation: State of the art. Journal of Internal Medicine 266, 339–357. Gai, H., Nguyen, D.M., Moon, Y.J., Aguila, J.R., Fink, L.M., Ward, D.C., Ma, Y., 2010. Generation of murine hepatic lineage cells from induced pluripotent stem cells. Differentiation 79, 171–181. Hara, H., Campanile, N., Tai, H.C., Long, C., Ekser, B., Yeh, P., Welchons, D., Ezzelarab, M., Ayares, D., Cooper, D.K., 2010. An in vitro model of pig liver xenotransplantation – Pig complement is associated with reduced lysis of wild-type and genetically modified pig cells. Xenotransplantation 17, 370–378.
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Hepatic differentiation of porcine induced pluripotent stem cells in vitro Rajagopal N. Aravalli a,⇑, Erik N.K. Cressman a, Clifford J. Steer b,c a
Department of Radiology, University of Minnesota Medical School, 420 Delaware Street SE, Minneapolis, MN 55455, USA Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455, USA c Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA b
a r t i c l e
i n f o
Article history: Accepted 19 May 2012
Keywords: Hepatocyte iPS Differentiation Porcine Stem cell Liver
a b s t r a c t Porcine hepatocytes are potentially important in liver regeneration and in the treatment of humans with acute and chronic liver diseases. Induced pluripotent stem (iPS) cells are a valuable source of hepatocytes for these applications as they have unlimited potential to propagate in vitro. An efficient and robust differentiation of iPS cells generated from porcine fetal fibroblasts into functional hepatocyte-like cells in vitro is reported. The methodology followed a three-step differentiation protocol using several growth factors, namely, activin A, basic fibroblast growth factor, bone morphogenetic protein-4, and oncostatin M. Porcine iPS cell-derived hepatocyte-like (piPS-Hep) cells were characterized by morphological analysis and were tested for the expression of hepatocyte-specific genes using RT-PCR. Functional analyses for albumin production and glycogen storage were also carried out. These differentiated hepatocyte-like cells could represent a valuable source for studies of drug metabolism and for cell transplantation therapy for a variety of liver disorders. Ó 2012 Elsevier Ltd. All rights reserved.
Introduction Liver failure is associated with high morbidity and mortality in humans and liver transplantation is the only viable treatment option for hepatic failure (Lee, 1993). At present, because of the scarcity of donor livers for transplantation, efforts are being made to develop alternate strategies, such as artificial liver support systems (Sen and Williams, 2003). Hepatocyte transplantation is also currently being studied to bridge the gap to transplantation until an organ becomes available (Fitzpatrick et al., 2009; Zhang et al., 2010). However, xenotransplantation from pigs could eliminate the shortage of livers. In addition, studies are being conducted on drug metabolism in hepatic cells in order to develop novel therapeutics for liver diseases, and more generally in predicting pharmacokinetics of new drug candidates (Langsch et al., 2009). The pig is the animal model for these efforts, since it is close to humans in terms of anatomy, physiology and size of the liver (Nieuwoudt et al., 2006; Hara et al., 2010; Caperna et al., 2011; Fondevila et al., 2011). Liver therapy using porcine hepatocytes is gaining significant attention because hepatocytes can be isolated in large numbers and can be propagated in cell culture and bioreactors (De Bartolo and Bader, 2001; Maringka et al., 2010). However, due to high costs and concerns about health and safety issues, attempts are being made to obtain functional hepatocytes from other sources such as stem cells (Zhang et al., 2010). Stem cells are pluripotent in nature ⇑ Corresponding author. Tel.: +1 612 626 5550. E-mail address: [email protected] (R.N. Aravalli). 1090-0233/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2012.05.013
and can be differentiated into hepatocyte-like cells and other liver cell types. Nevertheless, a number of hepatic stem/progenitor cell types isolated from different animal species have proven to be difficult not only to isolate in large numbers, but also to maintain in culture for prolonged periods of time (Aravalli et al., 2010). The discovery of induced pluripotent stem (iPS) cells (Takahashi and Yamanaka, 2006) and their subsequent generation using many mammalian somatic cell types have made iPS cells an attractive source of hepatocytes for liver transplantation and for metabolic studies (Asgari et al., 2010). To date, four reports have been published on the generation of porcine iPS cells by expressing reprogramming factors in porcine fibroblasts using retroviral vectors (Esteban et al., 2009), lentiviral vectors (Ezashi et al., 2009; Wu et al., 2009), and a polycistronic plasmid (Montserrat et al., 2011). However, none of these iPS cells have been differentiated into hepatic cells. In this study, we generated porcine iPS cells using fetal fibroblasts by expressing three human reprogramming factors, namely, sex determining region Y-box 2 (Sox2), Krüppellike factor 4 (Klf4) and octamer-binding transcription factor 4 (Oct4), from a non-integrating episomal vector. Here, we report, for the first time, the differentiation of porcine iPS cells into functional hepatocyte-like cells in vitro. Materials and methods Fibroblast cultures Porcine fetal fibroblasts (PFFs) isolated from fetuses of Yucatan pregnant sows at 25–40 days post-fertilization were kindly provided by Dr. Scott Fahrenkrug (University of Minnesota). Fetal viscera and heads were removed prior to mincing
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of tissue with scalpel and mechanical disruption. Minced tissue was then added to a 50 mL tube containing 5 volumes of growth medium to tissue mass (DMEM enriched with 10% fetal bovine serum [FBS] and 2 antibiotic/antimycotic solution). Type I Collagenase (Worthington Biochemical) reconstituted at 2000 U/mL in phosphate-buffered saline (PBS) was added at a final concentration of 200 U/mL prior to 2–4 h of incubation at 37 °C with periodic mechanical disruption. Digested material was allowed to settle for 5 min and cells were pelleted by centrifugation. Cells were then re-suspended in growth medium and plated onto several plates at densities to achieve 100% confluence in 1–2 days. PFFs thus obtained were cultured continuously in DMEM medium (Invitrogen) with 10% FBS and penicillin/streptomycin. Generation of iPS cells PFFs were seeded at a density of 2 105/cm2 in 6-well plates and were transfected with 3 lg of episomal vector pEP4EO2SET2K (Yu et al., 2009) purchased from Addgene using the Fugene 6 transfection reagent (Roche). This vector contains open reading frames for three human reprogramming factors (Oct4, Sox2 and Klf4) under the control of a strong elongation factor alpha promoter. Following transfection, cells were grown in mouse embryonic stem cell medium (DMEM, 15% FBS, 1 mM glutamine, 0.1 mM minimal non-essential amino acids, penicillin–streptomycin, 55 lM 2-mercaptoethanol, and no leukemia inhibitory factor). The medium was changed every 2 days. Subsequently, iPS cells were maintained in Knockout-DMEM medium (Invitrogen) with 20% knockout serum (Invitrogen) and ampicillin/streptomycin in gelatin-coated plates, and on feeder layers of Gibco mouse embryonic fibroblasts (MEFs) that were inactivated by gamma irradiation (Invitrogen). Alkaline phosphatase staining Alkaline phosphatase (AP) specifically stains undifferentiated iPS cells and is commonly used to visualize these cells in culture. In contrast, the feeder layer of differentiated cells does not stain. StemTAG AP kit (Cell Biolabs) was used for staining according to the manufacturer’s recommendations. Differentiation of porcine iPS cells Following the disassociation with trypsin and collagenase IV, porcine iPS cells (piPSCs) were placed in suspension culture in 12-well HydroCell plates (Thermo Fisher Scientific) at the density of 1 106 per well in mouse ES medium. They were then aggregated into embryoid bodies and were plated on gelatin-coated plates in RPMI medium (Invitrogen) with B27 supplements (Sigma) on an MEF STO cell line (kindly provided by Dr. Neil Talbot, USDA) that was mitotically inactivated either by gamma irradiation or by mitomycin C (10 lg/mL). For hepatic differentiation, the human iPS differentiation method was used with few modifications (Si-Tayeb et al., 2010). Briefly, cells were grown as embryoid bodies. Two days after plating on STO feeders, growth medium was replaced with RPMI medium containing B27 supplements and activin A (100 ng/mL) to induce the formation of endoderm. Cells were grown for 5 days at 37 °C with 5% CO2, and the medium was replaced with RPMI/B27 containing 10 ng/mL fibroblast growth factor-2 (FGF-2), 10 ng/mL bone morphogenetic protein-4 (BMP-4) and 20 ng/mL hepatocyte growth factor (HGF). Cultures were grown for 5 days and the medium was replaced with RPMI/B27 containing oncostatin M. After 5 days in this medium, hepatocyte-like cells were clearly visible in plates. They were collected with trypsin and were subjected to biochemical and morphological analyses. In subsequent experiments, piPSC-derived hepatocyte-like cells (piPSC-Hep) were cultured in plates coated with matrigel (BD Biosciences) under feeder-free conditions and propagated in STO-conditioned medium. RT-PCR analysis Total RNA was isolated from cells using the Trizol reagent (Invitrogen) following the manufacturer’s recommendations. Reverse transcription was carried out using 2 lg of total RNA with Superscript III Reverse Transcription Kit (Invitrogen) in a total volume of 20 lL. Resulting cDNA was diluted 10 times in RNAse- and DNAse-free water, and 1 lL of it was used per reaction to perform PCR using porcine-specific primers as described previously (Caperna et al., 2011). Primer sequences for b-actin were: forward: 50 -CCGTGAGAAGATGACCCAGATCATGT-30 ; reverse: 50 CGTGATCTCCTTCTGCATCCTGTC-30 ; and GAPDH sequences were: forward: 50 -ACTCACGGCAAATTCAACGGC-30 ; reverse: 50 -ATCACAAACATGGGGGCATCG-30 . PCR was performed as follows: initial denaturation at 94 °C for 3 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing for 1 min at 60 °C, and elongation for 1.5 min at 72 °C. Immunohistochemistry piPSC-Hep cells were tested for production of albumin by immunohistochemistry using porcine-specific anti-albumin antibody (Abcam; ab79960). Cells were fixed in 4% paraformaldehyde for 10 min on cover plates. They were permeabilized with 0.1% Triton X-100, washed three times with PBS and stained overnight with
albumin antibody (dilution, 1:1000). Cells were washed again with PBS and albumin was detected with Dylight 549 secondary antibody (Jackson ImmunoResearch Laboratories). Cells were visualized using a Nikon DXM1200C microscope. Periodic acid Schiff assay Glycogen storage by differentiated piPSC-Heps was evaluated using the periodic acid Schiff (PAS) assay. Cells were fixed on cover slips with 4% paraformaldehyde for 10 min and were stained with PAS assay reagent (Sigma–Aldrich) according to the manufacturer’s recommendations. Cytochrome P450 ethoxyresorufin-O-deethylase (EROD) activity assay piPSC-Hep cultures were cultured in 96-well plates and were incubated with 5 lM 3-methylcholanthrene (3-MC, Sigma–Aldrich) in DMEM medium for 48 h to induce CYP1A1 activity. Cells were then exposed to 8 lM 7-ethoxyresorufin (Santa Cruz Biotechnology) for 30 min, as described previously (Donato et al., 1993). The medium was harvested and the concentration of the fluorescent product, resorufin, was assayed in the presence and absence of b-glucuronidase/arylsulfatase (Sigma– Aldrich) to determine the extent of possible conjugation reactions. Activity is presented as pmol resorufin product formed per 30 min/mg cell protein in cultures treated with and without 3-MC.
Results Generation of induced pluripotent stem cells To generate iPS cells, 1 106 PFF cells (passage 1) were transfected with the episomal vector using the Fugene 6 reagent, and plated in 6-well plates with mouse ES medium (Fig. 1A). The transfection efficiency was determined with a dsRed expression vector (Fig. 1B) and was found to be in the range of 10–12%. After propagating cells for 7–10 days, small iPS cell colonies developed (Fig. 1C), which displayed morphological characteristics of piPSC colonies consistent with previous reports (Esteban et al., 2009; Ezashi et al., 2009; Wu et al., 2009). In total, we generated six piPSC colonies. These colonies were isolated by mechanical disruption and were transferred onto mitomycin-treated MEF feeder layers (Fig. 1B). After 7 days of growth on feeder cells, secondary colonies were formed. These colonies stained positively for AP (Fig. 1D), thereby, demonstrating that piPSCs were successfully generated from PFF cells. Characterization of porcine iPS cells RT-PCR was used to determine whether the piPSCs express the reprogramming factors. PCR was performed with human primers for three reprogramming genes as well as with porcine-specific primers for other known reprogramming factors (Nanog and Lin28). As shown in Fig. 1E, piPSCs expressed all these factors demonstrating that they were indeed stem-like cells. Differentiation of iPS cells into hepatocyte-like cells To determine whether the porcine iPSCs are capable of differentiating into hepatocyte-like cells, we subjected them to a differentiating protocol based on the human iPSC differentiation method with some modifications (Si-Tayeb et al., 2010). A three-step protocol was used in this study to induce hepatic differentiation of piPSCs (Fig. 2). It involved the use of activin A to induce the formation of definitive endoderm, followed by the propagation of cells in the medium with FGF-2, BMP-4 and HGF to differentiate cells into hepatic lineage. In the final step, cells were grown in the presence of oncostatin M to induce maturation of the immature hepatocytelike cells. In this protocol, piPSCs were grown on STO feeder layers and were successfully differentiated into hepatocyte-like cells. In the first step, they slowly began to migrate and differentiate in RPMI with activin A (Fig. 2A).
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A
B
C
D
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Fig. 1. Generation of porcine inducible pluripotent stem (iPS) cells. Pig fibroblasts (A) were transfected with pEP4EO2SET2K, which contains the open reading frames of three human reprogramming factors (Klf4, Oct4 and Sox2) using the Fugene 6 reagent. pCAGGS-DsRed construct expressing the Ds-Red ORF was used as a control to determine transfection efficiency (B). About 5–7 days after the initial transfection, small iPS colonies began to develop (C). These colonies tested positive for alkaline phosphatase (D). After growing the colonies for 3 more days, they were mechanically dispersed and total RNA was isolated for RT-PCR using primers specific for all three human reprogramming factors, as well as for two porcine genes, Nanog and Lin28 (E). Porcine GAPDH was used as a positive control. As shown here, all these genes were expressed by iPS cells but not by the porcine fetal fibroblast (PFF) cells.
the presence of BMP-4, FGF-2 and HGF, and matured in medium with oncostatin M by displaying typical hepatocyte-like morphology (Fig. 2D and E). DAPI staining of these porcine iPSC-derived cells (piPSC-Hep) revealed that a large number of the cells were binucleated (Fig. 3B), a distinct feature of mature hepatocytes that strongly supported successful differentiation of the piPSCs. Functional characterization of iPS cell-derived hepatocytes
Fig. 2. Schematic representation of the porcine induced pluripotent stem cells (piPSC) differentiation protocol. piPSC colonies that developed on MEF feeders were transferred to matrigel-coated plates and cultured in RPMI medium until they grew between 50 and 100 lm in diameter. They were then grown in the presence of activin A to initiate differentiation of the definitive endoderm. After 5 days in this medium, cells were grown for an additional 5 days in medium containing BMP-4, FGF2, and HGF. This step allows for the proliferation of cells that have committed to the hepatic lineage. Finally, they were grown for 5 days in the presence of oncostatin M to induce their maturation into hepatocyte-like cells (piPSC-Hep). Binucleated cells are indicated by arrows (C).
After 4–5 days in culture, they transformed into a large vacuolelike structure (Fig. 2B). The cells became binucleated (Fig. 2C) in
To determine functionality, the hepatocyte-like cells were evaluated for expression of albumin by immunocytochemistry. Albumin production is another unique feature of hepatocytes. By using a porcine albumin-specific antibody, we showed that our piPSC-Heps produced albumin (Fig. 3C). RT-PCR was also performed to study the expression of hepatocyte-specific markers with porcine primers for albumin, a-fetoprotein (AFP), c-Met, hepatocyte nuclear factors 1A and 4 (HNF1A and HNF4), cytokeratins 8 and 18 (CK8 and CK18), transferrin, and transthyretin (TTR). As shown in Fig. 4 (left panel), piPSC-Heps expressed all these hepatocyte markers. A PAS assay was carried out to further study the hepatic function of differentiated iPSC-Heps, since mature hepatocytes synthesize and store glycogen. Most of the differentiated cells tested positive for glycogen storage (Fig. 4, right panel). piPSC-Heps were then assayed for inducible cytochrome P450IA1 (CYPIA1) activity following 3-MC treatment by measuring EROD activity, i.e., conversion of 7-ethoxyresorufin (7-ER) to the fluorescent product resorufin. piPSC-Heps converted 7-ER to resorufin at rates of nearly 250 pmol/mg cell protein per 30 min, suggesting that piPSC-Heps express functional CYPIA1 protein (Fig. 5). Most notably, EROD activity was negligible in the piPSC-Heps that were not induced by exposure to 3-MC (control). The human liver cancer cell line HuH-7 was used as a positive control for this study. Collectively, these findings demonstrated that our piPSC-Heps possess hepatocyte-specific functions. Discussion The discovery that somatic cells can be converted into pluripotent stem cells by expressing four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) has revolutionized biomedical research (Takahashi and Yamanaka, 2006). Since then, other groups have successfully generated iPS cells using these and other reprogramming
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Resorufin (pmol/mg protein/30 min)
Fig. 3. Albumin production in porcine induced pluripotent stem hepatocyte-like cells (piPSC-Heps). Expression of albumin is a hallmark of hepatocytes. To test whether piPSC-Heps express albumin, they were grown on gelatin-coated plates (A) and were fixed with 4% paraformaldehyde. Many cells in culture were binucleated, as seen with DAPI staining (B and inset). They were then stained with porcine antibody against albumin and were visualized using a Dylight 549 fluorescent secondary antibody (C). Differentiated piPSC-Heps expressed albumin and stained positively. Merged image (D). Scale bar, 50 lm.
300 250 200 150 100 50 0 Control
Fig. 4. Functional characterization of porcine induced pluripotent stem hepatocytelike cells (piPSC-Heps). RT-PCR (left panel). piPSC-Heps were collected on day 15 following the differentiation procedure, and total RNA was isolated from them (Lane 2). RT-PCR was performed using primers specific for several hepatocyte markers: albumin, alpha fetoprotein (AFP), transthyretin (TTR), hepatocyte nuclear factors 1A and 4 (HNF1A, HNF4A), cytokeratins 8 and 18 (CK8, CK18), c-Met, and transferrin; house-keeping gene, b-actin. piPSC-Heps expressed all these markers in contrast to porcine induced pluripotent stem cells (piPSCs). Total RNA from piPSCs was used as negative control (Lane 1), while total RNA from primary porcine hepatocytes was used as positive control (Lane 3). Glycogen storage in piPSC-Heps (right panel). piPSC-Heps (A) were tested for their ability to store glycogen using a periodic acid Schiff (PAS) assay. piPSC-Heps stained positive for glycogen (B and inset). Original magnification, 10; inset, 60; scale bar, 50 lm.
factors (Wu et al., 2011). These findings have opened a new avenue to obtain hepatocytes for cell transplantation and for studying liver diseases.
piPSC-Hep
HuH-7
Fig. 5. Cytochrome P450IA1 ethoxyresorufin-O-deethylase (EROD) activity in porcine induced pluripotent stem hepatocyte-like cells (piPSC-Heps). Resorufin production was measured after exposing piPSC-Heps to 5 lM 3-methylchloranthrine (3-MC) for 48 h. Control, porcine induced pluripotent stem cells (piPSCs) without addition of 3MC and b-glucoridinase/arylsulfatase; piPSC-Hep (negative control); piPSC cells with 3-MC and b-glucoridinase/arylsulfatase; HuH-7, human liver cancer cell line with 3-MC and b-glucoridinase/arylsulfatase (positive control). Values represent the mean ± SEM (n = 6). P < 0.05 as determined by ANOVA.
The ability of iPS cells to differentiate into most mammalian cell types represents an attractive source of hepatocytes for the study and treatment of liver diseases. iPS cells can proliferate indefinitely under appropriate culture conditions in vitro and can be differentiated into hepatocytes for a prolonged period time. However, the major challenge remains the generation of iPS cells using methods that involve minimal modification to the genome of the
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primary somatic cells. To date, four reports have been published on the generation of porcine iPS cells by expressing various reprogramming factors with retroviral vectors (Esteban et al., 2009), lentiviral vectors (Ezashi et al., 2009; Wu et al., 2009), and a single plasmid (Montserrat et al., 2011). However, none of these iPS cells were differentiated into any liver-specific cell type(s). In all cases, integration of transgene sequences into the host chromosome did occur, making them undesirable for liver studies. In the present study, we used three ‘Yamanaka factors’ (Sox2, Klf4, and Oct4) to generate iPS cells with PFFs, but omitted the fourth factor c-Myc based on the report that it is not required to produce iPS cells (Nakagawa et al., 2008). Furthermore, c-Myc expression during the generation of iPS cells renders them tumorigenic (Okita et al., 2007). We used a single non-integrating episomal vector (Yu et al., 2009) to express the reprogramming factors so these iPS cells were free from vector and transgene sequences and are ideal candidates to generate hepatocyte-like cells to study liver-specific functions. To our knowledge, this is the first report to describe hepatic differentiation of piPSCs. We have demonstrated robust differentiation of piPSCs into hepatocyte-like cells. To achieve our objective, we adopted a three step differentiation protocol: (1) growth of iPS cells for 5 days in the presence of activin A to induce the differentiation of definitive endoderm; (2) growth in medium supplemented with BMP-4, FGF and HGF for an additional 5 days to obtain hepatocyte-like cells; and (3) growth in medium for 5 days containing oncostatin M. After 15 days, >90% of the cells in the culture medium displayed hepatocyte-specific features such as the production of albumin, glycogen storage, and a large number of bi-nucleated cells. Several laboratories have reported the differentiation of rodent and human iPSCs into hepatocyte-like cells using different methods by employing various cytokines (Duan et al., 2007; Song et al., 2009; Li et al., 2010; Gai et al., 2010; Si-Tayeb et al., 2010; Sullivan et al., 2010; Iwamuro et al., 2010; Sancho-Bru et al., 2011). However, these protocols are lengthy (15–20 days) and need to be fine-tuned to obtain hepatocyte-like cells in sufficient numbers for autologous cell therapy. In order to achieve optimal differentiation efficiency, it is necessary to use the appropriate cell type as a feeder layer. Feeder layers secrete growth factors and cytokines that will influence stem cells. Our efforts to differentiate piPSCs on an MEF feeder layer resulted in a very low level of hepatic differentiation, and it never reached more than 50% efficiency (data not shown). However, when MEFs were replaced with mouse STO fibroblast feeder cells (Martin and Evans, 1975), differentiation efficiency improved dramatically, and over 90% efficiency was achieved. Furthermore, piPSC-Heps grew rapidly when they were replenished with STO-conditioned medium. Earlier studies have reported that the STO feeder cells produce an extracellular matrix containing collagen, laminin, and proteoglycans (Horák and Fléchon, 1998; Talbot et al., 2002; Caperna et al., 2011). In addition to the three-dimensional extracellular matrix environment, STO cells provide interaction with cell-bound and soluble proteins that may support hepatocyte differentiation (Caperna et al., 2011). These cells have been shown to secrete a number of growth factors that promote hepatocyte morphogenesis and function, including insulin-like growth factor (IGF)-binding proteins (Caperna et al., 2011), stem cell factor (Pesce et al., 1997), HGF (Montesano et al., 1991), leukemia inhibitory factor (LIF) (Rathjen et al., 1990), and BMP-4 (Qi et al., 2004). Thus, it is likely that these cells play a critical role in the differentiation of piPSCs into hepatocyte-like cells. We have shown that piPSC-derived hepatocyte-like cells express albumin, AFP, c-Met, HNF-1a, HNF-4A, TTR, CK8, CK18, transferrin, and CYP1A1 and can store glycogen. Hengstler et al. (2005) have described specific characteristics that define a ‘hepatocyte-like cell’
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that include both quantitative analysis of enzymatic activities as well as qualitative analyses such as RT-PCR and immunohistochemistry. As shown here, piPSC-Heps displayed both qualitative and quantitative traits of hepatocytes. Furthermore, morphological analysis clearly demonstrated that a large number of differentiated cells were binucleated, a hallmark of hepatocytes, and formed structures both on STO feeders as well as under feeder-free conditions. Lack of porcine-specific antibodies and reagents currently preclude further studies such as those on phase II metabolism. However, the analysis conducted in this study clearly demonstrated that piPSC-Heps possess hepatocyte-specific functions. Conclusions piPSC-Heps displayed qualitative, morphologic and quantitative traits of hepatocytes and possessed hepatocyte-specific functions. The porcine hepatocyte-like cells generated from transgene-free piPSCs may be ideal candidates to study drug metabolism and may have potential for cell therapy of liver diseases. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgments We thank Dr. Scott Fahrenkrug for porcine fetal fibroblasts, Dr. Neil Talbot for STO-feeder cells and primary porcine hepatocytes, and Phillip Wong for assistance with the RT-PCR studies. This work was supported by the start-up funds from the Department of Radiology to R.N.A. References Aravalli, R.N., Sahin, M.B., Cressman, E.N., Steer, C.J., 2010. Establishment and characterization of a unique 1 lm diameter liver-derived progenitor cell line. Biochemical and Biophysical Research Communications 391, 56–62. Asgari, S., Pournasr, B., Salekdeh, G.H., Ghodsizadeh, A., Ott, M., Baharvand, H., 2010. Induced pluripotent stem cells: A new era for hepatology. Journal of Hepatology 53, 738–751. Caperna, T.J., Blomberg, L.A., Garrett, W.M., Talbot, N.C., 2011. Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media. In Vitro Cell and Developmental Biology Animal 47, 218–233. De Bartolo, L., Bader, A., 2001. Review of a flat membrane bioreactor as a bioartificial liver. Annals of Transplantation 6, 40–46. Donato, M.T., Gomez-Lechon, M.J., Castell, J.V., 1993. A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Analytical Biochemistry 213, 29–33. Duan, Y., Catana, A., Meng, Y., Yamamoto, N., He, S., Gupta, S., Gambhir, S.S., Zern, M.A., 2007. Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo. Stem Cells 25, 3058–3068. Esteban, M.A., Xu, J., Yang, J., Peng, M., Qin, D., Li, W., Jiang, Z., Chen, J., Deng, K., Zhong, M., Cai, J., Lai, L., Pei, D., 2009. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. Journal of Biological Chemistry 284, 17634–17640. Ezashi, T., Telugu, B.P., Alexenko, A.P., Sachdev, S., Sinha, S., Roberts, R.M., 2009. Derivation of induced pluripotent stem cells from pig somatic cells. Proceedings of the National Academy of Sciences of the USA 106, 10993–10998. Fondevila, C., Hessheimer, A.J., Flores, E., Vendrell, M., Muñoz, J., Escobar, B., Calatayud, D., Taurá, P., Fuster, J., García-Valdecasas, J.C., 2011. Step-by-step guide for a simplified model of porcine orthotopic liver transplant. Journal of Surgical Research 167, e39–e45. Fitzpatrick, E., Mitry, R.R., Dhawan, A., 2009. Human hepatocyte transplantation: State of the art. Journal of Internal Medicine 266, 339–357. Gai, H., Nguyen, D.M., Moon, Y.J., Aguila, J.R., Fink, L.M., Ward, D.C., Ma, Y., 2010. Generation of murine hepatic lineage cells from induced pluripotent stem cells. Differentiation 79, 171–181. Hara, H., Campanile, N., Tai, H.C., Long, C., Ekser, B., Yeh, P., Welchons, D., Ezzelarab, M., Ayares, D., Cooper, D.K., 2010. An in vitro model of pig liver xenotransplantation – Pig complement is associated with reduced lysis of wild-type and genetically modified pig cells. Xenotransplantation 17, 370–378.
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