Abstract
Induced pluripotent stem (iPS) cells are pluripotent and are able to unlimitedly proliferate in vitro. This technical breakthrough in creating iPS cells from somatic cells has noteworthy implications for overcoming the immunological rejection and the ethical issues associated with the derivation of embryonic stem cells from embryos. In the current work, we present an efficient hepatic differentiation of mouse iPS cells in vitro. iPS cells were cultured free floating to induce the formation of embryoid bodies (EB) for 5 days. EB were transferred to a gelatin-coated plate and treated with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF) for 3 days to induce definitive endoderm. Cells were further cultured for 8 days with 100 ng/ml hepatocyte growth factor (HGF) to generate hepatocytes. Characterization was performed by RT-PCR assay. Functional analysis for albumin secretion and ammonia removal was also carried out. iPS cell-derived hepatocyte-like cells (iPS-Heps) were obtained at the end of the differentiation program. Expression levels of a gestational hepatocyte gene and lineage-specific hepatic genes intensified in iPS-Heps. The production of albumin increased in a time-dependent manner. iPS-Heps were capable of metabolizing ammonia. We present here instant hepatic differentiation of mouse iPS cells using combined 3-day treatments of activin A and bFGF with subsequent 8-day HGF. Our study will be an important step to generate hepatocytes from human iPS cells as a new source for liver-targeted cell therapies.
Introduction
Hepatocytes have a wide range of functions in the body. Once hepatocytes are massively damaged, even temporally, the patients will be placed in a very lethal status. Orthotopic liver transplantation has been applied in the case of liver failure. Although the surgical techniques and the postoperative managements are improved, surgery-associated mortality is still considerable.
To overcome the complications associated with hepatic organ transplantation, several clinical trials of hepatocyte transplantation have been conducted. Partial improvements of bilirubin metabolism (1, 7, 8), a urea cycle disorder (10), glycogen storage disease type 1 (15), an inborn error in fatty acid metabolism (9, 19), and a clotting factor deficiency (6) have been reported. Furthermore, in patients with acute liver failure, hepatocyte allotransplants decreased cerebral perfusion pressure, lowered ammonia levels, and even improved an overall survival (5, 21). However, the major limitation of cell therapies for liver diseases is the donor liver shortage (11). Besides, chronic immunosuppression is still required for allotransplantation of hepatocytes. In general, the patient-derived cells are extremely attractive in cell therapies.
Historically, in 1992, autologous hepatocyte transplantation was first reported in 10 patients with chronic liver disease (14). Hepatocytes were directly transplanted into the spleen after isolating the cells from surgically resected recipient's left lateral segment of the cirrhotic liver. Although autotransplanted hepatocytes were detected in the spleen at 1–6 months, significant clinical effects were not obtained. The unsatisfactory results may include the site of hepatocyte transplantation, the number of the transplanted cells, and viability of the isolated cells.
Transplantation of the cells derived from extrahepatic sites of patients may be another potentially effective cell therapy for liver diseases. It has been reported that bone marrow cells can populate the damaged liver and differentiate into albumin-producing hepatocytes (18). The authors performed an autologous bone marrow cell infusion in nine liver cirrhotic patients and reported amelioration of albumin production (24). In animal models, mesenchymal stem cells originating from bone marrow (12) or adipose tissue (2, 26) are expected as an alternative cell source for obtaining differentiated hepatocytes.
Recently, induced pluripotent stem (iPS) cells had been successfully established from mouse and human fibroblasts using defined factors (13, 17, 23, 25). iPS cells are pluripotent and are able to unlimitedly proliferate in vitro. This technical breakthrough in creating iPS cells from somatic cells has noteworthy implications for overcoming the immunological rejection and the ethical issues associated with the derivation of embryonic stem (ES) cells from embryos.
In the current work, we present an efficient hepatic differentiation of mouse iPS cells in vitro. At the end of the differentiation program, iPS cell-derived hepatocyte-like cells (iPS-Heps) were obtained.
Materials and Methods
Culture of Undifferentiated Mouse iPS Cells
The mouse iPS cells were kindly provided by Riken Cell Bank (Cell No. APS0001, Cell name iPS-MEF-Ng-20D-17, Lot No. 006) (16). Mouse iPS cells of passages 11–20 were maintained by culture on a feeder layer of mouse embryo fibroblasts (MEF) (Dainippon Pharmaceutical, Osaka, Japan) inactivated by mitomycin C (Biomol, Enzo Life Sciences International, PA) on gelatin (Specialtymedia, Chemicon International, MA)-coated plates. iPS cells were cultured with Complete ES cell medium (Specialtymedia, Chemicon International) supplemented with 1000 U/ml recombinant leukemia inhibitory factor (LIF) (ESGRO™, Chemicon International) at 37°C in 5% CO2. Every passage was carried out before cells reached confluency (22).
In Vitro Differentiation of Hepatocyte-Like Cells From Undifferentiated iPS Cells
Differentiation was carried out in three stages, as shown in Figure 1.
Differentiation scheme and morphological changes in generating hepatocyte-like cells from mouse iPS cells. (A) Stage 1: undifferentiated iPS cells were differentiated in free-floating culture to form cellular aggregates, so-called EB. Stage 2: EB was transferred to a gelatin-coated plate and was treated with 100 ng/ml activin A and 100 ng/ml bFGF for 3 days. Stage 3: iPS cell-derived aggregates were further cultured for 8 days with DMSO and HGF. (B) Undifferentiated iPS cells formed round colonies on a mouse embryonic fibroblast. (C) After 5 days of flee-floating culture, cells assembled and formed EB. (D) EB was attached to a plate soon after being transferred to the gelatin-coated plate, and cells started to proliferate from the periphery of the cellular clusters. (E) The differentiation protocol generated cells morphologically similar to hepatocytes, being polygonal in shape with cytoplasmic granules and multiple nuclei (arrow). Abbreviations: EB, ambryoid body; FGF, fibroblast growth factor; DMSO, dimethyl sulfoxide; HGF, hepatocyte growth factor.
Stage 1: Formation of Embryoid Bodies (Days 0–4)
iPS cells growing on feeder cells were dispersed by treatment with trypsin-EDTA (Sigma-Aldrich Japan, Tokyo, Japan) and collected by centrifugation at 800 rpm for 3 min. Cells were then resuspend in Knockout DMEM (Gibco, Invitrogen, CA) supplemented with 15% knockout serum replacement (KSR) (Gibco), 1% nonessential amino acids (MP Biomedicals, CA), 1% 2-mercaptoethanol (Gibco), 1% penicillin/streptomycin (Sigma-Aldrich Japan), and 1% l-glutamic acid (DS Pharma Biomedical, Osaka, Japan). iPS cells were then transferred to ultra-low attachment six-well plates (Corning, NY) and cultured free floating in the culture medium at a density of 2 × 105 cells/2 ml/well to induce the formation of embryoid bodies (EB) (at days 0). Half of the medium was replaced daily.
Stage 2: Induction of Definitive Endoderm (Days 5–7)
At days 5, EB were collected from the single well and centrifuged at 1000 rpm for 3 min. EB were then resuspend in 9 ml of Knockout DMEM, 1% penicillin/streptomycin, 1% l-glutamic acid, 100 ng/ml activin A (R&D Systems, MN), 100 ng/ml basic fibroblast growth factor (bFGF) (Peprotech, NJ) to induce definitive endoderm (DE). In each well of a gelatin-coated 12-well plate, 1.5 ml of the medium containing EB was seeded. EB were cultured for additional 3 days. KSR was supplemented at days 6–7 and the concentrations were varied: 0.2% for days 6 and 2.0% for days 7. Half of the medium was replaced daily.
Stage 3: Differentiation of Hepatocyte-Like Cells (Days 8–16)
At day 8, the medium was replaced by Knockout DMEM supplemented with 10% KSR, 1% nonessential amino acids, 1% l-glutamic acid, 1% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Irvine, UK), and 100 ng/ml hepatocyte growth factor (HGF) (Peprotech, NJ) to induce hepatocyte-like cells. Half of the medium was replaced daily. The differentiation program was finished at day 16.
Isolation of RNA and Reverse-Transcription Polymerase Chain Reaction
Reverse-transcription polymerase chain reaction (RT-PCR) was carried out at various points of the differentiation program to determine the degree to which iPS cells differentiated toward iPS-Heps. RNA was extracted using a TRIzol reagent (Invitrogen). Complementary DNA was prepared using a MuLV reverse transcriptase (Applied Biosystems, CA) and RNase inhibitor (Applied Biosystems) from 2 μg of total RNA. RT-PCR was performed with AmpliTaq gold DNA polymerase, Gene Amp PCR gold buffer, and MgCl2 solution (Applied Biosystems). Primers used for RT-PCR are listed in Table 1. PCR products were resolved on 2.5% agarose gels and visualized by ethidium bromide staining. A real-time PCR was performed on LightCycler 1.5 Instrument (Roche Applied Science, IN) with LightCycler FastStart DNA master SYBR green I (Roche Applied Science, IN) as per the manufacturer's instructions. Primers are listed in Table 2.
RT-PCR Primers Used in the Present Study
AFP, α-fetoprotein; Alb, Albumin; Trf, transferring; PCK1, phosphoenolpyruvate carboxykinase 1; CPS, carbamylphosphate synthetase.
Real-Time RT-PCR Primers Used in the Present Study
AFP, α-fetoprotein; Alb, Albumin; Trf, transferring; PCK1, phosphoenolpyruvate carboxykinase 1; CPS, carbamylphosphate synthetase.
Measurement of Albumin Production of Mouse iPS Cells by ELISA
In the process of endodermal and subsequent hepatic differentiation of mouse iPS cells, the supernatant of mouse iPS cell culture was collected at regular intervals. The amount of albumin secreted into the culture medium was measured by an albumin enzyme-linked-immunosorbent assay kit (ALBUWELL M; Exocell, Philadelphia, PA), as per the manufacturer's instructions.
Measurement of Ammonia-Metabolizing Capacities of Mouse iPS Cells
At the end of hepatic differentiation, ammonia-metabolizing capacities of iPS-Heps were measured. Ammonium sulfate (Sigma-Aldrich, Irvine, UK) were added into the culture medium at the final concentration of 0.56 mM. The concentration of each reagent was measured 24 h later to estimate the metabolic capacities. Ammonium concentration was determined using an ammonium concentration assay kit (Wako Pure Chemical, Osaka, Japan), as per the manufacturer's instructions. iPS cells in free-floating culture were used as controls.
Statistical Analysis
Student's t-test was used to estimate the difference and p < 0.05 was deemed statistically significant.
Results
Mouse iPS Cells Cultured with Activin A, bFGF, and HGF Showed Morphological Characteristics of Hepatocytes
Mouse iPS cells were cultured with activin A (100 ng/ml) and bFGF (100 ng/ml) for 3 days and then with HGF (100 ng/ml) for 8 days in a gelatin-coated culture plate. Morphologically, the cells revealed polygonal shape with two nucleoli and enriched cytoplasmic granules, which are compatible with the characteristics of normal mouse hepatocytes in culture (Fig. 1E).
Mouse iPS Cells Cultured with Activin A, bFGF, and HGF Expressed the Genes of Albumin
RT-PCR analysis showed that the expression of albumin, a marker for matured hepatocytes, progressively increased over the course of differentiation program (Fig. 2). Gene expression levels of hepatocyte-enriched markers (3), such as urea cycle enzyme carbamylphosphate synthetase (CPS), cytosolic key control enzyme of gluconeogenesis phosphoenolpyruvate carboxykinase 1 (PCK1), and transferrin (Trf) also intensified in a time-dependent manner. iPS-Heps expressed asialoglycoprotein receptor (ASGR) gene, which is also called hepatic lectin (data not shown). The result confirmed progressive differentiation toward hepatocytes in a population of cells. α-Fetoprotein (AFP) expression, which is present in endoderm but is not expressed by mature hepatocytes, was still detected 16 days after culture, indicating that differentiation toward a mature hepatocyte was not uniform.
RT-PCR and real-time RT-PCR analysis in iPS-derived hepatocytes. RT-PCR (A) and real-time analysis (B) was performed to determine gene expression level during the various stages of differentiation. Messenger RNA levels were normalized relative to β-actin and iPS-Heps. Expression levels of a gestational hepatocyte marker α-fetoprotein (AFP), lineage-specific hepatic markers [albumin (Alb), transferrin (Trf), phosphoenolpyruvate carboxykinase 1 (PCK1), carbamylphosphate synthetase (CPS)], and a pan-endodermal marker Sox17 progressively increased in the differentiation program.
Mouse iPS Cells Cultured with Activin A, bFGF, and HGF Produced Albumin and Metabolized Ammonia
iPS-Heps secreted albumin at 396.0 ± 65.5 pg/ml/24 h/1 × 105 cells and metabolized 18.1 ± 5.9% of loaded ammonia (Fig. 3). The result showed that iPS-Heps hold a metabolic activity as functional hepatocytes.
Metabolic activities of iPS-derived hepatocytes. (A) Albumin secretion by iPS-Heps was determined in vitro during differentiation (n = 4). (B) Removal of loaded ammonia was confirmed in iPS-Heps, whereas ammonium concentration increased in the medium of undifferentiated iPS cells (n = 3).
Discussion
The liver is one of the largest organs in the body and performs numerous functions that are vitally important to maintain metabolic homeostasis. These functions include synthesis of serum proteins, regulation of nutrients, production of bile, and the metabolism and conjugation of compounds for excretion in the bile or urine. Hepatocytes, which are the predominant cell type within the liver, account for two thirds of the liver mass. The liver is normally able to regenerate after acute injury and regain its function under appropriate physiological stimuli. However, when the normal regenerative process is compromised and the residual functional capacity of the damaged liver is unable to sustain life, liver failure occurs. The two principal causes of the liver failure are cirrhosis and fulminant hepatic failure. Liver cirrhosis, whose etiology includes alcoholism and chronic hepatitis, is an irreversible process that occurs when fibrotic tissue replaces normal liver tissue as a result of chronic injury. Fulminant hepatic failure is a clinical syndrome, defined by impaired mental and neuromuscular function, whose etiology includes chemical and viral hepatitis. It occurs as a result of massive hepatocyte necrosis and is the most severe manifestation of liver insufficiency, with mortality rates greater than 80%. The ability to obtain unlimited numbers of human hepatocytes may improve the development of cell therapies for liver diseases.
ES cells are pluripotent, and potentially can differentiate into any cell type. We have previously shown that mouse ES cells can be differentiated by sequential culture in activin A, bFGF, HGF, and dexamethasone, and can be isolated by an albumin promoter-based cell sort to generate functional hepatocytes (20). Such ES cell-derived hepatocytes express liver-specific genes, but not genes representing other lineages. We have successfully applied the differentiation protocol to human ES cells with a minor modification (4). Such studies provide a foundation for efficient development of the cells with numerous characteristics of hepatocytes from both mouse and human cells.
Generation of functional hepatocytes from precursors derived from individual patients using an iPS cell establishment technology will lead to the development of individualized patient specific drug regimens and finally may be used to overcome the need for life-long immune suppression after transplantation of the cells. Therefore, in the present study, we applied our strategy to mouse iPS cells and found that the resultant iPS cells showed the expression of hepatocyte-enriched genes, albumin secretion, and potential for metabolizing ammonia. ASGR-based cell sorting is now under investigation for the enrichment of the hepatic population. Further studies are needed to determine whether the differentiation protocol and enrichment strategy can be scaled for use in humans and can be modified to eliminate the risk of the contamination of undifferentiated cells and the risk of teratoma formation after transplantation.
In the near future, successful repopulation of the livers of animals, such as uPA/SCID mice, with human iPS-derived hepatocytes could be used to better understand many human liver diseases that cannot presently be modeled in animals, which includes alcoholic liver disease, α-1-antitrypsin deficiency, urea cycle disorders, and hepatitis-mediated liver failure. Such studies will generate unique new therapies, which include 1) transplantation of iPS cell-derived hepatocytes without immunological barrier, 2) in vitro determination of a toxicity of the newly developed drugs, and 3) development of the personalized health care by evaluating drugs for efficacy and toxicity on patient-specific hepatocytes.
In conclusion, we have here presented instant hepatic differentiation of mouse iPS cells using combined treatments of activin A (100 ng/ml) and bFGF (100 ng/ml) with subsequent 8-day HGF (100 ng/ml). Our study will be an important step to generate hepatocytes from human iPS cells as a new source for liver-targeted cell therapies.