Rat iPSC line M13 was provided by the research group led by Professor Lei Xiao of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences[23]. Rat iPSCs were cultured with a feeder layer of irradiated mouse embryonic fibroblasts (MEF) in iPSC medium (DMEM/F12 containing 20% knockout serum replacement, 2 mmol/L L-glutamine, 1% nonessential amino acids, 0.1 mmol/L β-mercaptoethanol (all from Invitrogen/Gibco, Grand Island, NY, United States) in a humid atmosphere containing 5% CO₂. Before differentiation, the cells were cultured on gelatin-coated tissue culture dishes with MEF-conditioned medium.

During the first 5 d, rat iPSCs were cultured in the Roswell Park Memorial Institute 1640 medium (Invitrogen/Gibco) with 25 ng/mL Wnt3a and 100 ng/mL Activin A (both from R and D Systems, Minneapolis, MN, United States) for endodermal induction. Next, the inductive factors were replaced with 30 ng/mL fibroblast growth factor 4 (FGF4, R and D Systems) and 20 ng/mL bone morphogenetic protein 2 (BMP2, R and D Systems) for 5 d. Finally, during the maturation step, the differentiated cells were incubated in the same basal medium containing 10 ng/mL Oncostatin M (OSM, R and D Systems), 20 ng/mL hepatocyte growth factor (HGF, R and D Systems), and 0.1 μmol/L dexamethasone (Dex, Sigma-Aldrich, St. Louis, MO, United States) for another 10 d.

On day 5 after endodermal induction, differentiated cells were tested for SRY-box containing gene 17 (SOX17) and forkhead box protein A2 (FOXA2). On day 20, they were tested for the other markers. Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% TrionX-100, and blocked with 10% goat serum (Sigma-Aldrich) for 1 h at room temperature. Then the cells were incubated overnight with primary antibodies at 4 °C. The primary antibodies against rat FOXA2 (1:200, Chemicon/Millipore, Billerica, MA, United States), SOX17 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, United States), albumin (ALB, 1:500), α-fetoprotein (AFP, 1:200), α1-antitrypsin (AAT, 1:200), cytokeratin 18 (CK18, 1:200, all from Santa Cruz Biotechnology), cytochrome P450 1A2 (CYP1A2, 1:200, Abcam, La Jolla, CA, United States) and cytochrome P450 3A4 (CYP3A4, 1:200, Abcam) were obtained. Then the cells were incubated with TRITC-conjugated secondary antibody (Invitrogen, Carlsbad, CA, United States) for 1 h at room temperature. The nuclei were then counterstained with 4,6-diamidino-2-phenylindole (DAPI, Roche, Mannheim, Germany). The cells were analyzed using a fluorescence microscope (IX71, Olympus, Japan).

To assess endodermal and hepatic markers, differentiated cells were tested for FOXA2 and SOX17 on day 5 and for other markers on day 20. Trizol reagent was used to extract total RNA from differentiated iPSCs (Invitrogen). Reverse transcription-PCR was used to produce cDNA. GenBank accession numbers, primer sequences, and amplicon size are listed in Table 1 for each gene. The PCR procedure was as follows: cDNA denaturation at 94 °C for 5 min followed by 28 cycles (ALB, 40 cycles) of denaturation at 94 °C for 15 s, annealing at 66 °C for 10 s, and extension at 72 °C for 20 s. The final extension took place at 72 °C and lasted 7 min. PCR products were separated by electrophoresis on agarose gels. The GAPDH housekeeping gene was used as an internal control.

The urea production test was performed using a colorimetric QuantiChrom Urea Assay Kit (BioAssay Systems, Hayward, CA, United States). Undifferentiated cells and differentiated cells on days 5, 10, 15, and 20 were trypsinized and counted with a hemocytometer. The assay was performed in accordance with the manufacturer’s instructions. The sample supernatants were stored at -20 °C. Absorbance was measured using a microplate reader. Urea production was normalized to the total number of cells.

The concentration of rat albumin in the supernatant was determined using a Rat Albumin ELISA Quantitation Kit (ICL, Portland, OR, United States) in accordance with the manufacturer’s instructions. Undifferentiated iPSCs and differentiated cells were trypsinized and counted on days 5, 10, 15, and 20 using a hemocytometer. Albumin secretion was normalized to the total number of cells.

On day 20, differentiated iPSCs were stained using a Periodic acid-Schiff (PAS) staining system (Sigma-Aldrich). The cells were first fixed in 4% paraformaldehyde for 20 min and oxidized with 1% periodic acid solution for 5 min. Then they were washed. The cells were treated with Schiff’s reagent for 15 min at room temperature. The cells were washed with PBS and then stained with hematoxylin for 90 s. They were then examined under a light microscope (IX50, Olympus, Japan).

After differentiation, cells were incubated for 1 h with indocyanine green (ICG, Sigma-Aldrich) in basal medium at 37 °C. Uptake of ICG was detected under a light microscope. The ability of these cells to take up low-density lipoprotein was determined using an LDL Uptake Cell-Based Assay Kit (Cayman Chemical, Ann Arbor, MI, United States) according to the manufacturer’s instructions. The cells were visualized under a fluorescent microscope.

Results are shown as mean ± SD. One-way ANOVA was used for statistical analysis. P < 0.05 was considered significant.

A simple three-stage differentiation method for iPSCs was developed. During the first five days, iPSCs were treated in a medium with Activin A and Wnt3a for endodermal induction. The cells gradually took on flatter and spikier shapes (Figure 1B). Then, the cells were cultured under feeder-free conditions with high concentrations of FGF4 and BMP2 for the next five days. Cells in these colonies had compact, polygonal shapes like those of differentiated hepatocytes (Figure 1C). The medium was replaced with mature medium supplemented with HGF, OSM, and Dex for an additional 10 d. The cells exhibited the morphology of mature hepatocytes with large amounts of cytoplasm and distinct, round nuclei (Figure 1D).

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Figure 1 Morphological changes of rat induced pluripotent stem cells at different stages of differentiation. A: Undifferentiated induced pluripotent stem cells; B: Differentiated cells on day 5; C: Differentiated cells on day 15; D: Differentiated cells on day 20. Scale bar = 100 μm.

Cell characteristics were determined at different stages of differentiation using immunofluorescence staining. After endodermal induction, approximately 70%-80% of the differentiated cells expressed endodermal markers FOXA2 and SOX17 at the protein level (Figure 2). These results indicate that rat iPSCs could form endoderm efficiently if exposed to Activin A and Wnt3a. After hepatic maturation at stage 3, immunostaining analysis showed that 50%-60% of the cells expressed hepatic markers, including AFP, ALB, CK18, and functional proteins such as AAT, CYP1A2 and CYP 3A4 (Figure 2).

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Figure 2 Protein expression in differentiated induced pluripotent stem cells on days 5 and 20 as indicated by immunofluorescence staining. A, B, C: FOXA2; D, E, F: SOX17; G, H, I: AFP; J, K, L: ALB; M, N, O: CYP1A2; P, Q, R: CYP3A4; S, T, U: AAT; V, W, X: CK18. Nuclei were stained blue with DAPI. Scale bar = 100 μm. FOXA2: Forkhead box protein A2; SOX17: SRY-box containing gene 17; AFP: α-fetoprotein; ALB: Albumin; CYP1A2: Cytochrome P450 1A2; AAT: α1-antitrypsin.

To further analyze the properties of iPSCs that developed into endodermal cells and HLCs, RT-PCR analysis was performed in the cells at various stages of differentiation. After induction of endodermal differentiation, endoderm markers FOXA2 and SOX17 were detected in the cells at stage 1 (Figure 3). This was consistent with the results of the immunostaining assay. After the cells differentiated into HLCs at stage 3, RT-PCR analysis indicated the expression of liver associated genes such as AFP, ALB, CK8, CK18, CK19, and transcription factor HNF-4α (Figure 3). These results collectively indicate that the cells had changed from endodermal to hepatic.

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Figure 3 RT-PCR analysis of differentiated induced pluripotent stem cells on days 5 and 20. Differentiated cells expressed endodermal markers FOXA2 and SOX17 after 5 d of endoderm induction. Liver associated genes AFP, ALB, CK8, CK18, CK19, and HNF-4α mRNA were expressed on day 20. H2O served as a negative control. FOXA2: Forkhead box protein A2; SOX17: SRY-box containing gene 17; AFP: α-fetoprotein; ALB: Albumin; iPSCs: Induced pluripotent stem cells.

In order to evaluate the liver-specific function of iPSC-derived differentiated cells, the functional properties of HLCs of urea synthesis and albumin secretion were analyzed. The rates of urea synthesis (20 d 1.202 ± 0.080 mg/dL vs 0 d 0.317 ± 0.021 mg/dL, P < 0.01; Figure 4A) and albumin secretion (20 d 1.601 ± 0.102 mg/dL vs 0 d 0.313 ± 0.015 mg/dL, P < 0.01; Figure 4B) increased significantly as differentiation progressed, indicating hepatic maturation.

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Figure 4 Functional analysis of differentiated induced pluripotent stem cells. A: Urea production of differentiated induced pluripotent stem cells (iPSCs); B: Albumin secretion of differentiated iPSCs; C: Glycogen synthesis of differentiated iPSCs as indicated by PAS staining on day 20; D: ICG uptake test of differentiated iPSCs; E: LDL uptake test of differentiated iPSCs. Scale bar = 100 μm. ^bP < 0.01 vs undifferentiated cells; ^dP < 0.01 vs differentiated cells on day 15.

The ability of the cells to store glycogen was tested. PAS staining was used to confirm the presence of stored glycogen. Differentiated cells showed glycogen staining, indicating glycogen accumulation (Figure 4C).

The iPSCs-HLCs were also found to take up ICG and LDL (Figure 4D and E). These results showed that the iPSC-HLCs had the same function as hepatocytes in vitro.

Recent studies have suggested that differentiated hepatocytes derived from transplanted stem cells may be a suitable form of therapy. Embryonic stem cells (ESCs) and bone marrow-derived liver stem cells can form mature hepatocytes and reduce the severity of hepatic fibrosis. However, the ethical issues of ESCs and the low purity of bone marrow stem cells limit their use in clinical settings. Induced pluripotent stem cells (iPSCs) sidestep most of the ethical issues surrounding ESCs. The differentiation of iPSCs into hepatic lineages has been observed in humans and mice. So far, no reports have been published on the differentiation of rat iPSCs into hepatocyte-like cells (HLCs).

The laboratory rat was the first mammalian species domesticated for scientific research, and the rat model is commonly used in research involving hepatic diseases. This study is the first to show the efficient generation of HLCs differentiated from the iPSCs of rats.

Hepatic differentiation was achieved using a three-step protocol. Rat iPSCs were differentiated into HLCs after 20 d of culture. These differentiated cells expressed hepatic markers and exhibited functional hepatic characteristics.

This study suggests that rat iPSCs can differentiate into HLCs in a rapid and efficient manner through a three-stage process involving the application of inductive factors. Differentiated cells may be an attractive resource for treatment of end-stage liver disease.

Induced pluripotent stem cells are a type of pluripotent stem cell that can be generated directly from adult cells. Pluripotent stem cells hold great promise in the field of regenerative medicine. Because they can propagate indefinitely, as well as give rise to every other cell type in the body, they represent a single source of cells that could be used to replace those lost to damage or disease.

The manuscript is the first to demonstrate the efficient generation of HLCs differentiated from iPSCs of rat species.