Stem Cell Reviews and Reports

, Volume 6, Issue 4, pp 622–632

Generation of Liver Disease-Specific Induced Pluripotent Stem Cells Along with Efficient Differentiation to Functional Hepatocyte-Like Cells

Authors

  • Arefeh Ghodsizadeh
    • Department of Stem Cells and Developmental BiologyRoyan Institute for Stem Cell Biology and Technology, ACECR
    • Department of Biotechnology, College of ScienceUniversity of Tehran
  • Adeleh Taei
    • Department of Stem Cells and Developmental BiologyRoyan Institute for Stem Cell Biology and Technology, ACECR
  • Mehdi Totonchi
    • Department of GeneticsRoyan Institute for Reproductive Biomedicine, ACECR
  • Ali Seifinejad
    • Department of Stem Cells and Developmental BiologyRoyan Institute for Stem Cell Biology and Technology, ACECR
  • Hamid Gourabi
    • Department of GeneticsRoyan Institute for Reproductive Biomedicine, ACECR
  • Behshad Pournasr
    • Department of Stem Cells and Developmental BiologyRoyan Institute for Stem Cell Biology and Technology, ACECR
  • Nasser Aghdami
    • Department of Stem Cells and Developmental BiologyRoyan Institute for Stem Cell Biology and Technology, ACECR
    • Department of Regenerative BiomedicineRoyan Institute for Stem Cell Biology and Technology, ACECR
  • Reza Malekzadeh
    • Digestive Disease Research Center, Shariati HospitalTehran University of Medical Sciences
  • Navid Almadani
    • Department of GeneticsRoyan Institute for Reproductive Biomedicine, ACECR
  • Ghasem Hosseini Salekdeh
    • Department of Molecular Systems BiologyRoyan Institute for Stem Cell Biology and Technology, ACECR
    • Department of Stem Cells and Developmental BiologyRoyan Institute for Stem Cell Biology and Technology, ACECR
    • Department of Developmental BiologyUniversity of Science and Culture, ACECR
Article

DOI: 10.1007/s12015-010-9189-3

Cite this article as:
Ghodsizadeh, A., Taei, A., Totonchi, M. et al. Stem Cell Rev and Rep (2010) 6: 622. doi:10.1007/s12015-010-9189-3

Abstract

The availability of disease-specific induced pluripotent stem cells (iPSCs) offers a unique opportunity for studying and modeling the effects of specific gene defects on human liver development in vitro and for testing small molecules or other potential therapies for relevant liver disorders. Here we report, for the first time, the derivation of iPSCs by the retroviral transduction of Yamanaka’s factors in serum and feeder-free culture conditions from liver-specific patients with tyrosinemia, glycogen storage disease, progressive familial hereditary cholestasis, and two siblings with Crigler-Najjar syndrome. Furthermore, they were differentiated into functional hepatocyte-like cells efficiently. These iPSCs possessed properties of human embryonic stem cells (hESCs) and were successfully differentiated into three lineages that resembled hESC morphology, passaging, surface and pluripotency markers, normal karyotype, DNA methylation, and differentiation. The hepatic lineage-directed differentiation showed that the iPSC-derived hepatic cells expressed hepatocyte-specific markers. Their functionality was confirmed by glycogen and lipid storage activity, secretion of albumin, alpha-fetoprotein, and urea, CYP450 metabolic activity, as well as LDL and indocyanin green uptake. Our results provide proof of principal that human liver-disease specific iPSCs present an exciting potential venue toward cell-based therapeutics, drug metabolism, human liver development and disease models for liver failure disorders.

Keywords

DifferentiationHepatocyteHuman induced pluripotent stem cellsLiver disease

Abbreviations

hiPSCs

Human induced pluripotent stem cells

hESCs

Human embryonic stem cells

TYR

Tyrosinemia

GSD

Glycogen storage disease

HER

Hereditary cholestasis

CNS

Crigler-Najjar syndrome

EB

Embryoid body

AFP

Alpha-fetoprotein

ALB

Albumin

LDL

Low-density lipoprotein

PAS

Periodic Acid-Schiff

ICG

Indocyanin green

HDFs

Human dermal fibroblasts

HLCs

Hepatocyte like cells

ALP

Alkaline phosphatase

Introduction

Recent advances for establishing human induced pluripotent stem cells (hiPSCs) through the expression of a combination of transcription factors has offered an unprecedented opportunity to use these somatic cell-derived stem cells as sources for drug screening, toxicology, cell replacement therapy, and generating disease models. However, several issues must be resolved before hiPSCs can be used in a clinical setting [1]. Currently, animal models and human cell cultures provide invaluable data for human congenital and acquired diseases; however, they have a limited representation of human pathophysiology. For example, mice carrying the same genetic deficiencies as Fanconi’s anemia patients do not develop spontaneous bone marrow failure [2] or most human cell lines carry genetic and epigenetic artifacts which arise from their long-term cultures and are generated either from malignant tissues or carry genetic modifications (for review see [3]). Additionally, most drugs rely on liver CYP450 activity for detoxification which cannot be tested in animal liver cells due to species differences [4].

Disease-specific hiPSCs have opened new possibilities to study human pathologies in vitro. Moreover, the embryonic stem cell (ESC)-like properties of hiPSCs allows for the investigation of early embryonic developmental phenomena within a culture system. This unique potential of hiPSCs offers an invaluable tool to study the abnormal early embryogenesis that may occur in these genetic disorders. Several groups have successfully generated a wide range of hiPSCs from patients suffering from a number of different diseases (for review see [1]). However, the generation of iPSCs from liver-specific disorders has not been reported yet.

Here, we show for first time the generation and characterization of hiPSCs lines in serum and feeder free-culture conditions from patients with tyrosinemia (TYR), glycogen storage disease (GSD), progressive familial hereditary cholestasis (HER), and two siblings diagnosed with Crigler-Najjar syndrome (CNS). Our hiPSCs are indistinguishable from hESCs with respect to colony morphology, passaging, surface and pluripotency markers, normal karyotype, DNA methylation, and differentiation potential. We also describe and illustrate the efficient directed differentiation of these cell lines into functional hepatocyte-like cells (HLCs) in vitro which are characterized with the use of a variety of experimental approaches.

Material and Methods

Generation of iPSCs from Patients’ Fibroblasts

Human skin biopsies were obtained in accordance with the Declaration of Helsinki and following the approval of the institutional review board and after obtaining informed consent from Iranian patients with liver disease (Table 1, ClinicalTrials.gov Identifier: NCT00953693).
Table 1

hiPSCs derived from somatic cells of patients with liver disease

Phenotype of disease

Sex-age at biopsy

Mutation

No. of derived lines

Characterized cell lines

Lines with normal karyotype

Tyrosinemia type 1 (TYR1)

F-6

FAH, p.Gln64His

7

TYR1-hiPSC1

TYR1-hiPSC1, 3, 5

Glycogen storage type Ib (GSD1)

M-7

SLC37A, c.1124-2A>G

7

GSD1-hiPSC7

GSD1-hiPSC5, 6, 7

Progressive familial hereditary cholestasis(HER1)

F-17

Multifactorial

6

HER1-hiPSC1

HER1-hiPSC1, 2, 4

Crigler-Najjar Syndrome (CNS1)

F-19

UGT1A1, p.Leu413Pro

9

CNS1-hiPSC10

CNS1-hiPSC5, 8, 10

Crigler Najjar Syndrome (CNS2)

M-21

UGT1A1, p.Leu413Pro

9

CNS2-hiPSC7

CNS2-hiPSC1, 5, 7

hiPSCs were established by transduction of viral vectors containing Oct4, Sox2, c-Myc and Klf4, as described earlier [5]. The transduced cells were passaged on serum and feeder free-culture medium or hESC medium supplemented with 100 ng/ml basic fibroblast growth factor (bFGF) on Matrigel (1:30, Sigma-Aldrich, E1270). The hESC medium contained DMEM/F12 medium (Gibco, 21331-020) supplemented with 20% knockout serum replacement (KOSR, Gibco, 10828-028), 2 mM L-glutamine (Gibco, 25030-024), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, M7522), 1% nonessential amino acids (Gibco, 11140-035), 1% penicillin and streptomycin (Gibco, 15070-063), 1% insulin-transferrin-selenite (ITS, Gibco, 41400-045) and 100 ng/ml basic-fibroblast growth factor (bFGF, Royan Institute). Cells were grown in 5% CO2 with 95% humidity. After approximately 2 weeks, a few colonies which showed hESC morphology were identified. Several colonies were selected for initial characterization. For passaging, the hiPSCs were washed once with PBS (Gibco, 14287-072) and then incubated with DMEM/F12 containing collagenase IV (1 mg/ml, Gibco, 17104-019), at 37°C, for 5–7 min. When colonies on the dish periphery began to dissociate from the base, the enzyme was removed and colonies were washed with PBS. The cells were gently pipetted out. The cell lines were passaged and maintained under feeder-free culture in hESC medium. The hiPSCs were frozen as explained before [6, 7].

Immunofluorescence Staining

The hiPSCs were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.1–0.2% Triton X-100 for 10–30 min and blocked in 10% goat serum in PBS for 1 h at 37°C. Incubation of cells with primary antibody was done for 1 h at 37°C, then washed and incubated with FITC-conjugated secondary antibodies, anti-mouse IgM (1:100, Sigma, F9259), anti-rat IgM (1:200, eBioscience, 11-0990) and anti-mouse IgG (1:200, Sigma, F9006) as appropriate, for 1 h at 37°C.

The following primary antibodies: anti-TRA-1-60 (1:100, Chemicon MAB4360), TRA-1-81 (1:100, Chemicon MAB4381), Oct4 (1:100, Santa Cruz Biotechnology, SC-5279) and SSEA-4 (1:100, Chemicon, MAB4304) for undifferentiated determination; anti-albumin (1:200, ALB, Santa Cruz, Sc-46293), cytokeratin18 (1:200, CK18, Chemicon, MAB16000) and CYP1A1 (1:200, Santa Cruz, Sc-48432), were used for hepatic lineage differentiation. Nuclei were counterstained with DAPI (Sigma, D8417) or propidium iodide (PI, Sigma, P4170) and cells were analyzed under a fluorescent microscope (Olympus, Japan).

Alkaline Phosphatase Staining

Alkaline phosphatase staining was conducted based on the manufacturer’s recommendations (Sigma, 86R).

RNA Isolation and Quantitative RT-PCR

Total RNA was isolated by Nucleospin RNAII kit (MN) and treated with DNaseI (Fermentas) to remove genomic DNA contamination. Two micrograms of total RNA was used for reverse transcription reaction with the RevertAid First Strand cDNA synthesis kit (Fermentas) and random oligo dT primer (Fermentas), according to the manufacturer’s instructions. Quantitative PCR reactions were set up in duplicate with the Power SYBR Green Master Mix (Applied Biosystems) and analyzed with the 7500 real-time PCR system (Applied Biosystems). Expression values were normalized to the average expression of the housekeeping genes (GAPDH for hiPSC lines and ActB for hepatic lineage cells) and relative to a calibrator [hESCs, Royan H6, [8] for hiPSC lines and undifferentiated hiPSC of the same cell-line for hepatic lineage cells] by the Comparative CT Method \( \left( {{{2}^{ - \Delta \Delta {\rm{ct}}}}} \right) \). The sequences of primers for the hiPSCs spontaneous differentiation analysis were presented before [5]. The primer sequences for HLCs analysis are presented in Supplementary Table 2.

Karyotype Analysis and Bisulfite Sequencing

Karyotype analysis was performed as described before [6]. Human Oct4 and Nanog gene promoter regions were amplified with PCR as previously described [5]. PCR products were subcloned into the InsTAclone PCR Cloning kit (Fermentas). Ten-twelve clones of each sample were sequenced with the M13 universal primers and analyzed using the BIQ Analyzer software [9].

Directed In Vitro Differentiation

The hiPSCs (CNS2hiPSC7, GSD1hiPSC7 and TYR1hiPSC1) at passages 18–25 were differentiated into hepatic lineage by the protocol of Basma et al. [10] with some modifications (Supplementary Fig. 1). Briefly, EBs were generated by plating collagenase/dispase-passaged cells at a density of 1–5 × 104 cells per cm2 on bacterial petri dishes for 48 h in DMEM/F12 supplemented with 20% KOSR, 1 mM nonessential amino acids and 2 mM L-glutamine. Then, EBs were plated on Matrigel-coated plates in DMEM/F12 supplemented with Activin A (100 ng/ml, R&D, 338-AC) for 3 days to induce definitive endoderm lineage. The concentration of KOSR was 0% for the first 24 h, 0.2% for the second 24 h and 2.0% for the final 24 h. Cells then were grown for 8 days in DMEM/F12 containing 2.0% KOSR, 1 mM nonessential amino acids, 2 mM L-glutamine, 1% dimethyl sulfoxide (Sigma-Aldrich) and 100 ng/mL HGF (R&D Systems, 294-HG), followed by culture for five additional days in DMEM/F12 containing 2% KOSR, 1 mM nonessential amino acids, 2 mM L-glutamine and 0.1 μM dexamethasone (Sigma-Aldrich, D-2915).

Flow Cytometric Analysis of Albumin Expression

Hepatic lineage directed differentiated cells underwent dissociation in 0.05% trypsin-EDTA (Sigma-Aldrich). Then, cells were washed twice in staining buffer (PBS supplemented with 1% heat-inactivated FBS, 0.1% sodium azide and 2 mM EDTA) and fixed in 4% paraformaldehyde for 15 min. For permeabilization, Triton X-100 0.1% (v/v) was used for 10 min at room temperature. Nonspecific antibody binding was blocked for 15 min at 4°C with the combination of 10% heat-inactivated goat serum in staining buffer. For each analysis, (1–5) × 105 cells were used per sample. Cells were incubated with anti-Alb (1:100) or the appropriate isotype matched controls (eBioscience, or Sigma-Aldrich) overnight at 4°C. The secondary antibody was goat anti mouse IgG (abcam, ab6785). Flow cytometric analysis was performed with a BD-FACS Calibur Flow Cytometer (Becton Dickinson). The experiments were replicated at least three times. Acquired data was analyzed with WinMDI software.

Albumin, AFP and Urea Secretion

24 h conditioned media obtained from the differentiated hiPSC-HLCs was collected at day 18 and stored at −20°C until assayed. The conditioned media were assayed for alpha-fetoprotein (AFP) secretion using a chemiluminescence immunoassay kit (Pishtaz-Teb); for ALB secretion using an ALB ELISA kit (Bethyl); and for urea secretion using a colorimetric assay kit (Pars Azmun) according to the manufacturers’ recommendations. Secretion was normalized to a defined cell number.

Periodic Acid-Schiff (PAS) Staining

Glycogen storage of hiPSC-HLCs was evaluated using PAS staining at day 18. Culture dishes containing the cells were fixed with 4% paraformaldehyde, then oxidized in 1% periodic acid for 5 min, washed and subsequently treated with Schiff’s reagent for 15 min, with subsequent color development in dH2O for 5–10 min and assessment under a light microscope (IX51, Olympus).

Cytochrome P450 Activity and Inducibility

Cytochrome P450-dependent pentoxyresorufin o-dealkylase activity (PROD) was evaluated using pentoxyresorufin substrate. Pentoxyresorufin is O-dealkylated by CYP and changes to resorufin, a fluorescent compound [11]. To evaluate the inducibility of cytochrome P450, 18-day HLCs were exposed to sodium phenobarbital for 3 days and subsequently washed. Then, an incubation mixture containing 7-pentoxyresorufin substrate (Sigma-Aldrich) and dicumarol (Sigma-Aldrich) in HBSS was added and plates were incubated at 37°C in a 5% CO2 incubator for 30 min. Nuclei were counterstained with DAPI and in situ assessment and detection of resorufin was performed by fluorescent microscopy. The percentages of positive cells of three independent experiments before and after induction for each line were counted.

Uptake of Low-Density Lipoprotein (LDL)

LDL uptake assay was performed using the DiI-Ac-LDL staining kit according to the manufacturer’s instructions (Biomedical Technologies, Stoughton, MA). Cells were visualized using a fluorescence microscope (IX71, Olympus).

Indocyanin Green Uptake and Release

Differentiated cells at day 18 were incubated with indocyanin green (ICG, Sigma-Aldrich) in basal medium for 1 h at 37°C. Uptake of ICG was detected with light microscopy (BX51, Olympus). ICG elimination from the positive cells was verified 6 h later.

Periodic Acid-Schiff (PAS) Staining

Glycogen storage of hiPSC-HLCs was evaluated using PAS staining at day 18. Culture dishes containing the cells were fixed with 4% paraformaldehyde, then oxidized in 1% periodic acid for 5 min, washed and then treated with Schiff’s reagent for 15 min, with subsequent color development in dH2O for 5–10 min and assessment under a light microscope (BX51, Olympus).

Oil Red Staining

Cells were assessed for lipid vesicle storage by Oil Red staining. Differentiated cells were fixed with 4% paraformaldehyde and then incubated for 1 h with Oil Red. Cells were washed afterwards and analyzed with a light microscope (IX51, Olympus).

Results

Verification of Disease-Specific Genotypes

Patients were sequenced with the purpose of identifying the mutations and disease-specific genotypes (Table 1). The results confirmed the specific disease types.

Generation of hiPSCs in Serum and Feeder-Free Conditions

All evaluated hiPSC lines exhibited hESC-like morphology and compact colonies and expressed high nucleus to cytoplasm ratios in addition to the presence of prominent nucleoli (Fig. 1). Six to nine colonies were selected from transduced human dermal fibroblasts (HDFs) per patient and expanded to give rise to stable cell lines of hESC-like morphology (Table 1). One hiPSC line per patient was selected for further analyses (Table 1). These lines were maintained in a continuous culture for several months by weekly passaging with a split ratio of 1:3 to 1:6.
https://static-content.springer.com/image/art%3A10.1007%2Fs12015-010-9189-3/MediaObjects/12015_2010_9189_Fig1_HTML.gif
Fig. 1

The characterization of established hiPSCs. Morphology, expression of different pluripotency and surface markers, and normal karyotypes were presented. The karyotype of other hiPSC lines was normal as indicated in Table 1. The lines are characterized after at least ten passages. Nuclei were stained with DAPI (blue)

Characterization of Established hiPSCs

Analysis of colonies with hESC-like morphology showed that the clones exhibited strong ALP activity and expressed Oct4, SSEA4, Tra-1-81 and Tra-1-60, all markers shared with hESCs. Additionally, all putative hiPSC lines also maintained a normal karyotype (Fig. 1).

The qRT-PCR analysis using primers specific for Oct4, Sox2, c-Myc, and Klf4 transgenes, indicated that the hiPSC clones silenced the expression of the retroviral transgenes with the exception of Oct4 transgene in the HER1-hiPSC1 line (Fig. 2a). We also analyzed gene expression by qRT-PCR and noted that the expressions of Oct4 and Nanog markedly increased over the respective fibroblast population, which were comparable to those in hESCs (Fig. 2b). Bisulfite genomic sequencing analyses were also performed to investigate the methylation status of the cytosine guanine dinucleotides (CpG) in the promoter regions of Oct4 and Nanog which revealed that these regions were highly unmethylated in hESCs and established hiPSCs, whereas the CpG dinucleotides of the regions were highly methylated in the parental HDFs (Fig. 2c). RT-PCR analysis of spontaneous differentiated cells showed gene expression of the lineage specific marker genes (Fig. 3a). Some differentiated EBs were characteristic of neural cells (Fig. 3b), and pigmented cells (Fig. 3c), whereas some of the EBs manifested spontaneous beating which demonstrated the formation of contractile cardiomyocytes (Fig. 3d and Supplementary Video).
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Fig. 2

The expression of hESC-marker genes in hiPSCs. a Expression levels of transgenes (relative to GAPDH) were assessed by qRT-PCR. The values from the infected HDFs (isolated 5d post-infection, HDF 5d) were set to 1. Uninfected HDF were used as negative controls. The error bar indicates standard deviation (SD). b The expression levels of endogenous factors quantified by qRT-PCR and plotted relative to GAPDH expression. c Bisulfite genomic sequencing of the promoter regions of Oct4 and Nanog. Open and closed circles indicate unmethylated and methylated CpGs

https://static-content.springer.com/image/art%3A10.1007%2Fs12015-010-9189-3/MediaObjects/12015_2010_9189_Fig3_HTML.gif
Fig. 3

The spontaneous differentiation of hiPSCs in vitro. a RT-PCR analyses of various differentiation markers for the three germ layers by EB-mediated differentiation “D” in comparison with the undifferentiated state “U”. Representative pictures of CNS1-hiPSC10. Phase contrast photomicrographs of neural cells (b), pigmented cells (c), beating cardiomyocytes (d). See Supplementary Video for beating cardiomyocytes

Directed Differentiation of hiPSCs to Hepatic Lineage Cells

We also examined the hepatic lineage-directed differentiation of selected liver disease-specific hiPSC lines (TYR1-hiPSC1, GSD1-hiPSC7 and CNS2-hiPSC7). Real-time RT-PCR was performed on hiPSCs and hiPSC-HLCs. Analysis included markers for gene expression of endoderm (SOX17 and AFP), hepatocyte (ALB, HNF4α, and CYP3A4) and undifferentiated hiPSCs marker (OCT4). As shown in Fig. 4a, endoderm and hepatocyte-specific gene expression increased whereas Oct4 expression decreased after differentiation induction. Morphological analysis of the cells revealed the presence of a polygonal epithelial morphology and large cytoplasms which contained various granules (Fig. 4b). To verify expression of hepatocyte specific genes at the protein level, immunofluorescence staining was performed for ALB, CK18, and CYP1A1. Flow cytometry analysis of differentiated cells showed that approximately 50% of the differentiated cells of each line expressed ALB (Fig. 4c). Additionally, the differentiated cells secrete albumin, AFP, and urea into the medium (Fig. 5a–c). To find out whether our cells expressed inducible CYP variants, we analyzed cytochrome p450 activity before and after induction with phenobarbital. We demonstrated an increased CYP activity between 1.3 and 2.1 fold (P < 0.05, Student’s t-test, Fig. 5d and Supplementary Fig. 2). These differentiated cells showed uptake of organic anion ICG and LDL in a fluorescence form (DiI-Ac-LDL, Fig. 5e). Glycogen and lipid storage, two other characteristics of hepatocytes, were visible on hiPSC-HLCs of the three cell lines as detected by PAS and Oil Red staining, respectively (Fig. 5f–h).
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Fig. 4

The directed differentiation of hiPSCs toward hepatic lineage. a Real time RT-PCR analysis of endoderm and hepatic markers. “D” and “U” depict the differentiated or undifferentiated states, respectively. HepG2 was used as a positive control. Messenger RNA expression levels were normalized relative to β-actin and HepG2. Relative expressions without normalization related to HepG2 and primer sequences that were used can be found in Supplementary Tables 1 and 2, respectively. b Morphology and immunofluorescence staining of hiPSC-HLCs confirming the expression of ALB, CYP1A1 and CK 18. Scale bars, 100 μm. c ALB expression was further confirmed and quantified by flow cytometry

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Fig. 5

Functional analysis of hiPSC-HLCs. ac ALB, AFP and urea secretion of the differentiated cells into the medium (results are the average of six experiments). d Cytochrome p450 induction. Pictures were taken before and after 72 h of exposure to phenobarbital and quantified. See supplementary Fig. 2 for CYP induction. Results are the average of three experiments. The increase in secretion of ALB, AFP and urea after differentiation, and increase in CYP activity after induction were statistically significant (P < 0.05, Student’s t-test). Data was shown as mean ± standard error of mean. e DiI-Ac-LDL uptake by HLCs. LDL vesicles are marked by arrows. f Uptake of ICG by the cells. After 6 h ICG was released, as depicted in the upper right corner. g Cells stored glycogen, as confirmed by PAS staining. h Lipid storage was assessed by Oil Red staining. Scale bars, 100 μm

Discussion

In this study, we showed the generation of hiPSCs from adult HDFs of patients with liver diseases by retroviral transduction of Oct4, Sox2, c-Myc, and Klf4 with the use of serum and feeder-free culture conditions. We analyzed colonies selected for hESC-like morphology, expression of ALP, Oct4, SSEA4, Tra-1-60, Tra-1-81, and Nanog—all markers shared with hESCs [12], and the epigenetic silencing of Oct4, Sox2, c-Myc and Klf4 transgenes in hiPSCs [1315]. Moreover, the promoter of Oct4 and Nanog was highly demethylated during reprogramming [16]. The established hiPSCs showed the expression of three embryonic germ layer markers after spontaneous differentiation. Animal models of these diseases have been previously reported (for review see [3]). Moreover, our results showed that the directed hepatic differentiation efficiency and functionality of liver disease-specific hiPSCs was comparable to that of the hESC lines [10]. Several recent papers have reported that regardless of somatic cell origin, the generated hiPSCs can be directed to hepatocytes in the same manner as hESCs [1720].

Since liver disease-specific hiPSCs can be generated with relative ease from any individual, the immediate potential benefit of hiPSCs will be in the exploration of disease etiology. Moreover, these data suggest that hiPSC-HLCs can provide us with a powerful resource for the generation of hepatocytes which can be used to study basic developmental mechanisms and for drug screening, as well as a cell source for the study of ethnic/polymorphic variation on xenobiotic metabolism. A hiPSC bank could be established through identification and reprogramming of human fibroblasts displaying different genetic and non-genetic liver disorders for different ethnic backgrounds. However, the demonstration of disease-related phenotypes and the ability to model pathogenesis remain challenging in this field [21].

In conclusion, our results open up a proof of concept that multiple liver-disease specific hiPSC lines can be generated and differentiated into functioning HLCs in a relatively simple and straightforward manner with high efficiency. Moreover, creation of hiPSC lines from patients with single-gene disorders will provide an opportunity to repair gene defects ex vivo.

Acknowledgements

This study was funded by a grant provided from Royan Institute and Iranian Stem Cell Council.

Financial support

This study was funded by a grant provided from Royan Institute and Iranian Stem Cell Council.

Disclosures

None of the authors have any conflicts of interest to disclose and all authors support submission to this journal.

Supplementary material

12015_2010_9189_MOESM1_ESM.doc (32 kb)
Supplementary Table 1Relative mRNA levels of lineage-specific hepatic markers [albumin (ALB), hepatic nuclear factor 4α (HNF4α) and CYP3a4], endoderm markers (SOX 17 and AFP), and markers for undifferentiated cells (Oct4) in differentiated and undifferentiated states. Data are normalized to β-actin and depicted as mean ± standard error of mean. (DOC 31 kb)
12015_2010_9189_MOESM2_ESM.doc (38 kb)
Supplementary Table 2Primer sequences and conditions of real time RT-PCR. (DOC 37 kb)
12015_2010_9189_MOESM3_ESM.doc (108 kb)
Supplementary Fig. 1A schematic view of protocol for the directed differentiation of hiPSCs toward HLCs. (DOC 107 kb)
12015_2010_9189_MOESM4_ESM.doc (501 kb)
Supplementary Fig. 2PROD assay on hiPSC-HLCs of three lines at day 18, before and after CYP450 induction with phenobarbital. (DOC 501 kb)
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