Hepatic population derived from human pluripotent stem cells is effectively increased by selective removal of undifferentiated stem cells using YM155
© The Author(s). 2017
Received: 3 September 2016
Accepted: 21 February 2017
Published: 17 April 2017
Pluripotent stem cells (PSCs) such as embryonic stem cells and induced pluripotent stem cells are promising target cells for cell regenerative medicine together with recently advanced technology of in-vitro differentiation. However, residual undifferentiated stem cells (USCs) during in-vitro differentiation are considered a potential risk for development of cancer cells and nonspecific lineage cell types. In this study we observed that USCs still exist during hepatic differentiation, consequently resulting in poor quality of the hepatic population and forming teratoma in vivo. Therefore, we hypothesized that effectively removing USCs from in-vitro differentiation could improve the quality of the hepatic population and guarantee safety from risk of teratoma formation.
Human PSCs were differentiated to hepatocytes via four steps. YM155, a known BIRC5 inhibitor, was applied for removing the residual USCs on the hepatic differentiation. After YM155 treatment, hepatocyte development was evaluated by measuring gene expression, immunostaining and hepatic functions at each stage of differentiation, and forming teratomas were confirmed by cell transplantation with or without YM155.
The selected concentrations of YM155 removed USCs (NANOG+ and OCT4+) in a dose-dependent manner. As a result, expression of endodermal markers (SOX17, FOXA2 and CXCR4) at stage II of differentiation and hepatic markers (ALB, AFP and HNF4A) at stage III was up-regulated by YM155 treatment as well as the hepatic population (ALB+), and functions (ALB/urea secretion and CYP450 enzyme activity) were enhanced at the final stage of differentiation (stage IV). Furthermore, we demonstrated that NANOG and OCT4 expression remaining until stage III (day 15 of differentiation) completely disappeared when treated with YM155 and teratoma formation was effectively prevented by YM155 pretreatment in the in-vitro study.
We suggest that the removal of USCs using YM155 could improve the quantity and quality of induced hepatocytes and eliminate the potential risk of teratoma formation.
KeywordsPluripotent stem cells Hepatic differentiation Residual undifferentiated stem cell YM155
Human pluripotent stem cell (PSC)-induced hepatocytes (iHeps) are a promising target for drug development and cell transplantation. Over the past 10 years, many protocols for hepatocyte induction from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been developed by recapitulating in-vivo hepatic development [1–6]. Acquiring pure hepatocytes is a crucial factor for hepatotoxicity screening of drug candidates and stem cell-derived hepatocyte therapy. Thus far, hepatocyte induction has enabled progress in acquiring highly purified hepatocytes and in enhancing hepatic functions [3, 7–9].
There are many previous reports about undifferentiated stem cells (USCs) that remain during in-vitro differentiation. The residual USCs exist long term in both in-vitro and in-vivo differentiation . This is a considerably critical concern in cell replacement therapy because the residual stem cells have inherent problems (i.e., teratoma formation) [11–13]. Therefore, eliminating the residual USCs is an indispensable step for safer cell therapy and efficient hepatotoxicity screening of drug candidates. Recently, selective removal of USCs has been tried to prevent tumorigenic potential and teratoma formation using antibody-based strategy in clinical fields [14–16]. In addition, small molecules have been suggested to effectively eliminate the risk of teratomas [17, 18]. Of these molecules, YM155 is one of the inhibitors targeting the BIRC5 (Survivin) gene, an anti-apoptotic factor, which is highly overexpressed in ESCs/iPSCs and cancer stem cells. Thus, this molecule can induce selective apoptotic cell death of USCs . Currently, the compound has been introduced in clinical applications for cancer therapy and interruption of teratoma formation after stem cell therapy .
In our former study, USCs remained in a small portion during hepatic differentiation and consequently developed nonhepatic phenotypes . Lee et al.  reported that YM155 effectively eliminated USCs without affecting differentiated counterparts. However, little research has been conducted to selectively remove the undifferentiated cells during in-vitro hepatic differentiation. In the present study, we investigated the effects of YM155 on hepatic differentiation using human PSCs concerning the effective treatment stage of differentiation and optimal concentration of YM155 and in-vivo teratoma formation. To our knowledge, this is the first report of applying YM155 to in-vitro hepatic differentiation.
Human ESCs (WA01) and iPSCs (iPS(IMR90)-1) were purchased from the WiCell Research Institute (WI, USA; http://wicell.org/). Human iPSCs (QIA7) were established from adipose tissue-derived stromal cells in our laboratory . These cells were mechanically passaged using Dispase (STEMCELL Technologies, Canada) and maintained on a Matrigel-coated plate (BD Bioscience, CA, USA) with mTeSR1 medium (STEMCELL Technologies). HepG2 (ATCC, VA, USA) was cultured in EMEM (ATCC) containing 10% FBS (Gibco-BRL, NY, USA) and 1% penicillin and streptomycin (Millipore, MA, USA). Human cryopreserved primary hepatocytes (p-Heps) were purchased as BD Gentest™ Inducible-Qualified Human CryoHepatocytes (Lot #178; BD Bioscience). Human hepatocytes were cultured in HMM medium using a SingleQuots™ kit (Lonza, MD, USA) on Coning BioCoat Collagen I-coated plates (BD Bioscience). Human adipose tissue-derived stromal cells (hAT-SCs) were maintained with DMEM containing 10% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin.
Differentiation of PSCs into hepatocytes
For the hepatic differentiation of human PSCs, a previously reported hepatic differentiation protocol designed for human ESCs  was applied with some modifications. Briefly, human PSCs were plated at a density of 2.5 × 105 cells/ml on Matrigel-coated six-well plates with mTeSR1 medium (STEMCELL Technologies) including ROCK inhibitor (Y27632; STEMCELL Technologies). The medium was replaced with definitive endodermal induction medium (DE) for 5 days (stage I). The DE medium consisted of RPMI 1640 (without l-glutamine; Gibco-BRL) supplemented with 2 mM l-glutamine (Millipore), 0.5 mg/ml albumin fraction V (Merck Millipore, Germany) and 100 ng/ml Activin A (PeproTech, NJ, USA). Afterward, the definitive endodermal cells were differentiated into hepatoblasts using the hepatic endodermal medium (Hep-1) for 5 days (stage II) followed by hepatic specification medium (Hep-2) for 5 days (stage III). Hep-1 and Hep-2 comprised the HBM SingleQuots™ kit (Lonza) supplemented with 30 ng/ml FGF4 (PeproTech) and 20 ng/ml BMP2 (Invitrogen, MD, USA) at stage II and 20 ng/ml HGF (PeproTech) and 20 ng/ml OSM (R&D Systems, MN, USA) at stage III, respectively. After hepatic specification, the cells were further matured using the HMM SingleQuots™ kit for 5 days (stage IV).
Total RNA was isolated from the cells using the RNeasy mini kit (Qiagen, Germany). cDNA was synthesized from 1 μg of total RNA primed with AcuPower cDNA synthesis premix (Bioneer, Republic of Korea). Primer sets are represented in Additional file 1: Table S1. Real-time PCR was performed using the CFX (Bio-Rad, CA, USA) instrument and the SYBR Green script premix (Bio-Rad). Normalization of samples was determined by GAPDH, and all sets of reactions were conducted in triplicate (n = 3). The relative expression levels are expressed as a fold-change of the indicated control.
For immunostaining, the cells were fixed with 4% paraformaldehyde (PFA; Thermo Scientific) for 5 min after washing with 1× phosphate-buffered saline (PBS). After rinsing with 1× rinse buffer (1× Tris–HCl including 0.05% Tween-20), permeabilization was performed with 0.1% Triton X-100 for 10 min. After blocking with blocking solution (1:20 diluent with 1× PBS) for 30 min at room temperature, each primary antibody was reacted by incubating overnight at 4 °C. After rinsing, secondary antibodies were applied in incubation for 1 h at room temperature. Information for antibodies is listed in Additional file 1: Table S2. The nuclei were stained for 1 min with Hoechst33258 (Invitrogen) diluted in 1× PBS (1:10,000). Immunofluorescence was detected under a fluorescence microscope (Axiovert; Carl Zeiss, Germany).
To measure cytotoxicity by YM155 treatment, QIA7 (stage 0), QIA7-iHeps (stage I and stage II) and hAT-SCs were seeded on Matrigel-coated 96-well plates (2 × 104 cells per well) and cultured for 24 h at 37 °C. Serial concentrations of YM155 (0–100 μM) were diluted with each culture medium as already described and incubated with the cells for 24 h. The culture medium was subsequently replaced with 100 μl of each fresh medium containing 10% (v/v) 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetraxolium (WST) reagent (DoGEN, Republic of Korea) and again incubated for 2 h at 37 °C. Absorbance was read using a luminometer (FlexStation III; Molecular Device, CA, USA) at 450 nm, and the results were expressed as the percent of nontreated control.
To detect apoptotic cell death, we used the Caspase-3 colorimetric activity assay kit (Millipore). QIA7, QIA7-iHeps (day 7 of differentiation) and hAT-SCs were seeded with a relevant cell number and incubated overnight at 37 °C. Each cell was treated by serial concentrations of YM155 (1–100 nM) for 16 h. After lysis with 1 × 106 cells, the supernatant was incubated with Caspase-3 substrate for 1 h at 37 °C. Absorbance was read using a luminometer (FlexStation III) at 405 nm, and the activities were expressed as a fold-increase against DMSO control.
Cells were dissociated in 0.05% Trypsin–EDTA (Invitrogen) and then resuspended in 10% FBS/DMEM (v/v). The collected cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and stained with an antibody against OCT4 and Albumin. The analyses were performed using FACSCalibur (BD Biosciences).
Functional assay for hepatocytes
To test hepatic functions with stem cell iHeps at the final day of stage IV (day 20 of differentiation), the differentiated cells were dissociated in Accutase for 30 min and seeded on Collagen I-coated plates with HMM medium. After overnight incubation at 37 °C, each functional test such as periodic acid Schiff (PAS) staining, low-density lipoprotein (LDL) uptake, albumin/urea secretion, CYP450 enzyme activity and drug metabolism was carried out as described in the following. HepG2 and p-Heps were used as control cells.
To detect glycogen storage, PAS staining was performed at day 21 of differentiation. The cells were fixed for 5 min with 4% PFA and oxidized for 5 min in periodic acid (Sigma-Aldrich, MO, USA) at room temperature. After rinsing with distilled water for 5 min, the cells were treated with Schiff’s reagent (Sigma-Aldrich) for 15 min.
To estimate uptake of low LDL, LDL-DyLight™ 550 (Abcam) was diluted with culture medium (1:100). Cells were incubated with LDL-DyLight™ 550 working solution for 4 h at 37 °C. The medium was replaced with fresh culture medium. Immunofluorescence was detected under a fluorescence microscope (Axiovert).
Albumin and urea secretion
The culture medium was changed at day 21 of differentiation, and the cells were additionally incubated for 24 h at 37 °C. The supernatants collected from each well were centrifuged for 5 min at 3000 × g to remove floating cells and stored at –20 °C until assay. The albumin and urea amounts in culture medium were measured using an Albumin Human ELISA kit (Abnova, CA, USA) and urea assay kit (Cell Biolabs, CA, USA), respectively, according to the manufacturer’s instructions. Absorbance was read on a luminometer (FlexStation III) at a wavelength of 450 nm for albumin and 630 nm for urea. The albumin and urea amounts were calculated using each standard curve and normalized by protein concentration (mg/ml).
CYP450 enzyme activity
CYP1A2 and CYP3A4 enzyme activities were measured using the CYP450-Glo™ assay kit (Promega, WI, USA) according to the manufacturer’s instructions. The supernatants were removed, and the cells were incubated with substrate (Luciferin-1A2 for CYP1A2 and Luciferin-IPA for CYP3A4) for 1 h. The supernatants of each well were transferred to white opaque 96-well plates. CYP450 activities were then measured using a luminometer (FlexStation III). The results were expressed as a relative activity for control.
To evaluate drug metabolism, 1 μM aflatoxin B1 (Sigma-Aldrich) and 100 μM acetaminophen (Sigma-Aldrich) diluted with HMM medium treated QIA7-iHeps for 24 h, and medium containing test drugs was used as control (no cells). The supernatants were collected, and the concentrations of each compound in the supernatants were determined by HPLC (Waters 2996; Waters, MA, USA). Drug clearance in p-Heps was also performed under the same method. The values were normalized by protein concentration (mg/ml) and expressed by the percentage of control.
For in-vivo cell transplantation, QIA7 and QIA7-iHeps (day 7 of differentiation) pretreated with and without YM155 (5 nM) were dissociated by Dispase and Accutase, respectively. Approximately 1 × 106 cells were prepared in DMEM/F12 (50 μl) and mixed with Matrigel (1:1) on ice. The mixture was injected into the testis of 6-week-old nude mice (BkINbt:BALB/c/nu/nu; NARA-Biotech, Republic of Korea). Six or seven weeks later, the teratomas were dissected. Tumor masses were fixed with 10% neutral buffered formalin (Sigma-Aldrich). Paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin (H&E) and analyzed in the Cell Imaging-Histology core facility at the Quarantine & Inspection Agency.
Results were expressed as the mean ± standard deviation (SD) for triplicate experiments (n = 3). The statistical significance was determined using Statistica5.5 (StatSoft, OK, USA) with one-way analysis of variance (ANOVA) and post-hoc comparisons between the control group and each treatment group using Duncan’s multiple comparison test. p < 0.05 was considered statistically significant.
Hepatic differentiation of human PSCs with the modified protocol
Characterization of nonhepatic lineage cells derived from USCs
Effects of YM155 on hepatic differentiation
Evaluation of hepatic induction followingYM155 treatment at stage II
YM155 induces selective cell death of USCs and prevents teratoma formation
In the present study, we confirmed that human PSCs differentiated into hepatocyte-like cells via four sequential steps modified from the protocol of Cai et al.  by demonstrating the expression of hepatic genes, such as HNF4A, AFP and ALB, and functional hepatic proteins, such as ALB and AFP. Contrary to our expectation, expression of ALB and HNF4A decreased at stage IV. This might be derived from a nonoptimal culture condition of stage IV (no evaluation in this study) or a relatively low ratio of hepatic purity due to expansion of nonhepatic lineage cells. Morphologically nonhepatic cells (A1) were actually observed at stage III and expanded rapidly during the differentiation. This cell population (A1) showed a distinct morphology against hepatic population as a counterpart (A2) and also contained USCs (Fig. 2d). Additionally, those cells were negative for albumin protein (mature hepatocyte marker), overexpressed AFP and IGF2 (fetal hepatic genes) and expressed mesoderm and ectoderm origin genes. These findings suggest that these cell clusters were derived from USCs and finally reduced cellular homogeneity at the final stage of hepatic differentiation. Therefore, the efficiency of hepatic differentiation could be improved when USCs are selectively eliminated during differentiation.
Small molecules target anti-apoptotic factors to remove residual USCs [17, 18, 22]. Of these small molecules, YM155 in particular triggers apoptosis of USCs but not that of their differentiated derivatives . Likewise, we also demonstrated that human PSCs were uniquely affected by YM155 within the specific ranges (1–10 nM) more than differentiating cells and somatic cells (Fig. 3a). However, it was difficult to apply YM155 at stage I because almost all cells were easily detached from the plate. Therefore, YM155 was applied at stage II. After YM155 treatment, we confirmed apoptotic cell death by apoptotic gene expression, Caspase-3 activity and Annexin V staining. This apoptosis was defined in USCs positive against TRA-1-60 and OCT4. Taken together, YM155 effectively removed USCs at stage II of the differentiation procedure. Finally, this selective elimination resulted in enhancing the expression of endodermal marker genes (CXCR4, SOX17 and FOXA2) at stage II and hepatic marker genes (ALB, AFP and HNF4A). We think these increases might be compensation following the decrease of nonspecific lineages.
Drug metabolization is one of the key functions of the liver. The role is primarily conducted by phase I enzymes (CYP450 enzymes) during xenobiotic exposure. Here, the activity of CYP1A2 and CYP3A4 in QIA7-iHeps was significantly increased by 5 nM of YM155 compared with no treatment due to the decrease of the nonhepatic population. However, drug clearance of QIA7-iHeps and WA01-iHeps was significantly lower than that of p-Heps. Aflatoxin B1 and acetaminophen used in this study were metabolized mainly by CYP1A2 and CYP3A4 in hepatocytes. In our previous study, the activity of CYP1A2 and CYP3A4 in QIA7-iHeps and WA01-iHeps was approximately half that of primary hepatocytes . The data are considerably correlated with drug clearance in this study. These lower metabolic activities in QIA7-iHeps and WA01-iHeps compared with p-Heps might be derived from the individual variation of CYP450 genes (i.e., single nucleotide polymorphism) and/or levels of hepatic purity and maturation. A combination of YM155 and additional factors [5, 7, 23, 24] as well as a 3D culture system [8, 25, 26] will be able to demonstrate these issues.
We confirmed that pluripotent genes (NANOG and OCT4) were completely down-regulated after YM155 treatment and were nearly prevented at the final day of stage II. Finally, expression was not detected at 15 day of differentiation. Under our optimized concentration of YM155 at stage II, USCs could efficiently induce apoptotic cell death. Also, YM155 effectively prevented teratoma formation in our in-vivo study. Interestingly, teratoma occurrence was observed in the transplantation of QIA7-iHeps without YM155. This might be derived from residual USCs on differentiation. Residual USCs accounted for approximately 20–30% of the whole differentiating cells based on the expression of pluripotent marker genes and proteins at stage II. Arithmetically, we can presume that approximately 2 × 105 cells remaining as USCs were injected into the testis. Finally, these cells induced teratoma (40%). Based on a previous study , this cell number is sufficient to induce teratoma formation. On the contrary, QIA7-iHeps pretreated with YM155 did not form anything. Regarding cell therapy, the elimination of residual USCs is one of the major concerns to guarantee safety. Based on our evidence, we will be able to utilize the differentiated cells safely as a donor in cell transplantation.
YM155 could selectively eliminate USCs with an effective treatment time point and optimal concentration on hepatic differentiation. The removal of residual USCs enhanced the efficiency of hepatic differentiation as well as reduced a potential risk of teratoma formation. To our knowledge, this is the first report to demonstrate the effects of YM155 on in-vitro hepatic differentiation. Based on recently advanced technology of hepatic differentiation, application of YM155 will allow generation of a pure hepatocyte population and safety of cell transplantation in regenerative medicine.
Bcl-2-like protein 4
B-cell lymphoma 2
Baculoviral inhibitor of apoptosis repeat-containing 5
Bone morphogenetic protein2
C-X-C chemokine receptor type 4
Dulbecco’s Modified Eagle Medium
Eagle’s minimum essential medium
Fetal bovine serum
Fibroblast growth factor 4
Forkhead box protein A2
Glyceraldehyde 3-phosphate dehydrogenase
Transcription factor GATA-4
Hepatocyte growth factor
Hepatocyte nuclear factor 4 alpha
Insulin growth factor2
Neural cell adhesion molecule
Octamer-binding transcription factor
Periodic acid Schiff
Paired box protein Pax-6
Rho-associated protein kinase
Sex determining region Y-Box 17
Cardiac muscle troponin T
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This project was supported by research funds from Animal and Plant Quarantine Agency (QIA), Republic of Korea.
Availability of supporting data
All data generated or analyzed during this study are included in this published article and its supplementary information files.
S-JK, H-GK, H-OK and J-YS were responsible for experimental design and data analysis. S-JK and H-GK performed the experiments, assembled and interpreted the data, and wrote the manuscript. Y-IP, HY and NT assisted with cell culture, PCR and immunostaining. S-RH was responsible for teratoma formation and animal care. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethical approval and consent to participate
All animal experiments were performed according to the Code of Laboratory Animal Welfare Ethics, Animal and Plant Quarantine & Inspection Agency (QIA), Republic of Korea. All surgeries and sacrifices were performed under anesthesia. Every effort was made to minimize animal suffering.
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