Suppressing Pitx2 inhibits proliferation and promotes differentiation of iHepSCs

Accepted Manuscript Title: Suppressing Pitx2 inhibits proliferation and promotes differentiation of iHepSCs Author: Fei Chen Ph.D Hao Yao Minjun Wang Bing Yu Qinggui Liu Jianxiu Li Zhiying He Yi-Ping Hu PII: DOI: Reference:

S1357-2725(16)30286-2 http://dx.doi.org/doi:10.1016/j.biocel.2016.09.024 BC 4996

To appear in:

The International Journal of Biochemistry & Cell Biology

Received date: Revised date: Accepted date:

28-6-2016 18-9-2016 29-9-2016

Please cite this article as: Chen, Fei., Yao, Hao., Wang, Minjun., Yu, Bing., Liu, Qinggui., Li, Jianxiu., He, Zhiying., & Hu, Yi-Ping., Suppressing Pitx2 inhibits proliferation and promotes differentiation of iHepSCs.International Journal of Biochemistry and Cell Biology http://dx.doi.org/10.1016/j.biocel.2016.09.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Suppressing Pitx2 inhibits proliferation and promotes differentiation of iHepSCs

Authors: Fei Chena,1, Hao Yaoa,1, Minjun Wanga,1, Bing Yua, Qinggui Liua, Jianxiu Lia, Zhiying Hea, Yi-Ping Hua,* aDepartment

of Cell Biology, Center for Stem Cell and Medicine, Second

Military Medical University; 1These

authors contributed equally to this work;

*Correspondence

author.

Address reprint requests to: Yi-Ping Hu, Ph.D., Department of Cell Biology, Second Military Medical University, 800 Xiangyin Road, Shanghai, 200433, P.R. China. Tel: +86-21-81870943. Fax: +86-21-81870948. E-mail: [email protected]

Highlights: 

Pitx2 knockdown decreased the expression of hepatic stem cell markers in iHepSCs

 

Pitx2 knockdown significantly inhibited the proliferation of iHepSCs Proliferation inhibition was caused by G1/S transition arrest through p53-p21 pathway



Pitx2 knockdown promoted iHepSCs differentiation rapidly and efficiently

Abstract Induced hepatic stem cells (iHepSCs) have great potential as donors for liver cell therapy due to their abilities for self-renewal and bi-potential differentiation. However, the molecular mechanism regulating proliferation and differentiation of iHepSCs is poorly understood. In this study, we provide evidence that the homeodomain transcription factor, Pitx2, is essential to maintain iHepSCs stem cell characteristics. Suppressing Pitx2 expression in iHepSCs by lentivirus mediated specific shRNA markedly reduced the expression of the hepatic stem cell-associated genes (Lgr5, EpCAM, and Sox9) with concomitant inhibition of proliferation by blocking the G1/S phase transition, and these phenotypic changes were reversed upon re-expression of Pitx2. Pitx2 knockdown also resulted in up-regulation of the p53-induced Cdk inhibitor p21, and down-regulation of its downstream effector CDK2-Cyclin E kinase complex. Furthermore, we observed that iHepSCs were more efficiently induced to differentiate into both hepatocytes and cholangiocytes when Pitx2

expression was suppressed, as compared to unmanipulated iHepSCs. These findings reveal that Pitx2 expression may be leveraged to control the status of iHepSCs during expansion in vitro to provide a strategy for further application of iHepSCs in liver cell therapy.

Abbreviations Pitx2, paired-like homeodomain transcription factor 2; iHepSCs, induced hepatic stem cells; shRNA, small hairpin RNA; Alb, albumin; DAPI, 4’, 6-diamidino-2-phenylindole; DiI-ac-LDL, DiI-labelled acetylated low-density lipoprotein; Aat, α-anti-trypsin; Ttr, tranthyretin; G-6-p, glucose 6-phosphate; qRT-PCR, quantitative real-time polymerase chain reaction.

Keywords: iHepSCs, Pitx2, knockdown, proliferation, differentiation

1. Introduction Hepatic stem/progenitor cells are tissue-specific stem cells in the liver endowed with the ability to self-renew and the bi-potential ability to differentiate into hepatocytes and cholangiocytes (Cardinale et al., 2012; Miyajima et al., 2014). Current efforts to obtain hepatic stem cells have met with little success (Grompe, 2014; Hindley et al., 2014; Huch, 2015; Miyajima et al., 2014). We previously reported generating induced hepatic stem cells (iHepSCs) from mouse embryonic fibroblasts by two confirmed transcription factors, Hnf1β and Foxa3 (Yu et al., 2013). These iHepSCs possess hepatic stem cell characteristics, can be expanded for at least 30 passages in vitro, express hepatic stem cell-associated genes (e.g. Afp, Alb, CK19, Sox9, EpCAM), and have bi-potential differentiation ability. The generation of iHepSCs opens an avenue for cell therapy and provides a utility in disease modeling (Miyajima et al., 2014; Yu et al., 2013). However, major obstacles exist for further application. For instance, there is no known way to generate significant amounts of iHepSCs that retain stem cell characteristics. Furthermore, it remains problematic to efficiently induce iHepSCs to differentiate when needed. These are problems not unique to hepatic stem cells but common for other stem cells as well (Grompe, 2012). In general, little is known about the mechanism of maintaining stem cell identity and controlling the fate of stem cell differentiation (Hindley et al., 2014; Miyajima et al., 2014). Pitx2 is a homeodomain transcription factor that is required for the

development of the eyes, teeth, heart, lungs, gut and central nervous system (Kieusseian et al., 2006). Pitx2 is involved in various functions, including regulation of cell growth and differentiation, but the function of Pitx2 in the liver remains unclear (Heldring et al., 2012; Hirose et al., 2011). Jochheim-Richter et al (Jochheim-Richter et al., 2006) reported that Pitx2 is highly expressed in the fetal liver at embryonic day (ED) 9.5 and also in the adult liver at 24 hours after partial hepatectomy, which are the times when hepatic stem/progenitor are mostly likely to contribute to liver development and regeneration (Furuyama et al., 2011; Si-Tayeb et al., 2010). We therefore hypothesized that Pitx2 functions in liver by regulating stem cell proliferation or differentiation. By analysis of iHepSCs and hepatocytes, we found that Pitx2 was highly expressed in iHepSCs but expression was negligible in hepatocytes. Therefore, we aimed to identify the role of Pitx2 in regulating the fate of iHepSCs. In this study, we suppressed Pitx2 in iHepSCs by lentivirus-mediated small hairpin RNA interfering technology (Manjunath et al., 2009). It was found that Pitx2 suppression resulted in a number of observable consequences in iHepSCs: the expression of hepatic stem cell-associated genes (Lgr5, EpCAM and Sox9) was significantly reduced; the proliferation ability and colony formation efficiency were also markedly inhibited when Pitx2 was down-regulated. Furthermore, iHepSCs with diminished Pitx2 could be rapidly and efficiently differentiated into both hepatocytes and cholangiocytes. These observations demonstrate a direct role of Pitx2 in maintaining stem cell characteristics of

iHepSCs and provide a strategic axis to manipulate iHepSCs for potential clinical benefit (Daley, 2012).

2. Materials and Methods 2.1. Cell Culture Mouse induced hepatic stem cells (iHepSCs) and 293T were from our own laboratory. The iHepSC culture medium was prepared as previously described (Yu et al., 2013) and 293T cells were cultured in DMEM (Thermo Fisher, USA) with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco). Medium was changed every other day. Appropriate density of iHepSC was applied for the lentivirus infection and further experiments. 2.2. Isolation of hepatocytes Hepatocytes were isolated by in situ liver perfusion as previously described (Wang et al., 2014; Wang et al., 2003). Generally speaking, after mouse was anaesthetized, the liver was perfused by a two-step perfusion with collagenase D (Roche) for 30-40 min at 37°C by using a pump. The digestion mix was filtered through a 70-μm nylon mesh to get a single cell suspension. After washing twice with DMEM (Gibco) containing 10% fetal bovine serum, the final cell suspension was collected. 2.3. Lentivirus production and administration Three shRNA sequences for the Pitx2 gene were chosen (all from

Sigma-Aldrich) for the lentivirus vector and unrelated control scrambled sequence was chosen (listed in the supplemental information). The sequence encoding mouse Pitx2 was amplified by PCR using primers listed in the supplemental information. Lentiviruses were generated in 293T cell by adding pMD2.G and pSPAX2 vectors with each 3 interfered vector or control vector and also overexpressing vector into 5 independent dishes, respectively. The lentiviral particles were harvested 48 hours after co-transfection and filtered through a 0.45 mm cellulose acetate filter (All plasmids were supplied by Addgene, and protocols were detailed in the website of Addgene: http://www.addgene.org/tools/protocols/). 2.4. Quantitative real-time PCR Total RNA from cells was isolated with TRIzol reagent (Invitrogen, USA) according to the manufacture’s instruction. 2 ug of RNA was reversed to cDNA using the SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Mouse specific primers including Pitx2, Sox9, EpCAM, Lgr5, Ttr, Hnf4a, Gja1, Ggt, G6P, Cyp7a1, CK19, Alb, Abcg2, Aat and GAPDH were designed by Oligo 7 (listed in the supplemental information). Quantitative real-time PCR(qRT-PCR) was performed in three repeats of each sample using ABI-7900 (Applied Biosystems, USA) with SYBR Green Premix Ex Taq (Takara). Fold change was calculated by the 2-△△CT method (Wang et al., 2014). 2.5. Western Blotting

Total protein was extracted from cells using Total Protein Extraction Kit (Merck Millipore, Germany) according to manufacturer’s protocols. Proteins (50 µg) were separated on 10% or 12% SDS–polyacrylamide gels and electroblotted onto polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were blocked with blocking buffer for 1hour at room temperature and then incubated with primary antibodies at 4℃ overnight. Then, membranes were washed three times with PBST and incubated with HRP-conjugated secondary antibody at 37℃ for 30min. Protein bands were detected by the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher). 2.6. Immunofluorescent staining For immunofluorescent staining, cells were fixed with 4% paraformaldehyde (PFA) for 20 min, washed by PBS-Tween (3 times per 5 minutes) and blocked with blocking buffer (PBS containing 0.1% Tween 20, 1% BSA) for 30 minutes at room temperature. Cells were then incubated with primary antibody at 4 °C overnight, followed by fluorescence-labeled secondary antibodies at 37 °C for 30 min. Nuclei were stained with DAPI (Molecular Probes, USA). Images were acquired with a 50i Nikon fluorescence microscope (Nikon). The primary and secondary antibodies were listed in the supplemental information. Images were processed with Adobe Photoshop CS5 software. 2.7. Cell cycle analysis Flow cytometry for assessing the distributions of cell cycle was performed

by using Becton, Dickinson FACS Aria (BD Biosciences, USA). Cells were digested into single cell suspension by EDTA-Trypsin (Gibco), washed in PBS, fixed in 70% ice-cold ethanol at 4℃ overnight. Then the cells were stained with propidium iodine (PI, 50ng/ml, BD Biosciences) at 37℃ for 15 min at room temperature. Cell cycle analysis was carried out with FlowJo software. 2.8. Colony formation assay Cells were digested by EDTA-Trypsin and suspended as single cell by culture medium. Then cells were plated into 60 mm dish (Corning, USA) at the density of 200 cells per dish. Cells were fixed with 4% PFA for 15 minutes at room temperature and stained by Giemsa (Sigma, USA) solution for 30 minutes at room temperature. Clones were counted (an individual clone >50 cells) at low magnification. The colony forming efficiency (CFE) was calculated by the following formula: CFE= (clone number/plated cell number) × 100%. 2.9. Cell Counting Kit 8 assay Cell proliferation ability was assessed by cell counting kit-8 (CCK8, DOJINDO, Japan). The procedure was followed by the manufactures instruction. 2.10. In vitro differentiation Both hepatic and cholangiocytic were performed as previously described (Yu et al., 2013). 2.11. Low-density lipoprotein (LDL) uptake assay DiI-labelled acetylated low-density lipoprotein (DiI-ac-LDL, Molecular

Probes) was added to the culture medium. 2 h later, the cells were washed with medium and imaged by fluorescence microscopy. 2.12. Statistical Analysis All experiments were performed at least three times. Data were expressed as the mean ±standard deviation (S.D.). Statistical analyses were carried out with GraphPad Prism 5.0. The differences between groups were analyzed by using a Student’s t-test or one-way ANOVA. p values<0.05 was considered statistically significant.

3. Results 3.1. Pitx2 is highly expressed in iHepSCs In our previous study (Yu et al., 2013), we showed that iHepSCs generated from mouse embryonic fibroblasts possessed typical hepatic stem cell features including self-renewal ability. These iHepSCs could be stably expanded in vitro and differentiate into hepatocytes and cholangiocytes. However, how the iHepSCs maintained their stem cell characteristics and controlled differentiation remains poorly understood. Jochheim-Richter et al. reported that homeodomain transcription factor Pitx2 was highly expressed in both fetal and adult livers 48 hours post partial hepatectomy (Jochheim-Richter et al., 2006), indicating that Pitx2 might play a vital role in the liver development or injury repair, both of which may be associated with hepatic stem cell behavior (Furuyama et al., 2011; Si-Tayeb et al., 2010). Thus, we

detected Pitx2 expression in both iHepSCs and hepatocytes by Western blotting assay, and found that Pitx2 was highly expressed in iHepSCs (two colonies of iHepSCs which were confirmed in previously studies were selected, Yu et al., 2013) while Pitx2 was rarely in hepatocytes (Fig. 1). According to these findings, we hypothesized that Pitx2 participated in maintaining hepatic stem cell features.

3.2. Down-regulation of Pitx2 by lentivirus mediates shRNA interfering To investigate the role of Pitx2 in iHepSCs, Pitx2 expression was suppressed by small hairpin RNA (shRNA) using lentivirus delivery. Three independent Pitx2 shRNA sequences were chosen from Sigma-Aldrich shRNA tools. Plasmid containing U6 promoter-driven Pitx2 shRNA-1, 2, 3 or an unrelated control scrambled sequence (named as pLKO) was co-transfected with packaging plasmids into 293T cells to generate independent lentivirus. iHepSCs were infected with the lentiviral constructs, and each group of infected cells was named as shPitx2-1, shPitx2-2 shPitx2-3 and pLKO, respectively. The knockdown efficiency of shPitx2 was evaluated by qRT-PCR and Western blotting. The mRNA expression level of Pitx2 in each interfered group significantly decreased to 0.51±0.022, 0.28±0.025 and 0.25±0.016 (p<0.001), as compared with the non-treated iHepSCs (related to 1) (Fig. 2A). The expression level of Pitx2 protein in each treated group decreased to 0.59±0.015, 0.29±0.0058 and 0.22±0.0042 as compared with the control group

(p<0.001, Fig. 2B and C). Based on these findings, shPitx2-2 and shPitx2-3 were chosen for further study, for their knockdown efficiency was more than 70% when evaluated on the suppression of both mRNA and protein levels.

3.3 Effect of Pitx2 knockdown on iHepSCs The iHepSC clones that had morphological phenotypes of epithelial cells expressed hepatic stem cell markers including Afp, Alb, CK8, CK19, Lgr5, Hnf4a, Sox9 and EpCAM (Yu et al., 2013). After Pitx2 knockdown, the morphology of cells in each group remained unchanged. However, qRT-PCR and immunofluorescent staining assay showed that hepatic stem cell-associated genes Lgr5, Sox9 and EpCAM (Huch et al., 2013) were dramatically decreased in the Pitx2 knockdown groups (Fig. 3). In particular, Sox9, a member of the Sry HMG box transcription factors known to play vital roles in the liver development and liver stem cells (Furuyama et al., 2011; Vanderpool et al., 2012) was barely detected (Fig. 3). On the other hand, the expression of other stem cell-associated genes did not undergo significant change (data not shown). These results indicate that Pitx2 knockdown caused the loss of some hepatic stem cell markers of iHepSCs without affecting cellular morphology. iHepSCs showed the self-renewal capacity and remained stable after multiple cell divisions. After Pitx2 knockdown, the proliferation ability was evaluated by cell counting kit-8 assay, immunofluorescent staining, cell

doubling time and colony formation experiments. The proliferation rate was decreased in shPitx2 group as compared with the control group (p<0.001) (Fig. 4A). The doubling time of Pitx2 knockdown iHepSCs rose to 26.2±0.42 hours in shPitx2-2 group and 27.8±0.40 hours in shPitx2-3 group vs. 22.2±0.37 hours in the control group (p<0.001) (Fig. 4B). Ki67 staining showed that the ratio of the proliferated cells were significantly decreased to 0.59±0.034 in shPitx2-2 group and 0.62±0.037 in shPitx2-3 group, as compared with the control (related to 1, p<0.001) (Fig. 4C). Moreover, the colony formation efficiency of the two shPitx2 groups dramatically deceased to 0.63±0.054 (shPitx2-2) and 0.43±0.021 (shPitx2-3) as compared with the control (p<0.001) (Fig. 4D). These results strongly implicate that suppression of Pitx2 expression resulted in a significant decrease of iHepSC proliferation. To eliminate the possibility that inhibition of iHepSC proliferation resulted from off-target effect of shPitx2, rescue assays to restore Pitx2 expression by lentivirus were performed (Supplemental Fig. 1A and B, Kieusseian et al., 2006), Ki67 staining and colony formation assays indicated that the growth inhibition induced by Pitx2 knockdown was rescued (Supplemental Fig. 1C-F), along with restored expression of hepatic stem cell markers (Supplemental Fig. 1G). Thus, restoring Pitx2 expression resulted in a reversion of the effects induced by Pitx2 knockdown, confirming a role for Pitx2 in iHepSC proliferation.

3.4 Pitx2 knockdown induces cell cycle arrest through p53-p21 pathway in iHepSCs To investigate the mechanism of proliferation inhibition by Pitx2 knockdown in iHepSCs, cell cycle distributions were analyzed in all the groups by flow cytometry. The results indicate that Pitx2 knockdown caused an increase in the G1 phase (0.57±0.015 and 0.59±0.016 vs. 0.48±0.014, p<0.001, Fig. 5A) and a reduction in S phase (0.26±0.010 and 0.27±0.0088 vs. 0.34±0.0097, p<0.001, Fig. 5A) as compared with the control group. The data support the idea that Pitx2 knockdown resulted in growth inhibition of iHepSCs by interfering with G1/S phase transition (Bartek et al., 2001; Heldring et al., 2012; Pope et al., 2014). The p53-induced Cdk inhibitor p21 is known as a critical pathway required to block cell cycle in G1/S transition (Wu et al., 1997). Furthermore, the p53-p21 pathway could control the G1-S phase transition by two downstream effectors, including CDK4/6-Cyclin D and CDK2-Cyclin E kinase complexes (He et al., 2005; Neganova et al., 2009). All these related proteins were analyzed by western blotting assay. As shown in Fig. 5B, 1) the expression level of p53 and p21 was increased significantly in the Pitx2 knockdown group compared with the control (p<0.001, Fig. 5B), and 2) the protein expression levels of CDK4, 6, and Cyclin D remained unchanged significantly (Fig. 5B). On the other hand, CDK2 and Cyclin E were reduced significantly (p<0.001) (Fig. 5B). These results showed that Pitx2 knockdown induced cell cycle arrest at G1/S phase transition, thus increasing G1 phase

and reducing S phase by down-regulating the CDK2-Cyclin E expression via the p53-p21 pathway.

3.5 Pitx2 knockdown promotes iHepSCs differentiation rapidly iHepSCs possess the bi-potential differentiation ability, and Pitx2 has potential as target for further application of iHepSCs. We investigated the effect of Pitx2 knockdown on iHepSCs on hepatic and cholangiocytic differentiation. To our surprise, Pitx2 knockdowned iHepSCs could be rapidly induced into hepatocytes or cholangiocytes under certain conditions (Li et al., 2010; Yu et al., 2013). According to our previously study, iHepSCs formed 100um aggregates after 6 days and this phenomenon ended at day 12 under hepatic differentiation condition. Meanwhile, iHepSCs formed branch-like structures at day 9 post cholangiocytic induction. In contrast, Pitx2 knockdown iHepSCs took less time to differentiate into both hepatocytes (3 days on average and stop forming aggregates at Day 7) and cholangiocytes (3 days on average) (Fig. 6A). The efficiency of hepatic and cholangiocytic differentiation was raised to 79.5% ±6.7% and 4.25% ±1.5% vs. 56.4% ± 14.7% and 2.5% ±0.7% in the control group. Several experiments were performed to confirm that Pitx2 knockdowned iHepSCs were effectively differentiated. First, the expression of bi-potential differentiation related genes were monitored by qRT-PCR. The results revealed that hepatocyte related genes (Alb, Hnf4a, Aat, Ttr, Cyp7a1 and G6p,

Fig. 6B) and cholangiocyte related genes (CK19, Gja1, Ggt, Abcg2, EpCAM and Sox9, Fig. 6C) were up-regulated during differentiation. Second, Periodic Acid-Schiff (PAS) staining showed significant glycogen storage as mature hepatocytes (Hu et al., 2016) (Fig. 6D). Third, DiI-labelled acetylated lowdensity lipoprotein (DiI-ac-LDL) uptake revealed that the induced cells gained hepatocyte function (red, Fig. 6E). Immunofluorescent staining of albumin and CK19 indicated that cells became albumin-positive and CK19-negative after hepatic induction as hepatocytes (Zhang et al., 2015) (Fig. 6F). Meanwhile, cholangiocytic differentiation induced branch-like structures (Li et al., 2010) that strongly expressed CK19 and EpCAM (Fig. 6G-I). Taken together, Pitx2 knockdown iHepSCs could be rapidly and efficiently induced to differentiation.

4. Discussion In this study, we demonstrated that Pitx2, a member of the homeodomain transcription factor family, was required for the maintenance of stem cell characteristic in iHepSCs. Lentivirus mediated shRNA knockdown of Pitx2 not only strikingly inhibited the proliferative ability of iHepSCs, but also augmented iHepSC differentiation into hepatocytc and cholangiocytic lineages. The possibility that proliferative inhibition was the result of an off-target effect of the shRNA procedure was discounted when the proliferative block could be removed by re-expression of Pitx2.

Pitx2 is known to be involved in the behavior of many other stem cells, including neural stem cells, myogenic precursor and hematopoietic stem cells by regulating their proliferation or differentiation (Kieusseian et al., 2006). The role of Pitx2 in liver embryonic development or in adult liver regeneration remained previously uninvestigated, although there is evidence that Pitx2 was highly expressed in the embryonic day 9.5 fetal liver and adult liver 24 hours after hepatectomy (Kieusseian et al., 2006; Zhang et al., 2006). Here, we showed data that suppressing Pitx2 strikingly inhibited iHepSC proliferation and promoted their differentiation. Since Pitx2 has a known regulatory effect on many types of stem cells, in addition to iHepSC, manipulation of Pitx2 may be a useful approach to control stem cell status in general. The mechanism of cell cycle regulation has been well studied. Blocking G1-S entry is one of the most important mechanisms underlying proliferation inhibition controlled by the p53-p21 pathway (Bartek et al., 2001). Knowing that the expression level of Pitx2 could regulate the expression of the cell cycle inhibitor p21 (Heldring et al., 2012; Zhang et al., 2006), we studied the mechanism of proliferation inhibition when Pitx2 expression was suppressed in iHepSCs. The data showed that proliferation inhibited iHepSCs increased G1 phase and reduced S phase by down-regulating Cyclin E and CDK2 via the p53-p21 pathway. Furthermore, there is evidence that Pitx2 is a downstream effector of Wnt signaling pathway (Li et al., 2013), which is associated with stem cell proliferation, migration, differentiation and survival (Lade et al., 2011),

suggesting that Wnt signaling pathway may be also involved in affecting the proliferation or differentiation of hepatic stem cells through targeting Pitx2, though it needs to be confirmed by further study. Stem cells are characterized by self-renewal, multipotency and the distinct cell status (Grompe, 2012; Hsu and Fuchs, 2012). They proliferate rapidly until the induction of differentiation, resulting in the inhibition of cell cycle progression. There seems to be a ‘balance’ between proliferation and differentiation (Graham et al., 2003; Takeishi and Nakayama, 2014). Learning how to break or manipulate this ‘balance’ may be beneficial for the translational application of stem cells. Our results revealed that suppressing Pitx2 not only inhibited iHepSC proliferation, but also drives iHepSCs into a ‘sensitive’ state that could be induced to differentiation easily. The mechanism of Pitx2 knockdown on iHepSCs may help uncover the mechanisms underlying self-renewal in stem cells, the balanced regulation between proliferation and differentiation, and the maintenance of ‘stemness”. These are factors of critical importance in the development of stem cells for therapy.

Acknowledgments We thank Dr Joseph T.Y. Lau (Roswell Park Cancer Institute) for language editing of the manuscript. This work was funded by the National Natural Science Foundation of China (31601101, 31401166, 31171309), the Shanghai Committee of Science and Technology, China (15JC1403900), and by Science and Research Fund of Shanghai Municipal Commission of Health and Family Planning (20164Y0013).

Reference Bartek, J., and Lukas, J. (2001). Pathways governing G1/S transition and their response to DNA damage. FEBS letters 490, 117-122. Cardinale, V., Wang, Y., Carpino, G., Mendel, G., Alpini, G., Gaudio, E., Reid, L.M., and Alvaro, D. (2012). The biliary tree--a reservoir of multipotent stem cells. Nature reviews Gastroenterology & hepatology 9, 231-240. Daley, G.Q. (2012). The promise and perils of stem cell therapeutics. Cell stem cell 10, 740-749. Furuyama, K., Kawaguchi, Y., Akiyama, H., Horiguchi, M., Kodama, S., Kuhara, T., Hosokawa, S., Elbahrawy, A., Soeda, T., Koizumi, M., et al. (2011). Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nature genetics 43, 34-41. Graham, V., Khudyakov, J., Ellis, P., and Pevny, L. (2003). SOX2 functions to maintain neural progenitor identity. Neuron 39, 749-765. Grompe, M. (2012). Tissue stem cells: new tools and functional diversity. Cell stem cell 10, 685-689. Grompe, M. (2014). Liver stem cells, where art thou? Cell stem cell 15, 257-258. He, G., Siddik, Z.H., Huang, Z., Wang, R., Koomen, J., Kobayashi, R., Khokhar, A.R., and Kuang, J. (2005). Induction of p21 by p53 following DNA damage inhibits both Cdk4 and Cdk2 activities. Oncogene 24, 2929-2943. Heldring, N., Joseph, B., Hermanson, O., and Kioussi, C. (2012). Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells. CNS & neurological disorders drug targets 11, 884-892. Hindley, C.J., Mastrogiovanni, G., and Huch, M. (2014). The plastic liver:

differentiated cells, stem cells, every cell? The Journal of clinical investigation 124, 5099-5102. Hirose, H., Ishii, H., Mimori, K., Tanaka, F., Takemasa, I., Mizushima, T., Ikeda, M., Yamamoto, H., Sekimoto, M., Doki, Y., et al. (2011). The significance of PITX2 overexpression in human colorectal cancer. Annals of surgical oncology 18, 3005-3012. Hsu, Y.C., and Fuchs, E. (2012). A family business: stem cell progeny join the niche to regulate homeostasis. Nature reviews Molecular cell biology 13, 103-114. Hu, M., Li, S., Menon, S., Liu, B., Hu, M.S., Longaker, M.T., and Lorenz, H.P. (2016). Expansion and Hepatic Differentiation of Adult Blood-Derived CD34+ Progenitor Cells and Promotion of Liver Regeneration After Acute Injury. Stem cells translational medicine. Huch, M. (2015). Regenerative biology: The versatile and plastic liver. Nature 517, 155-156. Huch, M., Dorrell, C., Boj, S.F., van Es, J.H., Li, V.S., van de Wetering, M., Sato, T., Hamer, K., Sasaki, N., Finegold, M.J., et al. (2013). In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247-250. Jochheim-Richter, A., Rudrich, U., Koczan, D., Hillemann, T., Tewes, S., Petry, M., Kispert, A., Sharma, A.D., Attaran, F., Manns, M.P., et al. (2006). Gene expression analysis identifies novel genes participating in early murine liver development and adult liver regeneration. Differentiation; research in biological diversity 74, 167-173. Kieusseian, A., Chagraoui, J., Kerdudo, C., Mangeot, P.E., Gage, P.J., Navarro, N., Izac, B., Uzan, G., Forget, B.G., and Dubart-Kupperschmitt, A. (2006). Expression of Pitx2 in stromal cells is required for normal hematopoiesis. Blood 107, 492-500. Lade, A.G., and Monga, S.P. (2011). Beta-catenin signaling in hepatic development and progenitors: which way does the WNT blow? Developmental dynamics : an official publication of the American Association of Anatomists 240, 486-500. Li, F., Liu, P., Liu, C., Xiang, D., Deng, L., Li, W., Wangensteen, K., Song, J., Ma, Y., Hui, L., et al. (2010). Hepatoblast-like progenitor cells derived from embryonic stem cells can repopulate livers of mice. Gastroenterology 139, 2158-2169 e2158. Li, X., Florez, S., Wang, J., Cao, H., and Amendt, B.A. (2013). Dact2 represses PITX2 transcriptional activation and cell proliferation through Wnt/beta-catenin signaling during odontogenesis. PloS one 8, e54868. Manjunath, N., Wu, H., Subramanya, S., and Shankar, P. (2009). Lentiviral delivery of short hairpin RNAs. Advanced drug delivery reviews 61, 732-745. Miyajima, A., Tanaka, M., and Itoh, T. (2014). Stem/progenitor cells in liver development, homeostasis, regeneration, and reprogramming. Cell stem cell 14, 561-574.

Neganova, I., Zhang, X., Atkinson, S., and Lako, M. (2009). Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells. Oncogene 28, 20-30. Pope, P.A., Bhaduri, S., and Pryciak, P.M. (2014). Regulation of cyclin-substrate docking by a G1 arrest signaling pathway and the Cdk inhibitor Far1. Current biology : CB 24, 1390-1396. Si-Tayeb, K., Lemaigre, F.P., and Duncan, S.A. (2010). Organogenesis and development of the liver. Developmental cell 18, 175-189. Takeishi, S., and Nakayama, K.I. (2014). Role of Fbxw7 in the maintenance of normal stem cells and cancer-initiating cells. British journal of cancer 111, 1054-1059. Vanderpool, C., Sparks, E.E., Huppert, K.A., Gannon, M., Means, A.L., and Huppert, S.S. (2012). Genetic interactions between hepatocyte nuclear factor-6 and Notch signaling regulate mouse intrahepatic bile duct development in vivo. Hepatology 55, 233-243. Wang, M.J., Chen, F., Li, J.X., Liu, C.C., Zhang, H.B., Xia, Y., Yu, B., You, P., Xiang, D., Lu, L., et al. (2014). Reversal of hepatocyte senescence after continuous in vivo cell proliferation. Hepatology 60, 349-361. Wang, X., Foster, M., Al-Dhalimy, M., Lagasse, E., Finegold, M., and Grompe, M. (2003). The origin and liver repopulating capacity of murine oval cells. Proceedings of the National Academy of Sciences of the United States of America 100 Suppl 1, 11881-11888. Wu, R.C., and Schonthal, A.H. (1997). Activation of p53-p21waf1 pathway in response to disruption of cell-matrix interactions. The Journal of biological chemistry 272, 29091-29098. Yu, B., He, Z.Y., You, P., Han, Q.W., Xiang, D., Chen, F., Wang, M.J., Liu, C.C., Lin, X.W., Borjigin, U., et al. (2013). Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors. Cell stem cell 13, 328-340. Zhang, H.Z., Degar, B.A., Rogoulina, S., Resor, C., Booth, C.J., Sinning, J., Gage, P.J., and Forget, B.G. (2006). Hematopoiesis following disruption of the Pitx2 homeodomain gene. Experimental hematology 34, 167-178. Zhang, M., Sun, P., Wang, Y., Chen, J., Lv, L., Wei, W., Jin, C., and Li, W. (2015). Generation of Self-Renewing Hepatoblasts From Human Embryonic Stem Cells by Chemical Approaches. Stem cells translational medicine 4, 1275-1282.

Figure legends Fig. 1. Expression level of Pitx2 in iHepSCs and hepatocytes. (A) Western blotting analysis showed protein expression level of Pitx2 in iHepSCs and hepatocytes. (B) Quantitative analysis of Pitx2 protein level as compared with tubulin. All quantitative data are shown as the mean±S.D. (**, p<001). Fig. 2. Expression level of Pitx2 after shRNA interfered in iHepSCs. (A) qRT-PCR analysis showed that Pitx2 was down-regulated in the shPitx2 group. (B) Western blotting analysis showed that Pitx2 protein level was down-regulated in the shPitx2 group. (C) Quantitative analysis of Pitx2 protein level in each group. control, iHepSCs; pLKO, iHepSCs infected with control pLKO-lentivirus; shPitx2-1, 2 and 3, iHepSCs infected with Pitx2-shRNA lentivirus-1, -2 and -3, respectively. All quantitative data are shown as the mean±S.D. (**, p<001). Fig. 3. Hepatic stem cell-associated genes were down-regulated after Pitx2 was knockdown. (A) qRT-PCR analysis showed that Lgr5, Sox9 and EpCAM were significantly down-regulated. All quantitative data are shown as the mean±S.D. (**, p<001). (B) Immunofluorescent staining showed that the number of Lgr5, Sox9 and EpCAM-positive cells was decreased after Pitx2 knockdown in iHepSCs. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bars=100μm. Fig. 4. The proliferation ability of iHepSCs was inhibited after Pitx2 was knockdown. (A) Cell counting kit-8 assay showed that the proliferation of

iHepSCs was suppressed in the Pitx2 knockdown group. (B) The cell doubling time of each group. (C) Immunofluorescent staining and quantitative analysis showed that the number of Ki67-positive cells was significantly reduced after Pitx2 knockdown. Scale bars=100μm. (D) The colony formation assay and quantitative analysis showed that the colony formation efficiency was diminished after Pitx2 knockdown. All quantitative data are shown as the mean±S.D. (**, p<001). Fig. 5. Pitx2 knockdown induced blocking of the G1/S phase transition in iHepSCs. (A) Flow cytometry and quantitative analysis of cell cycle distributions in each group. (B) Western blotting assay and quantitative analysis were performed to assess the cell cycle-related protein levels. GAPDH was used as a control. All quantitative data are shown as the mean±S.D. (**, p<001). Fig. 6. iHepSCs could be rapidly and successfully differentiated into hepatocytes and cholangiocytes. (A) Diagram of iHepSCs and Pitx2 knockdown (Pitx2-KD) iHepSCs induced to differentiation. (B and C) qRT-RCR analysis revealed dynamic gene expressions during hepatic (B) and cholangiocytic (C) induction of iHepSCs after Pitx2 was down-regulated. All quantitative data are shown as the mean±S.D. (**, p<001). (D-F) PAS staining (D), DiI-ac-LDL uptaking (E) and double-immunofluorescent staining of Albumin and CK19 (F) revealed mature hepatocyte characteristic in hepatic induction of Pitx2-knockdown iHepSCs. (G-I) Typical branching structures (G)

as well as immunofluorescent staining of CK19 (H) and EpCAM (I) related to cholangiocyte characteristic in cholangiocytic induction of Pitx2-knockdown iHepSCs. The branch structure was also stained with F-actin (red). Scale bars=100μm.

Fig 1

Fig 2

Fig 3

Fig 4

Fig 5

Fig 6