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DNA Methyltransferases Modulate Hepatogenic Lineage Plasticity of Mesenchymal Stromal Cells Chien-Wei Lee,1,14 Wei-Chih Huang,3 Hsien-Da Huang,3,4,5,6 Yi-Hsiang Huang,7 Jennifer H. Ho,8 Muh-Hwa Yang,9,10,11,12,13 Vincent W. Yang,17 and Oscar K. Lee2,14,15,16,* 1Program

in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei 11221, Taiwan City Hospital, Taipei 10341, Taiwan 3Institute of Bioinformatics and Systems Biology 4Department of Biological Science and Technology 5Center for Bioinformatics Research National Chiao Tung University, HsinChu 30010, Taiwan 6Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan 7Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, Taipei 11221, Taiwan 8Center for Stem Cell Research, Taipei Medical University-Wan Fang Hospital, Taipei 11031, Taiwan 9Institute of Biotechnology in Medicine 10Genome Research Center 11Immunity and Inflammation Research Center National Yang-Ming University, Taipei 11221, Taiwan 12Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei 11217, Taiwan 13Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan 14Institute of Clinical Medicine, National Yang-Ming University 15Stem Cell Research Center, National Yang-Ming University 16Department of Medical Research, Taipei Veterans General Hospital Taipei 11221, Taiwan 17Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stemcr.2017.05.008 2Taipei

SUMMARY The irreversibility of developmental processes in mammalian cells has been challenged by rising evidence that de-differentiation of hepatocytes occurs in adult liver. However, whether reversibility exists in mesenchymal stromal cell (MSC)-derived hepatocytes (dHeps) remains elusive. In this study, we find that hepatogenic differentiation (HD) of MSCs is a reversible process and is modulated by DNA methyltransferases (DNMTs). DNMTs are regulated by transforming growth factor b1 (TGFb1), which in turn controls hepatogenic differentiation and de-differentiation. In addition, a stepwise reduction in TGFb1 concentrations in culture media increases DNMT1 and decreases DNMT3 in primary hepatocytes (Heps) and confers Heps with multi-differentiation potentials similarly to MSCs. Hepatic lineage reversibility of MSCs and lineage conversion of Heps are regulated by DNMTs in response to TGFb1. This previously unrecognized TGFb1-DNMTs-MSC-HD axis may further increase the understanding the normal and pathological processes in the liver, as well as functions of MSCs after transplantation to treat liver diseases.

INTRODUCTION Hepatocytes possess de-differentiation and re-differentiation potential to achieve liver regeneration (Tarlow et al., 2014). Such findings have challenged the long-standing belief that differentiation is an irreversible process in mammals (Jopling et al., 2011; Metcalf, 2007). However, to date detailed mechanisms of how somatic cells undergo de-differentiation and subsequent re-differentiation remain undefined, and whether lineage reversibility exists in adult stem cells remains controversial (Zhau et al., 2011). One useful model for studying lineage reversibility is the multi-potent mesenchymal stromal cells (MSCs) (Makino et al., 1999; Pittenger et al., 1999; Wislet-Gendebien et al., 2005), which can differentiate into hepatocyte-like cells (dHeps) (Jiang et al., 2014; Kuo et al., 2008; Lee et al., 2004) that possess therapeutic potentials for liver dis-

eases. Currently, whether and how differentiated hepatic progenies from MSCs can be de-differentiated is unknown. Studies do show, however, DNA methylation plays an important role in controlling MSC differentiation (Tsai et al., 2012). DNA methyltransferases (DNMTs) control gene transcription and cellular phenotypic changes during liver organogenesis (Snykers et al., 2009). It has been reported that inhibition of DNMTs increases liver-specific gene expression to maintain a hepatic fate; a concomitant decrease of DNMT1 and increase of DNMT3 expression is associated with hepatic maturation in mouse hepatoblasts (Gailhouste et al., 2013). DNMT1 is the key to methylation maintenance, while DNMT3 is responsible for the initiation of de novo methylation (Li, 2002). However, the function roles of each DNMT in hepatic lineage commitment of MSCs are unknown. The purpose of this study is to investigate the hepatic lineage reversibility in MSCs as well as the

Stem Cell Reports j Vol. 9 j 247–263 j July 11, 2017 j ª 2017 247 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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underlying molecular mechanisms. We hypothesize that alteration of culture conditions may achieve hepatic lineage reversibility between MSCs and their differentiated hepatic progenies.

RESULTS Mouse MSC-Derived Differentiated Hepatocyte-like Cells Possess De-differentiation Potential Mouse MSCs were induced into dHeps via our previously reported two-step protocol (Jiang et al., 2014; Kuo et al., 2008; Lee et al., 2004). During 4 weeks of hepatogenic differentiation (HD), the bipolar fibroblast-like morphology of the MSCs changed into cobblestone-like shapes from day 14 and then progressively changed into hexagonal shapes, a typical epithelial morphology at day 28 (Figure 1A). Expression of hepatocyte-specific genes was significantly upregulated after differentiation induction in a time-dependent manner (Figures 1B–1D). dHeps possessed hepatocyte surface markers similar to those of mouse Heps (Figure 1E). The in vitro functional characteristics were further investigated by periodic acid-Schiff (PAS) staining (Figure 1F), measurements of glycogen, albumin, and urea production (Figure 1G), as well as low-density lipoprotein (LDL) uptake assays (Figure 1H). All the hepatic function markers significantly and progressively increased during differentiation. In general, although slightly lower, dHeps expressed function markers similar to those of Heps (Figures 1B–1H). Expression levels of hepatic-specific genes of dHeps are close to those of embryonic day 12.5 (E12.5) fetal livers and FL83B cells, and differ relatively from those of Heps (Figure S2). Compared with Heps, dHeps also expressed higher levels of Sca-1 (Figure 1E), Dlk1, and Cd24a (Figure S2), which are hepatic progenitor markers (Miyajima et al., 2014; Petersen et al., 2003; Qiu et al., 2011). These

findings indicated that dHeps are similar to hepatic progenitors. Although dHeps are not identical to terminally differentiated mature Heps, they are committed hepatic cells. After 28 days of HD, we de-differentiated dHeps by replacing differentiation medium with mouse MSC-maintenance medium for 28 days. Following hepatogenic de-differentiation (dHD), cellular morphology reverted back to fibroblast-like shapes (Figure 1A). Furthermore, liver-specific gene expression, surface marker presentation, and hepatic functions all switched back to the original levels observed in original MSCs (Figures 1B–1H). We next examined cell-cycle profiles during HD and dHD. After differentiation for 28 days, the cells arrested at the G0/G1 phase but re-entered the cell cycle after de-differentiation for 28 days (Figure 1I). The reversibility of the cell cycle was accompanied by changes in expression of genes encoding cell-cycle checkpoint regulators as well as proliferation markers Ki67 and Pcna (Figures 1J and 1K). The results indicated that hepatic lineage reversibility in MSCs was accompanied by cell-cycle exit and re-entry. To explore the transcriptomic and proteomic changes during HD and dHD, we performed microarray analysis and high-density antibody arrays on MSCs (HD 0), dHeps (HD 28), and de-differentiated cells from dHeps (ddHeps, dHD 28). Hierarchical clustering (Figures 2A and 2B) showed that ddHeps clustered closely together with MSCs and were separated from dHeps and Heps. The profiles of genes involved in fibroblast markers, MSC markers (Figure 2C), lipid, glycolysis, cholesterol, and drug metabolism (Figure 2D) were very similar between dHeps and Heps, whereas ddHeps and MSCs displayed similar gene profiles. Collectively, these results indicated that the ddHep transcriptome and proteome had indeed reverted back to the MSC state. Using the KEGG database, gene ontology was analyzed to understand the functional significance of differential gene expression during HD and dHD. Pathways involved in

Figure 1. Hepatogenic Differentiation and De-differentiation of Mouse Mesenchymal Stromal Cells (A) Representative morphological changes in MSCs during hepatogenic differentiation (HD) and hepatogenic de-differentiation (dHD). Scale bar, 100 mm. (B–D) Results of (B) qRT-PCR (n = 4 independent experiments), (C) a representative immunoblot, and (D) immunocytochemical staining for hepatogenic-specific gene expression. b-Actin was used as an internal control. Scale bar, 50 mm. (E) Flow-cytometry analysis for surface phenotypic characterization. (F) PAS (periodic acid-Schiff) staining for glycogen storage. (G and H) Hepatic functions were measured via (G) albumin production (n = 7 independent experiments), glycogen storage (n = 4 independent experiments), urea production (n = 3 independent experiments), and (H) LDL-uptake assays. LDLR, LDL receptor. (I) Results of flow-cytometry analysis showing the relative percentage of cells in various cell-cycle phases (n = 5 independent experiments). (J and K) Results of (J) qRT-PCR (n = 4 independent experiments) and (K) a representative immunoblot for cell-cycle-related genes. ‘‘HD 0’’ refers to MSCs; ‘‘HD 28’’ refers to MSC-derived dHeps (hepatocyte-like cells) after 28 days of HD; ‘‘dHD 28’’ refers to dHep-derived ddHeps (de-differentiated cells) after 28 days of dHD; ‘‘Heps’’ refers to mouse primary hepatocytes. All quantitative data are presented as means ± SD. Statistical analyses were performed using Student’s paired t test, with significance set at **p < 0.001. NS, not significant. See also Figures S1–S5 and S7. Stem Cell Reports j Vol. 9 j 247–263 j July 11, 2017 249

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both general (e.g., cell cycle) and specific functions (e.g., lysosome and metabolism) were demonstrated (Table S1). A substantial overlap of KEGG pathways between HD and dHD was also observed. Moreover, microarray results revealed that upregulated pathways during HD substantially overlapped with downregulated pathways during dHD (Figure 2E, left panel), and vice versa (Figure 2E, right panel). These patterns indicated that dHD involved the pathways associated with HD and is essentially a reversal of the latter process. ddHeps Exhibit Potential for Multi-lineage Differentiation To investigate whether ddHeps regained multi-lineage differentiation potential, an essential function of MSCs, we examined their hepatogenic, osteogenic, and adipogenic differentiation capabilities. Hepatic induction caused ddHeps to become functional dHeps, evidenced by morphological changes, the capacity for glycogen storage and albumin production (Figure 3A), as well as the reexpression of hepatogenic-specific genes. Positive function assays, including glycogen storage, albumin production, and generation of urea, and downregulation of Ki67 also supported the manifestation of hepatic re-differentiation (Figures 3B and 3C). Next, osteogenic differentiation of ddHeps was evidenced by morphologic change, alkaline phosphatase activity, and calcium mineralization of the extracellular matrix (Figure 3D), visible through von Kossa and alizarin red S staining. Osteogenic-specific gene expression was also increased significantly (Figure 3E). Finally, ddHeps differentiation into adipocytes was shown by the accumulation of neutral lipid vacuoles (Figure 3F), positive oil red O staining, and increased adipogenic-specific gene expression (Figure 3G). To further confirm dHD and to rule out the possibility of incomplete HD, we sorted dHeps by staining for CD26, which is only expressed on mature hepatocytes but not in MSCs or immature hepatic progenitor cells. After dHD for 4 weeks, ddHeps derived from CD26high dHeps expressed a typical MSC phenotype (Figures S3A–S3D) and regained multi-lineage differentiation potentials (Figures S3E–S3G). Expression of lineage-specific genes in ddHeps upon various lineage induction is similar to that of primary

MSCs (Figures S3E–S3G). ddHeps lacked cell-surface antigens CD45 and CD90 (Figure S3D) and did not express markers of liver progenitors reported in the literature (Chen et al., 2012; Oikawa et al., 2009). Thus, ddHeps are indeed closer to MSCs rather than committed liver progenitor cells, oval cells, or hepatoblasts. Hepatic Lineage Reversibility Is Associated with MET and EMT Mesenchymal-to-epithelial transition (MET) and epithelial-to-mesenchymal transition (EMT) are reversible processes that dictate cell morphology, migration, and differentiation (Thiery and Sleeman, 2006). To examine whether MET and EMT were involved during HD and dHD, we surveyed the expression of mesenchymal and epithelial marker genes. During HD of MSCs, epithelial markers such as Cdh1, Epcam, Gja1, and Tjp1 were upregulated, whereas mesenchymal markers such as Vim, Fn1, Acta1, and Acta2, as well as the master EMT regulators Tgfb1 and Snai1, were downregulated. A reverse pattern was observed during dHD (Figures S4A–S4C). In addition, migratory capacity was decreased after HD and recovered upon dHD (Figure S4D). We concluded that HD is associated with MET, whereas dHD is associated with EMT. Reversible HD Also Occurs in Human MSCs We applied the same approach to induce HD and dHD in human MSCs. Reversible changes of morphology, hepatogenic-specific gene profile, hepatic functions, MET markers, and migration capacity (Figures S5A–S5F) were also observed in human MSCs. Moreover, human ddHeps retained multi-lineage differentiation potential (Figures S5G–S5I). Overall, the results from mouse and human MSCs were consistent; the fate of human MSCs could also be reversed after differentiation into the hepatic lineage. DNA Methyltransferases Regulate HD of MSCs and dHD To investigate the role of epigenetic effects during HD and dHD of MSCs, we used real-time qRT-PCR to screen the expression of 19 epigenetic modulators in DNA and histone methylation pathways (Figure 4A). We found that

Figure 2. Microarrays and High-Density Antibody Arrays of Transcriptomes and Proteomes Reveals Hepatogenic Differentiation and De-differentiation (A–D) Hierarchical clustering via correlations in the transcriptome (A) and the proteome (B). Expression patterns of genes involved in fibroblast, MSC, and EMT markers (C). Expression patterns of genes involved in lipid, glucose, cholesterol, and drug metabolism (D). Each column represents a single array sample. (E) Venn diagrams showing the overlap of up- and downregulated KEGG pathways within the transcriptome during HD and dHD. ‘‘HD 0’’ refers to MSCs; ‘‘HD 28’’ refers to MSC-derived dHeps after 28 days of HD; ‘‘dHD 28’’ refers to dHep-derived ddHeps after 28 days of dHD; ‘‘Heps’’ refers to mouse primary hepatocytes; ‘‘MLCs’’ refers to MSC-like cells derived from Heps. See also Tables S1 and S2. Stem Cell Reports j Vol. 9 j 247–263 j July 11, 2017 251

Figure 3. dHeps Can Also De-differentiate and Regain Multi-lineage Differentiation Potentials (A) Representative morphological changes and staining (PAS and albumin [ALB]) of ddHeps during hepatogenic re-differentiation (reHD). (B) qRT-PCR of hepatogenic-specific genes inddHep-derived dHeps (n = 3 independent experiments). (C) Hepatic functions of ddHep-derived dHeps were evaluated with albumin production (n = 3 independent experiments), glycogen storage (n = 6 independent experiments), and urea production (n = 3 independent experiments). (legend continued on next page) 252 Stem Cell Reports j Vol. 9 j 247–263 j July 11, 2017

DNMT1 expression was decreased during HD but was strongly increased after dHD (Figures 4A and 4B), to levels higher than the nascent MSCs (HD 0). In contrast, DNMT3a and DNMT3b expression was increased during HD and returned to the original levels similar to MSCs after dHD. In addition, pretreatment with 5-azacytidine (a panDNMT inhibitor) reduced global DNA methylation in MSCs (Figure 4C). Over time, increasing doses of 5-azacytidine enhanced hepatogenic-specific gene expression and hepatic functions (Figures 4D and 4E). These results indicated that DNMTs are involved in the HD of MSCs. We then performed loss-of-function assays to determine the roles of individual DNMTs in HD. Small interfering RNAs (siRNAs) against DNMT1, DNMT3a, and DNMT3b were added individually during HD (Figures 4F and 4G). The results revealed that in HD 14, DNMT1 knockdown significantly enhanced the expression of hepatogenicspecific genes, MET/EMT markers (Figure 4H), and the metabolically important cytochrome P450 (CYP) family members (Figure 4I). Furthermore, hepatic functions were enhanced (Figure 4J). An acceleration of HD via DNMT1 knockdown was observed: HD 14 cells with DNMT1 knockdown are similar to HD 28 cells in terms of gene expression and hepatic functions (Figures 4H–4J). Knockdown of DNMT3a only downregulated several hepatogenic markers (Figure 4H), but significantly reduced the expression of all CYP enzymes examined (Figure 4I). Knockdown of DNMT3b had no influence on HD (Figures 4H–4J). Simultaneous silencing of DNMT3a and DNMT3b did not further repress HD at day 14 (Figures 4H–4J). In terms of morphological changes during dHD, DNMT1 knockdown maintained epithelial morphology and increased albumin expression compared with parental and control groups. In contrast, DNMT3a knockdown promoted a fibroblast-like morphology and decreased albumin expression (Figures 4K and 4L). Collectively, these data showed that DNMT1 extensively regulated hepatic lineage commitment but DNMT3a specifically regulated the CYP family during the HD of MSCs. Moreover, DNMT1 silencing accelerated HD.

15 candidate genes, four (Tgfb1, Thbs1, Thbs2, and Thbs3) were involved in the TGFb signaling pathways. Among these four, Tgfb1 was highly expressed in both arrays (Figure 5A). Increased TGFb1 expression was strongly associated with reversible differentiation (Figures 5B and S5E). To test how DNMTs responded to TGFb1, we altered the concentrations of TGFb1 during HD. As TGFb1 dosage increased, DNMT1 expression also increased in dHeps, whereas DNMT3a and DNMT3b expression decreased (Figure 5C). These effects were eliminated by LY-364947, an Alk-5 (TGFbRI/II) inhibitor, indicating that extracellular TGFb1 signaling is Alk-5 dependent. To identify the signaling pathways involved in TGFb1-regulated DNMT expression during HD, we tested both Smad and nonSmad signaling pathway inhibitors (Figure 5D). We showed that PD0325901 and SIS3 treatment reversed DNMT1 upregulation, SB203580 and LY294002 treatment reversed DNMT3a/3b downregulation, and SP600125 treatment did both. Therefore, TGFb1 may regulate DNMT1 and DNMT3a/3b together via the JNK pathway, whereas it separately regulates DNMT1 via the ERK1/2 and canonical Smad2/3 pathways, as well as DNMT3a/3b via the p38 and PI3K-Akt pathways. Of note, neither DNMT knockdowns (Figures 5E and 5F) nor 5-azacytidine pretreatment (Figure 5G) affected TGFb1 expression. These results demonstrated that DNMTs regulate HD in response to TGFb1 signaling. Moreover, a feedback loop does not appear to exist in the TGFb1-DNMT axis during the HD of MSCs. To determine whether the downregulation of TGFb1 signaling is essential for HD, we treated MSCs with TGFb1 during HD. Our results showed that TGFb1 repressed morphologic changes (Figure 5H), hepatogenicspecific gene expression (Figure 5I), and hepatic functions (Figure 5J), whereas it increased cell migratory capacity (Figure 5K). All effects from TGFb1 treatment were reduced or eliminated by LY-364947. Our results confirmed that TGFb1 signaling must be downregulated for the HD of MSCs.

Identification of Signaling Pathways Involved in DNMT-Modulated HD and dHD To identify the signaling pathways involved in DNMTdependent HD, we integrated the microarray and protein array data associated with DNMT1 expression. Among

Murine Hepatocytes Can Be Converted to Functional MSC-like Cells Since dHeps are similar to hepatic progenitors and are different from Heps, we further investigate whether similar phenomenon of de-differentiation from dHeps to MSCs can

(D) Representative morphological changes and staining (alkaline phosphatase [ALP], von Kossa, and alizarin red S) of ddHeps during osteogenic re-differentiation (reOD). (E) qRT-PCR of osteogenic-specific genes in ddHep-derived osteoblasts (n = 3 independent experiments). (F) Representative morphological changes and staining (oil red O) of ddHeps during adipogenic re-differentiation (reAD). (G) qRT-PCR of adipogenic-specific genes in ddHep-derived adipocytes (n = 3 independent experiments). All quantitative data are presented as means ± SD. Statistical analyses were performed using Student’s paired t test, with significance set at *p < 0.05, **p < 0.005. NS, not significant. Scale bars represent 100 mm for phase contrast and staining. See also Figures S5 and S7. Stem Cell Reports j Vol. 9 j 247–263 j July 11, 2017 253

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also occur in Heps. To investigate whether TGFb1 can induce lineage conversion in hepatocytes, we treated primary hepatocytes with TGFb1 or TGFb1 receptor inhibitor (LY-364947) for 3–7 days. Treatment of Heps with TGFb1 for 7 days resulted in a fibroblast-like morphology and a loss of hepatogenic-specific genes. These effects were eliminated by LY364947 administration (Figure S7). Expression of hepatogenic-specific genes was increased in LY-364947-treated Heps compared with medium-only control on day 7 (Figure S7). Blocking of the endogenous TGFb1 signaling repressed the loss of hepatocyte characteristics, supporting that TGFb1 is necessary and sufficient to trigger hepaticmesenchymal lineage conversion. Currently known actions of TGFb1 include inducing apoptosis, de-differentiating fetal hepatocytes to liver progenitors, and mediating the acquisition of mesenchymal morphology (Caja et al., 2011; Tu et al., 2014). Additionally TGFb1 is involved in the myofibroblastic (Kakudo et al., 2012), chondrogenic (Toh et al., 2005), and cardiomyogenic differentiation (Mohanty et al., 2013) of MSCs. Therefore, we attempted to convert purified CD26high Heps to MSC-like cells (MLCs) by treating them with stepwise reduction of TGFb1 (Figure 6A). This stepwise protocol converted CD26high Heps to an MSC-like status in terms of morphology, hepatogenic-specific gene expression, cell-surface phenotype, hepatic functions (Figures 6B–6G), and EMT markers (Figures 6H and 6I). MLCs re-entered into cell cycle and increased the cell number in culture, and re-thawed MLCs retained proliferative capacity (Figures 6J and 6K). The transcriptomes and proteomes of MLCs and MSCs were similar (Figures 2A and 2B). The two cell types also exhibited similarities in the expression of metabolismrelated genes (Figure 2D) and other gene sets (Figure 2C). Gene ontology analyses revealed substantial overlap of enriched pathways during Hep lineage conversion to MLCs and during reversible HD of MSCs (Figure 2E and

Table S2). After treatment with TGFb1 we noted the induction of endogenous TGFb1 expression in MLCs, which was maintained after exogenous TGFb1 removal (Figures 6H and 6L). In addition, DNMT1 knockdown prevented TGFb1-induced loss of hepatogenic-specific genes in Heps (Figure 6M), and DNMT expression patterns were consistent between Hep-derived MLCs and MSCs (Figures 6N and 6O), providing evidence that DNMTs mediated the TGFb1-induced lineage conversion of Heps. Finally, MLCs possessed multi-lineage potentials comparable with those of MSCs (Figures 7A–7G); while they exhibited higher hepatic potential, particularly in the expression of CYP family enzymes (Figure 7B). Together, these results indicated that Hep-converted MLCs are very similar to primary MSCs. Reversible Differentiation Does Not Induce Tumorigenicity Long-term culture expansion, changes to methylation states, and DNMT deficiency may cause genomic instability and lead to malignant transformation (Foudah et al., 2009; Josse et al., 2010; Karpf and Matsui, 2005; Miura et al., 2006; Rosland et al., 2009). Moreover, DNMT1 expression has been associated with hepatocellular carcinomas (Raggi et al., 2014). We found a deletion of Y chromosome and a duplication of chromosome 16 after the long-term differentiation and de-differentiation process revealed by comparative genomic hybridization arrays (n = 1, data not shown). To test whether differentiation and de-differentiation process induces tumorigenicity, we subcutaneously inoculated nude mice with mouse MSCs, ddHeps, dHeps with siRNA against DNMT1, and MLCs. We observed no tumor formation after 13 weeks, indicating that tumorigenicity is not induced by long-term reversible differentiation, genetic manipulation of DNMTs, or lineage conversion (Figures 7H and 7I).

Figure 4. DNA Methyltransferases Regulate Hepatogenic Differentiation and De-differentiation of Mouse MSCs (A) A heatmap summarizing relative mRNA levels of 19 epigenetic modulators (n = 4). (B) Representative immunoblot for DNA methyltransferases (DNMTs) expression in MSCs during HD and dHD. (C–I) DNMT-inhibitor (5-azacytidine [5-aza]) treatment inhibited global DNA methylation (n = 3 independent experiments). After 14 days of HD, enhancement from pan-DNMT inhibitor pretreatment occurred in hepatogenic-specific gene expression, as visualized with qRT-PCR (n = 4 independent experiments) (D), and in hepatic functions (E), as represented by albumin production (n = 2 independent experiments, four technical replicates), glycogen storage (n = 2 independent experiments, 12 technical replicates), and urea production (n = 2 independent experiments, five technical replicates). Symbols indicate treatments with different 5-aza concentrations (+, 0.5 mM; ++, 1 mM; +++, 2 mM). Efficiency of DNMT knockdown determined with qRT-PCR (F) (n = 6 independent experiments) and immunoblot (G) at HD 14. Knockdown of DNMTs altered hepatogenic-specific genes, EMT/MET markers (H), and CYP enzymes (I) at HD 14 (n = 6 independent experiments). (J–L) Knockdown of DNMTs altered hepatic functions at HD 14, as represented by albumin production (n = 6 independent experiments), glycogen storage (n = 6 independent experiments), and urea production (n = 6 independent experiments). Knockdown of DNMTs altered cellular morphology at dHD 3 (K) and modulated albumin expression, as seen in the representative images of immunocytochemical staining (L). All quantitative data are presented as means ± SD. Statistical analyses were performed using Student’s paired t test, with significance set at *p < 0.05, **p < 0.005. NS, not significant. Scale bars represent 100 mm for phase contrast and staining. See also Figure S7. Stem Cell Reports j Vol. 9 j 247–263 j July 11, 2017 255

Figure 5. TGFb1 Regulates DNMTs and Represses Hepatogenic Differentiation of Mouse MSCs (A) Schematic illustration of signaling pathways. PCC, Pearson’s correlation coefficient. (B) ELISA results for TGFb1 expression of MSCs during HD and dHD (n = 5 independent experiments). (C) qRT-PCR of DNMTs from MSCs treated with TGFb1 during HD (n = 6 independent experiments). Symbols indicate treatments of different TGFb1 concentrations (+, 1 ng/mL; ++, 10 ng/mL; +++, 100 ng/mL). (D–F) qRT-PCR of DNMTs from MSCs treated with TGFb1 and various inhibitors during HD (n = 4 independent experiments) (D). LY-364947, Alk-5 inhibitor, 400 nM; SIS3, Smad2/3 inhibitor, 10 mM; SP600125, JNK inhibitor, 30 mM; SB203580, p38 inhibitor, 30 mM; PD0325901, MEK-ERK1/2 inhibitor, 1 mM; and LY294002, PI3K-Akt inhibitor, 25 mM. The mRNA expression from each treatment was statistically (legend continued on next page) 256 Stem Cell Reports j Vol. 9 j 247–263 j July 11, 2017

DISCUSSION In this study, we show that hepatic lineages are reversible between adult MSCs and hepatocytes. Such reversibility is regulated by the TGFb1-DNMT axis. To the best of our knowledge, this is also the demonstration that hepatocytes can be converted into multi-potent MLCs without ectopic gene delivery. Our findings support the occurrence of lineage reversibility in mammalian cells, indicating that such a phenomenon is not restricted to plants and non-mammalian vertebrates (Brockes, 1997; Brockes and Kumar, 2002; Weigel and Jurgens, 2002). Our findings demonstrate a role of DNMTs in hepatic lineage reversibility of MSCs, with individual DNMTs exerting opposite effects; DNMT1 repressed HD and promoted dHD; while DNMT3a promoted HD and repressed dHD. The two enzymes seem to have both unique and overlapping target cytosine-phosphate-guanine (CpG) sites, as shown by the promotion of all hepatogenic-specific genes and hepatic functions via DNMT1 silencing, but a more specific repression of the CYP family via DNMT3a silencing (Figure 4). In addition, the inhibitory effect of DNMT1 appears to be more predominant than the activating effect of DNMT3a, because a pan-DNMT inhibitor resulted in the promotion of HD (Figure 4). Although DNMT3b expression is also strongly associated with HD and responded to TGFb1 stimulation, DNMT3b knockdown fails to affect hepatic gene expression and function. While more data are necessary to clarify the function of DNMT3b in HD, we speculate that the enzyme may cooperate with other chromatin modifiers. Epigenetic effects exerted by these modifiers may have been sufficient for HD regulation, explaining why we do not observe significant changes resulting from DNMT3b knockdown (Baubec et al., 2015; Morselli et al., 2015). In addition to demonstrating the importance of DNMTs, we show that TGFb1 differentially regulated DNMTs, and that TGFb1 is both necessary and sufficient to induce lineage conversion of hepatocytes into MLCs with phenotypes and multipotency resembling MSCs. Upon withdrawal of TGFb1 on day 28, we observed that Hep-converted MLCs

possessed MSC-like DNMTs expression patterns (Figures 6N and 6O) due to the induction of endogenous TGFb1 expression (Figures 6H and 6L). This outcome supports the existence of an MSC-specific methylome that maintains MLC phenotypes and multipotency. a-Fetoprotein (AFP) is a marker indicating early hepatic fate. Although AFP is not detectable in hepatocytes in vivo, expression of AFP may be detected in the in vitro culture conditions owing to a loss of contact (Gleiberman et al., 1989). Moreover, TGFb1 downregulated albumin and AFP expression and induced fibroblast morphology and mesenchymal marker expression (mesenchymal fate) in Hep3B cells (Caja et al., 2011). Therefore, AFP may serve as a marker to distinguish hepatic fate from mesenchymal fate and allows us to track cellular conversion from the former to the latter. Fibrosis is often a consequence of various chronic liver diseases and leads to hepatic failure as well as cancer. TGFb1, a master inducer of EMT, orchestrates fibrogenesis. Persistent TGFb1 stimulation converts both hepatocytes and MSCs into myofibroblasts and participates in liver fibrosis, and even contributes to the development of tumor (Sekiya et al., 2016). While blocking TGFb signaling in hepatocytes alone attenuates liver fibrosis (Dooley et al., 2008) and facilitates liver regeneration (Karkampouna et al., 2016), MLCs may be at an intermediate state during the transition to myofibroblasts. Therefore, inhibition of the TGFb1-DNMT axis in vivo may convert Hepderived myofibroblasts back into MLCs or hepatocytes, and may attenuate liver fibrosis and facilitate liver regeneration. Our previous studies established a protocol for functional dHeps with therapeutic potentials when transplanted in vivo (Jiang et al., 2014; Kuo et al., 2008; Lee et al., 2004). However, dHeps required 28 days of in vitro differentiation, a lengthy duration that hampers their clinical application. In this study, we successfully accelerate differentiation to 14 days by silencing DNMT1 (Figure 4). Transplantation of dHeps with TGFb receptor inhibitor or DNMT1 antagonist may stabilize hepatic characteristics of transplanted dHeps. On the other hand, although transplanted dHeps may de-differentiate

compared with a TGFb1-treated group. Control refers to MSC-derived dHeps after 14 days. Results from qRT-PCR (E) (n = 6 independent experiments) and ELISA (F) (n = 3 independent experiments) of TGFb1 expression at HD 14 after DNMT knockdown. (G) DNMT-inhibitor pretreatment did not affect TGFb1 expression (n = 3 independent experiments). Symbols indicate treatments with different 5-azacytidine concentrations (+, 0.5 mM; ++, 1 mM; +++, 2 mM). (H) Representative morphological changes of MSCs treated with TGFb1 during HD. (I) qRT-PCR of hepatogenic-specific genes in dHeps under TGFb1 treatment (n = 5 independent experiments). (J) Hepatic functions of dHeps under TGFb1 treatment, as represented by albumin production (n = 7 independent experiments), glycogen storage (n = 5 independent experiments), and urea production (n = 6 independent experiments). (K) Quantification of transwell migration assay (n = 3 independent experiments) of MSCs treated with TGFb1 during HD. All quantitative data are presented as means ± SD. Statistical analyses were performed using Student’s paired t test, with significance set at *p < 0.05, **p < 0.005. NS, not significant. Scale bars represent 100 mm for phase contrast and staining. See also Figures S6 and S7. Stem Cell Reports j Vol. 9 j 247–263 j July 11, 2017 257

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into ddHeps, ddHeps may promote liver regeneration like MSCs through immunomodulation and trophic support (Christ et al., 2015). Hep-converted MLCs have proliferation ability and higher hepatic lineage potential (Figure 7B). Therefore, these cells may also serve as an alternative cell source for liver regeneration (Du et al., 2014; Takeuchi et al., 2015). In summary, hepatic lineage reversibility exists and is regulated by the previously unrecognized TGFb1-DNMT axis. The ability to convert Heps into MLCs with altered extracellular concentrations of TGFb1 suggests that terminally differentiated hepatocytes may exhibit considerably greater plasticity than previously believed. Our findings have not only offered therapeutic insights for use of MSCs or differentiated hepatic progenies for transplantation to treat liver diseases, but have also contributed to the development of approaches for achieving tissue regeneration through promoting de-differentiation and lineage conversion in vivo.

sion of surface markers and ability of multi-potent differentiation into osteoblasts, adipocytes, and chondroblasts in vitro (Figure S1). Mouse MSCs (mMSCs) were cultured in an mMSC-maintenance medium, consisting of low-glucose DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific); human MSCs (hMSCs) were cultured in hMSC-maintenance medium, consisting of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS, 10 ng/mL epidermal growth factor, and 10 ng/mL basic fibroblast growth factor (bFGF) (R&D Systems). Human primary hepatocytes (hHeps) were purchased from ScienCell and cultured in Hepatocyte Medium (ScienCell). Mouse hepatocytes were isolated from 10- to 12-week-old BALB/c mice with a protocol in the literature (Klaunig et al., 1981a, 1981b), and were cultured in Hep-maintenance medium consisting of DMEM/F12 supplemented with 10% FBS. FL83B cells were cultured in F-12K medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS. Low-glucose DMEM, IMDM, DMEM/ F12, 5-azacytidine, TGFb1, and LY-364947 were purchased from Sigma-Aldrich; SP600125, SB203580, PD325901, LY294002, and SIS3 were purchased from Abcam.

Hepatogenic Differentiation and De-differentiation

EXPERIMENTAL PROCEDURES All animal studies were approved by the Taipei Veterans General Hospital Institutional Animal Care and Use Committee (IACUC 2014-043).

Cell Culture and Reagents MSCs were obtained from bone marrow and were maintained in culture as previously described (Jiang et al., 2014; Lee et al., 2004). The characteristics of MSCs were confirmed by their expres-

HD was induced following our previously reported two-step protocol (Lee et al., 2004). In brief, 1.0 3 104/cm2 MSCs were seeded and cultured for 2 days in the maintenance medium. Two days post seeding, cells were treated with a step-1 differentiation medium, consisting of IMDM supplemented with 20 ng/mL hepatocyte growth factor and 10 ng/mL bFGF (R&D Systems), and 0.61 g/L nicotinamide (Sigma-Aldrich). Seven days later, cells were treated with a step-2 differentiation medium, consisting of IMDM supplemented with 20 ng/mL oncostatin M (Invitrogen), 1 mmol/L dexamethasone (Sigma-Aldrich), and 50 mg/mL ITS+ premix (BD Biosciences) for 3 weeks. The differentiation medium was changed

Figure 6. Stepwise TGFb1 Treatment Converts Mouse Primary Hepatocytes into MSC-like Cells though Initiating the Secretion of Endogenous TGFb1 (A) Schematic illustration of the experimental approach. (B) Representative flow-cytometry plots showing CD26 expression and the gating strategies used to isolate CD26low and CD26high hepatocytes. (C) Representative morphology and PAS staining for MLCs. (D and E) Loss of hepatogenic-specific gene expression in MLCs, analyzed with qRT-PCR (D) (n = 4 independent experiments) and immunoblot (E). (F) Loss of hepatic function in MLCs, as represented by albumin production (n = 4 independent experiments), glycogen storage (n = 3 independent experiments), and urea production (n = 4 independent experiments). (G) Surface phenotypic characterization analyzed with flow cytometry. (H and I) Gene expression of MET/EMT markers during de-differentiation of mouse Heps, analyzed with qRT-PCR (H) (n = 4 independent experiments) and immunoblot (I). (J and K) Cell numbers counted at each time point (n = 3 independent experiments) (J) and relative percentage of cells in various cell-cycle phases (n = 5 independent experiments) (K). ‘‘Re-thawed MLCs’’ refers to MLCs re-thawed from cryopreservation and cultured for another week at least. (L) ELISA of TGFb1 expression in Heps, MLCs, and MSCs (n = 4 independent experiments); the amount of TGFb1 from serum has been deducted. (M) qRT-PCR of hepatogenic-specific genes in mouse Heps under TGFb1 and siDnmt1 treatment for 3 days (n = 3 independent experiments). The plus and minus symbols respectively indicate treatments with and without TGFb1 (10 ng/mL), siControl (50 nM), and siDnmt1 (50 nM). (N and O) Expression of DNMTs in hepatocytes, MLCs, and MSCs, analyzed by qRT-PCR (N) (n = 4 independent experiments) and immunoblot (O). *p < 0.05, **p < 0.005. NS, not significant. Scale bars represent 100 mm for phase contrast and staining. See also Figures S6 and S7. Stem Cell Reports j Vol. 9 j 247–263 j July 11, 2017 259

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once per week. To induce dHD in MSC-derived dHeps, we replaced the step-2 medium with maintenance medium (low-glucose DMEM supplemented with 10% FBS). A protocol with stepwise reduction of TGFb1 was used to induce the lineage conversion of Heps. Specifically, Heps were cultured in an mMSC-maintenance medium with 10 ng/mL TGFb1 for the first 7 days, then with 5 ng/mL TGFb1 for another 7 days, and finally with 1 ng/mL TGFb1 for the following 14 days. After 28 days, cells were cultured in maintenance medium without TGFb1. Other experimental procedures and statistical analysis are described in Supplemental Experimental Procedures.

from Ministry of Education. The authors also acknowledge the technical support provided by Flow Cytometry Core Facility of National Yang Ming University and Imaging Core Facility of Nanotechnology of the UST-YMU. We acknowledge Prof. Ting-Fen Tsai from National Yang-Ming University, Taiwan for the kind gift of E12.5 fetal livers and thank Prof. Chi-Chang Juan from National Yang-Ming University, Taiwan for the kind gift of FL83B cells. Received: June 13, 2016 Revised: May 4, 2017 Accepted: May 5, 2017 Published: June 8, 2017

ACCESSION NUMBERS With regard to transcript profiling the accession numbers are: microarray, GEO: GSE73617; protein array, GEO: GSE73762.

SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and three tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr.2017. 05.008.

AUTHOR CONTRIBUTIONS C.-W.L. designed the study, performed experiments, analyzed data, and wrote the manuscript; W.-C.H. and H.-D.H. performed microarray and protein array analysis; Y.-H.H., J.H.H., and M.-H.Y. provided feedback for the manuscript; V.W.Y. and O.K.L. edited the manuscript and supervised the study.

ACKNOWLEDGMENTS This work was supported in part by the Novel Bioengineering and Technological Approaches to Solve Two Major Health Problems in Taiwan sponsored by the Taiwan Ministry of Science and Technology Academic Excellence Program under grant number MOST 105-2633-B-009-003. The authors acknowledge financial support from the Ministry of Science and Technology, Taiwan (MOST1032314-B-010-053-MY3, MOST 106-2321-B-010-008, MOST 1062911-I-010-502, and MOST 105-2633-B-010-002). This study was also supported by Aiming for the Top University Plan, a grant

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Supplemental Information

DNA Methyltransferases Modulate Hepatogenic Lineage Plasticity of Mesenchymal Stromal Cells Chien-Wei Lee, Wei-Chih Huang, Hsien-Da Huang, Yi-Hsiang Huang, Jennifer H. Ho, MuhHwa Yang, Vincent W. Yang, and Oscar K. Lee

Figure S1

A

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CD29

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Figure S1 Characterization of Mouse Mesenchymal Stromal Cells, Related to Figure 1.

Figure S2

A HD 28

FL83B

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Heps

Alb Tat Krt18 Dpp4 Hnf1β Hnf3β Hnf4α Asgr1 G6pc Cyp2b10 Αfp Dlk1 Cd24a

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Figure S2 Comparison of Mouse MSC-Derived Hepatocyte-Like Cells, FL83B Cell Line and Primary Hepatocytes with Regard to Their Hepatic-Specific Genes Expression, Related to Figure 1.

Figure S3 D mesoderm

CD26high dHeps

osteoblasts

Isolation by FASC with hepatogenic surface marker

endoderm

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dHeps

MSCs*

dHeps

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88.4%

100%

4%

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5.5%

100%

1.1%

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0.4%

5.8%

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41.5%

CD29

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ddHeps CD90.2

A

mesoderm

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0.2%

4.9%

0.7%

9.1%

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2.7%

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2.9%

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4%

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Hepatocyte markers VE marker

CD106

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C

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82%

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0%

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4.9%

84%

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reHD 0

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30 25 20 15 10 5 0

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0.5 0.4 0.3 0.2 0.1 0 reOD 0

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reOD 14

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Figure S3 CD26high dHeps Derived from mBM-MSCs Can De-Differentiate into Functional MSCs In Vitro, Related to Figure 1 to 3.

AD 14

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1.5

H D H 0 D H 14 D H 21 D d H 28 dH D D 7 dH 1 D 4 dH 2 D 1 2 H 8 ep s

H D H 0 D H 14 D H 21 D d H 28 dH D D 7 dH 1 D 4 dH 2 D 1 2 H 8 ep s

Relative mRNA expression 2.0

H D H 0 D H 14 D H 21 D dH 28 dH D D 7 dH 1 D 4 dH 2 D 1 2 H 8 ep s

H D H 0 D H 14 D H 21 D dH 28 dH D D 7 dH 1 D 4 dH 2 D 1 2 H 8 ep s

Cdh1

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CDH1

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Figure S4 B

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** ** ** ** **

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Figure S4 Mesenchymal-to-Epithelial Transition Was Involved in Reversible Hepatogenic Differentiation of Mouse MSCs, Related to Figure 1.

Oil Red O

Morphology

reAD 0

reAD 0

reAD 7

reAD 7

reAD 14

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COL1A2

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ADIPOQ

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2 1.5

SPARC

25

FABP4

100

1

20

0

0

14

**

14

**

D

BGLAP

14

8

7

0

D

5

*

D

** 2.5

SNAI1

2.0

1.5

1.0

0.5

Migrated Cells/ field

CYP3A4 activity assay

re O D

RUNX2 **

re O

CYP2B6

re A

** *

re O

10 120 80 40 8

7

UPP2

D

** **

re O

FN1

7

TGFβ2

**

D

** 50 40 30 15

**

re H D 0 re HD 14 re HD 28 re HD 42 hH ep s

ALB

**

re A

0

200

0

**

D

UPP2

40

30

50 45 40 35 20 15 10 5 0

** **

0.5

0.0

350

**

250

200

HD 0

**

**

**

HD 42

H H D0 D H 14 D H 21 D H 28 D H 35 D d H 42 dH D d HD 1 7 d H D 24 D 1 hH 2 ep 8 s

H H D0 D H 14 D H 21 D H 28 D H 35 D d 4 dHHD 2 d HD 1 7 d HD 24 D 1 hH 2 ep 8 s

0

re O

10

0

0

20

5

D

10

re O

50

0

0.5

H H D0 D H 14 D H 21 D H 28 D H 35 D d 4 dHHD 2 D dH 17 d HD 24 D 1 hH 2 ep 8 s

H H D0 D H 14 D H 21 D H 28 D H 35 D d 4 dHHD 2 d HD 1 7 d HD 24 D 1 hH 2 ep 8 s

ALB

D

150

Fold of CYP3A4 activity

H H D0 D H 14 D H 21 D H 28 D H 35 D d 4 dHHD 2 d HD 1 7 d HD 24 D 1 hH 2 ep 8 s

Relative mRNA expression 10000 7500 5000 15

re A

1.0

14

1.0

14

1.5

D

0.0

14

5

H D 0 H D 14 H D 28 H D 4 dH 2 D 1 dH 4 D 2 hH 8 ep s

100

D

0.5

7

1.0

D

1.0

re O D

** H H D0 D H 14 D H 21 D H 28 D H 35 D d 4 dHHD 2 D dH 17 dHD 24 D 1 hH 2 ep8 s

1.5

re O

10

re O

0.0

H H D0 D H 14 D H 21 D H 28 D H 35 D d 4 dHHD 2 D dH 17 d HD 24 D 1 hH 2 ep 8 s

0

7

15

re HD 0 re H D 14 re H D 28 re H D 42 hH ep s

42 14

**

D

** D 2 hH 8 ep s

dH

D

28

Albumin production

re A

300

dH

H D

0

14

20

re O

10

*

0

*

D

H H D

**

7

reOD 14 60

re AD

reOD 14 *

re O

reOD 14 **

0

reOD 7 reHD 42 25000 20000 15000 10000 10 8 6 4 2 0

D

0.5

re O

1.5 80

0

reOD 7 HD 42

re AD

TGFβ1

14

CDH2

14

2.0 **

14

0.0

re O D

0.5 **

D

1.5 40

re O

2.0 H D

C

D

reOD 0 reOD 7 dHD28

re A

reOD 0 400

μg/ 106 cells

150

7

reOD 0 reHD 28 reHD 42 **

D

reHD 0 reHD 28 **

7

0.5

Urea production

D

1.0 **

0

ACTA2 NS

re O

CDH1 dHD28

7

0

reHD 0 600

D

0

HD 42

re O

1.5 800

H

0

28 D 4 dH 2 D 1 dH 4 D 2 hH 8 ep s

50 200

D

*

H

Glycogen production

H H D0 D H 14 D H 21 D H 28 D H 35 D dH 42 dH D dHD 17 dHD 24 D 1 hH 2 ep 8 s

800 600 400 20 15 10 5 0 0

**

14

100 **

dHD21

0

0.0

H H D0 D H 14 D H 21 D H 28 D H 35 D d H 42 dH D d HD 1 7 d HD 2 4 D 1 hH 2 ep 8 s

D **

D

**

H

NS

H D

dHD 14

H

250

HD 28

re HD 0 re H D 14 re H D 28 re H D 42 hH ep s

0

14

dHD 7

H H D0 D H 14 D H 21 D H 28 D H 35 D d 4 dHHD 2 D dH 17 dHD 24 D 1 hH 2 ep 8 s

D

200

28 H D 4 dH 2 D 1 dH 4 D 2 hH 8 ep s

H

D

H D H

HD 14

D

0.0

H H D0 D H 14 D H 21 D H 28 D H 35 D d 4 dHHD 2 D dH 1 7 d H D 24 D 1 hH 2 ep 8 s

H H D0 D H 14 D H 21 D H 28 D H 35 D d 4 dHHD 2 dHD 17 dHD 24 D 1 hH 2 ep 8 s

HD 0 dHD21

re O

0.0

H H D0 D H 14 D H 21 D H 28 D H 35 D d H 42 dH D d HD 1 7 d HD 24 D 1 hH 2 ep 8 s

A

re AD

D

I

re O

Morphology

Morphology

dHD 14

Relative mRNA expression

PAS

PAS dHD 7 HD 28

re AD

Morphology

G HD 14

Relative mRNA expression

ALP

μg/ 106 cells

HD 0

Relative mRNA expression

Von kossa

Relative mRNA expression

Figure S5 B HNF4α

1500 1000 500 20 15 10 5 0

TAT

50

E

40

30

20

10 0

F

**

50 0

HD 0 HD 42

dHD 28 hHeps

2.0

VIM

1.5

1.0

Migration assay

300

** NS

**

150

100 50

0 dHD 28

CYP2B6 **

6

4

**

2

0

SP7

1.0

**

4

0.5

0.0

SPP1

20

**

PPARG

80

**

60

40

Figure S5 Hepatogenic Differentiation of Human MSCs Is a Reversible Process, Related to Figure 1 to 3. hHeps

Figure S6

A

Control+LY364947

TGFβ1

TGFβ1+LY364947

7 days

3 days

Control

B Alb 4 3.5

Relative mRNA expression

Hnf1β 5

**

3

**

1.5

*

1

TGFβ1 Alk-5 inhibitor

-

-

+

+

-

-

+

+

-

+

-

+

-

+

-

+

3d

0

**

1.2

-

-

+

+

-

-

+

+

-

+

-

+

-

+

-

+

0.6

3

0.4

2

0.2

1

0

TGFβ1 Alk-5 inhibitor

-

-

+

+

-

-

+

+

-

+

-

+

-

+

-

+

3d

7d

0

-

-

+

+

-

-

+

+

-

+

-

+

-

+

-

+

-

-

+

+

-

-

+

+

-

+

-

+

-

+

-

+

3d

Krt18

**

Dpp4 **

**

**

-

+

+

-

-

+

+

-

+

-

+

-

+

-

+

7d

0

**

4

**

3

**

2

0.5

-

*

5

** 1

*

7d

6

1.5

3d

0

7d

2

5 4

0.5

3d

**

6

*

0

7d

Tat

1

**

1

1

7

0.8

**

1.5

1.5

3d

*

2

*

0.5

7d

** *

2

**

Cyp2b10 1.4

**

2.5

1

0.5 0

3.5

*

2

*

Hnf4α 2.5

**

3

3

**

2

*

4

**

2.5

Hnf3β **

4

*

1

-

-

+

+

-

-

+

+

-

+

-

+

-

+

-

+

3d

7d

0

-

-

+

+

-

-

+

+

-

+

-

+

-

+

-

+

3d

Figure S6 TGFβ1 Induced De-Differentiation in Mouse Hepatocytes, Related to Figure 6.

7d

Figure S7 A

Figure 1C

Figure 1K

Figure 4B

CCND2

250

DNMT1

130

ALB

Figure 4D

250 130 100

DNMT3b

PCNA

DNMT1

130 250

250 β-actin αFP CK18

DNMT1

130

DNMT1

130 100

Longer exposure

130 100

DNMT3a

130 100

DNMT3a

DNMT3b

130 100

Shorter exposure

130

PCNA

55 43 34 26

β-ACTIN

DNMT3b

100

Longer exposure

Short exposure

β-actin

130 100

DNMT3a

55

β-ACTIN

35

Figure 6E

Figure 6I

170 130 100 72 55 43 34 26

ALB

Figure 6O 250

CDH1

130 130

Figure S4B

Figure S2C

DNMT1

DNMT3a

ALB

CDH1

100 CK18

250

CK18

FN1

130

DNMT3b

100 αFP

72 55 43 34 26

αFP

250

SNAI1 TJP1

72 55 43 34 26

β-ACTIN

DLK1

TJP1

FN1

SNAI1 β-ACTIN

130 β-ACTIN

72 55 43 34

β-ACTIN β-ACTIN

B Rabbit IgG

Mouse IgG2a, k

Mouse IgG1, k

MSCs

mHeps

Figure S7 Uncropped Scans of Western Blots and Isotype Control for Immunocytochemical staining, Related to Figure 1, 3, 4, 5, 6 and 7.

Supplemental Figure Legends

Figure S1 Characterization of Mouse Mesenchymal Stromal Cells, Related to Figure 1. (A) Surface phenotype of MSCs. (B) Alkaline-phosphatase staining at 14 days of osteogenic induction in MSCs. Scale bar at 50 μm. (C) Oil Red O staining at 28 days of adipogenic induction in MSCs. Scale bar at 50 μm. (D) Safranin O staining at 28 days of chrondrogenic induction in MSCs. Scale bar at 1 μm. Figure S2 Comparison of Mouse MSC-Derived Hepatocyte-Like Cells, FL83B Cell Line and Primary Hepatocytes with Regard to Their Hepatic-Specific Genes Expression, Related to Figure 1. (A) Cellular morphology of MSC-derived hepatocyte-like cells, FL83B and Heps. Scale bar, 100 μm. (B) A heat-map summarizing relative mRNA levels of hepatogenic-specific genes (n = 3 independent experiments). Result of qRT-PCR is normalized relative to MSCs. (C-D) Results of a representative immunoblot, and (D) immunocytochemical staining for hepatogenic-specific gene expression. Scale bar, 50 μm. “HD 28” refers to MSC-derived hepatocyte-like cells (dHeps). “FL83B” is a hepatocyte cell line from a 15-17 day old fetal mouse. “Heps” refers to mouse primary hepatocytes. All quantitative data are presented as means ± SD. Statistical analyses were performed using Student’s paired t-test, with significance set at P < 0.05. (*P < 0.05; **P < 0.005). Figure S3 CD26high dHeps Derived from mBM-MSCs Can De-Differentiate into Functional MSCs In Vitro, Related to Figure 1 to 3. (A) Schematic illustration of the experimental approach. (B) Representative flow-cytometry plots showing CD26 expression and the gating strategies used to isolate CD26low and CD26high dHeps. (C) qRT-PCR of hepatogenic-specific gene expression in CD26low and CD26high dHeps (n = 4 independent experiments). (D) Surface phenotypic characterization analyzed with flow cytometry during dedifferentiation of CD26high dHeps. CD26high dHeps-derived ddHeps retained hepatogenic (E), osteogenic (F), and adipogenic (G) differentiation potential in vitro (n = 3 independent experiments). Results from Alizarin red S staining are shown (n = 3 independent experiments). Scale bar at 100 μm for phase contrast and staining. All quantitative data are presented as means ± SD. Statistical analyses were performed using Student’s paired t-test, with significance set at P < 0.05. (*P < 0.05; **P < 0.005). “HD 28,” “OD 14,” and “AD 14” represent primary MSCs after lineage commitments to hepatocyte-like cells, osteocytes, and adipocytes, respectively. “reHD 0,” “reOD 0,” and “reAD 0” represent CD26high dHepsderived ddHeps before lineage commitments to hepatocyte-like cells, osteocytes, and adipocytes, respectively. All quantitative data are presented as means ± SD. Statistical analyses were performed using Student’s paired t-test, with significance set at P < 0.05. (*P < 0.05; **P < 0.005). Figure S4 Mesenchymal-to-Epithelial Transition Was Involved in Reversible Hepatogenic

Differentiation of Mouse MSCs, Related to Figure 1. Gene expression of MET-markers analyzed with qRT-PCR (n = 4 independent experiments) (A), immunoblot (B), and immunocytochemical staining (n = 3 independent experiments) (C). Scale bar, 50 μm. (D) Scratch wound-healing assay (upper panel) and trans-well migration assay (lower panels) to measure cell migration capacity (n = 3 independent experiments). Quantification of three independent experiments (lower right panel) from trans-well migration assay (lower left panel). Scale bar, 100 μm. All quantitative data are presented as means ± SD. Statistical analyses were performed using Student’s paired t-test, with significance set at P < 0.05. (*P < 0.05; **P < 0.005). Figure S5 Hepatogenic Differentiation of Human MSCs Is a Reversible Process, Related to Figure 1 to 3. (A) Representative morphological changes and PAS staining of human MSCs during hepatogenic differentiation and de-differentiation. (B) Characterization of hepatogenic-specific genes with qRT-PCR (n = 3 independent experiments). (C) Hepatic functions were measured via albumin production (n = 4 independent experiments), CYP3A4 activity (n = 2, six technical replicates for each point), glycogen storage (n = 2 independent experiments, six technical replicates for each point), and urea production (n = 3 independent experiments). (D) Gene expression of MET/EMT markers analyzed with qRT-PCR (n = 3 independent experiments). (E) Trans-well migration assays of cell migration. The lower left graph shows the quantification of three independent experiments. (F) TGFβ1 expression was measured with ELISA (n = 3 independent experiments); the amount of TGFβ1 from serum has been deducted from results. De-differentiated human MSCs retained hepatogenic (G), osteogenic (H), and adipogenic differentiation (I) potential in vitro (n = 3 independent experiments). Scale bar at 100 μm for phase contrast and staining. All quantitative data are represented as means ± SD. Statistical analyses were performed using Student’s paired t-test, with significance set at P < 0.05. (*P < 0.05; **P < 0.005). Figure S6 TGFβ1 Induced De-Differentiation in Mouse Hepatocytes, Related to Figure 6. (A) Representative morphology of mouse hepatocytes under TGFβ1 treatment. (B) qRT-PCR of hepatogenic-specific genes in mouse Heps under TGFβ1 treatment for 3 and 7 days (n = 4 independent experiments). Scale bar at 100 μm for phase contrast. The + and - symbols respectively indicate treatments with and without TGFβ1 (10 ng/mL) and Alk-5 inhibitor (400 nM). All quantitative data are presented as means ± SD. Statistical analyses were performed using Student’s paired t-test, with significance set at P < 0.05. (*P < 0.05; **P < 0.005). Figure S7 Uncropped Scans of Western Blots and Isotype Control for Immunocytochemical staining, Related to Figure 1, 3, 4, 5, 6 and 7. (A) Uncropped scans of western blots. (B) Rabbit IgG for αFP, CK18 and SNAI1 staining; mouse IgG2a, k for ALB and CDH1 staining; mouse IgG1, k for FN1 staining. Scale bar, 50 μm.

Supplemental Tables Table S1 Functional Enrichment of KEGG Pathways in Hepatogenic Differentiation and DeDifferentiation of Mouse MSCs, Related to Figure 2. KEGG pathway KEGG ID

Up-regulation in HD; down-regulation in dHD

P Value HD 0 to HD 28

HD 28 to dHD 28

mmu00982

Drug metabolism

1.40E-15

1.69E-15

mmu04610

Complement and coagulation cascades

1.91E-15

3.36E-17

4.76E-14

4.08E-13

mmu00980

Metabolism of xenobiotics by cytochrome P450

mmu04142

Lysosome

1.39E-10

3.27E-12

mmu01100

Metabolic pathways

8.93E-10

2.09E-09

mmu00140

Steroid hormone biosynthesis

1.24E-09

1.67E-07

mmu00830

Retinol metabolism

1.07E-08

1.38E-08

mmu03320

PPAR signaling pathway

2.43E-06

2.40E-06

mmu00380

Tryptophan metabolism

9.03E-06

4.65E-04

mmu00480

Glutathione metabolism

1.43E-05

1.91E-06

mmu00590

Arachidonic acid metabolism

1.59E-04

1.58E-04

mmu04146

Peroxisome

6.39E-04

5.84E-05

mmu00350

Tyrosine metabolism

6.66E-04

4.92E-03

mmu00120

Primary bile acid biosynthesis

9.39E-04

1.49E-03

mmu00591

Linoleic acid metabolism

1.45E-03

2.92E-03

mmu00360

Phenylalanine metabolism

1.46E-03

2.43E-03

KEGG pathway KEGG ID

Down-regulation in HD; Up-regulation in dHD

PValue HD 0 to HD 28

HD 28 to dHD 28

mmu03030

DNA replication

2.05E-21

4.62E-23

mmu04110

Cell cycle

2.08E-18

1.39E-17

mmu00240

Pyrimidine metabolism

4.30E-09

2.21E-09

mmu03440

Homologous recombination

1.01E-07

1.88E-07

mmu03430

Mismatch repair

1.01E-06

5.99E-09

mmu00230

Purine metabolism

3.73E-06

1.08E-05

mmu03013

RNA transport

1.45E-05

4.78E-11

mmu03420

Nucleotide excision repair

1.63E-05

2.79E-08

mmu04914

Progesterone-mediated oocyte maturation

5.99E-05

1.33E-04

mmu03410

Base excision repair

7.62E-05

5.07E-08

mmu00670

One carbon pool by folate

3.70E-04

2.32E-04

mmu03040

Spliceosome

2.23E-03

1.32E-08

mmu03008

Ribosome biogenesis in eukaryotes

5.06E-03

7.77E-04

mmu01130

Biosynthesis of antibiotics

6.14E-03

9.28E-02

mmu04114

Oocyte meiosis

6.51E-03

5.38E-03

mmu03018

RNA degradation

3.33E-02

2.03E-03

Table S2 Top 10 Functional Enrichment of KEGG Pathway during MSC-Like Cells Generation from Mouse Hepatocytes, Related To Figure 2.

KEGG ID

KEGG pathway Up-regulation

P Value

mmu04110

Cell cycle

8.55E-25

mmu03040

Spliceosome

3.22E-17

mmu04510

Focal adhesion

4.44E-15

mmu03013

RNA transport

9.02E-12

mmu03030

DNA replication

9.47E-10

mmu04151

PI3K-Akt signaling pathway

3.64E-09

mmu04114

Oocyte meiosis

8.99E-09

mmu04390

Hippo signaling pathway

1.44E-08

mmu00240

Pyrimidine metabolism

9.42E-08

mmu04810

Regulation of actin cytoskeleton

1.25E-07

KEGG ID

KEGG pathway Down-regulation

P Value

mmu01100

Metabolic pathways

9.49E-50

mmu04146

Peroxisome

4.63E-22

mmu00280

Valine, leucine and isoleucine degradation

2.87E-21

mmu04610

Complement and coagulation cascades

1.27E-18

mmu00140

Steroid hormone biosynthesis

1.83E-16

mmu00071

Fatty acid degradation

4.77E-16

mmu00830

Retinol metabolism

6.69E-16

mmu01130

Biosynthesis of antibiotics

5.85E-15

mmu00380

Tryptophan metabolism

1.04E-12

mmu00980

Metabolism of xenobiotics by cytochrome P450

2.56E-12

Table S3 Primer and Primary Antibodies List, Related to Figure 1, 3, 4, 5, 6 and 7. ICC, immunocytochemical assay; IB, immunoblot; FL, flow cytometry; M, mouse; H, Human; NA, not accessed. * The clone numbers are provided for all monoclonal antibodies used in this study. NA means that the antibodies are polyclonal.

Supplemental Experimental Procedures Osteogenic, adipogenic and chondrogenic differentiation We induced osteogenic and adipogenic re-differentiation via treating with osteogenic medium or adipogenic medium for two weeks. The differentiation medium was changed once per week. Mouse osteogenic medium consists of DMDM supplemented with 100 nM dexamethasone, 10 mM β-glycerol phosphate, and 50 μg/mL ascorbate-2-phosphate. Human osteogenic medium consists of IMDM supplemented with 100 nM dexamethasone, 10 mM β-glycerol phosphate, and 50 μg/mL ascorbate-2phosphate. Osteogenesis was assessed by qRT-PCR, alkaline phosphatase, and von Kossa and Alizarin red S stainings. Mouse adipogenic medium consists of DMDM supplemented with 0.5 mM 3-isobutyl1-methylxanthine, 1 μM dexamethasone, 50 μM indomethacin, 5 μg/mL Insulin, and 10% FBS (Gibco®, Thermo Fisher Scientific, Waltham, MA). Human adipogenic medium consists of IMDM supplemented with 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, 50 μM indomethacin. Adipogenesis was assessed by qRT-PCR and Oil Red O staining. To induce chrondrogenic differentiation, MSCs were transferred into a 15 mL polypropylene tube and centrifuged at 1000 rpm for 5 min, to form a pelleted micromass at the bottom of the tube, and then treated with chondrogenic medium for 4 weeks. Chondrogenic medium consists of high-glucose DMEM supplemented with 0.1 μM dexamethasone, 50 μg/mL ascorbic acid, 100 μg/mL sodium pyruvate, 40 μg/mL proline, 10 ng/mL hTGFβ1, and 50 mg/mL ITS+ premix (BD Bioscience, San Jose, CA). Chondrogensis was assessed by Safranin O staining. SigmaAldrich (St. Louis, MO) supplied dexamethasone, β-glycerol phosphate, ascorbate-2-phosphate, 3isobutyl-1-methylxanthine, indomethacin, Insulin, ascorbic acid, sodium pyruvate, proline and hTGFβ1. Quantitative real-time quantitative polymerase chain reaction RNA was extracted using an RNeasy Mini kit (Qiagen, Valencia, CA) or Trizol reagent (Sigma-Aldrich, St. Louis, MO) and reverse transcribed by Moloney murine leukemia virus reverse transcriptase (Epicentre, Madison, WI). Quantitative real-time quantitative polymerase chain reaction (qRT-PCR) was performed using a ABI StepOnePlus (Applied Biosystems, Waltham, MA). All quantifications were normalized to an endogenous control, Gapdh or RPS27a. Primers for mouse and human genes are listed in Supplemental Table S3. Hepatic functional assays Glycogen storage and urea concentrations were measured by colorimetric assays (Biovision, Milpitas, CA), cytochrome P450 3A4 (CYP3A4) activity was measured by cell-based P450-Glo™ assays (Promega, Madison, WI) through a luminescent method, and albumin secretion was measured by an ELISA assay (Immunology Consultants Laboratory, Inc., Portland, OR) per the manufacturers’ recommendations and analyzed with microplate readers (TECAN, Männedorf, Switzerland). Lowdensity lipoprotein (LDL) uptake by cells was assessed by LDL Uptake Cell-Based Assay kit (Cayman, Ann Arbor, MI) according to the manufacturer’s instructions. MSC-maintenance medium and Step-2 differentiation medium served as background controls for urea assay, CYP3A4 activity, and albumin

secretion assay. Cytochemical, Immunoblot and Immunocytochemical Analysis For glycogen storage determination (PAS staining), cells were fixed in 4% formaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, oxidized in 1% periodic acid for 5 min, rinsed 3 times with ddH2O, treated with Schiff’s reagent for 30 min, and rinsed 3 times with ddH2O. For alkaline phosphatase detection (ALP staining), cells were fixed with 4% formaldehyde and treated with NBT/BCIP RT in the dark for 1 hour, and rinsed 3 times in ddH2O. For evaluation of mineralization, cells were fixed with 4% formaldehyde and assayed by von Kossa staining using 1% silver nitrate under ultraviolet light for 45 min, and assayed by Alizarin Red S (ARS) staining using 40 mM Alizarin-red S solution pH 4.1 for 30 min. ARS staining extent was extracted by acetic acid and semi-quantified as previously described (Gregory et al., 2004). For Oil Red O staining, cells were fixed with 4% formaldehyde, treated with 60% isopropanol for 5 min, and stained with Oil Red O for 10 min. Samples were assessed under an optical microscope. For Safranin O staining, paraffin section were deparaffinized by xylene and gradient alcohol treatment and were hydrated by distilled water. Samples were stained with hematoxylin for 3 min and washed in running tap water for 10 min. Samples were rinsed with 1% acetic acid for 10 s and washed in running tap water. Samples were stained in 0.1% Safranin O solution for 5 min. Samples were dehydrated with 95% ethyl alcohol, absolute ethyl alcohol, and xylene, using 2 changes each, 2 min each time. For immunoblotting, cells were lysed in cell lysis buffer containing protease inhibitors and phosphatase inhibitors. Quantified samples were separated by polyacrylamide gels and transferred to PVDF membranes. For immunocytochemical staining, cells were fixed in 4% formaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 for 10 min. After blocking, samples were incubated with primary antibodies against target proteins overnight at 4⁰C, followed by a secondary antibody at room temperature for 1 hour. Sigma-Aldrich (St. Louis, MO) supplied Triton X-100, periodic acid, Schiff’s reagent, formaldehyde, NBT/BCIP, silver nitrate, Alizarin-red S, Oil Red O, Safranin O, cell lysis buffer, protease inhibitor, phosphatase inhibitors, and secondary antibody. Primary antibodies for immunoblotting and immunocytochemical staining were as follows: anti-Fn1 (BD Bioscience, San Jose, CA); anti-CK18 (Epitomic, Burlingame, CA); anti-Cdh1 (Cell Signaling, Danvers, MA). Abcam (Cambridge, UK) supplied anti-Albumin (western blotting), anti-αFP, anti-Cd26, anti-Pcna, anti-Snai1, anti-Tjp1, anti-DNMT1, anti-DNMT3a, and anti-DNMT3b. Sigma-Aldrich (St. Louis, MO) supplied anti-Ccnd2 and anti-β-actin. R&D Systems (Minneapolis, MN) supplied anti-Albumin (for immunocytochemical staining and flow cytomerty). For cell surface phenotyping, cells were detached and stained with fluorescein- or phycoerythrin-coupled antibodies; for cell cycle analysis, cells were detached and stained with propidium iodide per the manufacturer’s recommendations (BD Biosciences, San Jose, CA) and analyzed with FACS Calibur. Primary antibodies for flow cytometry are as follows: BD Biosciences (San Jose, CA) supplied anti-Cd117, anti-Cd44, anti-Cd106, anti-Cd26, anti-Sca-1, antiCd31, anti-Cd90.1, anti-Cd34, anti-Cd45, anti-Cd90.2). eBioscience (San Diego, CA) supplied antiCd29, anti-Cd73, anti-Cd105. Primary antibodies list are listed in Supplemental Table S3 in detail.

Cell growth curve MSCs, MLCs and re-thawed MLCs were seeded at density of 1.8 x 104 and Heps were seeded at a density of 1 x 105 in 6-well plate. The cells were detached and the numbers of cells were counted at each time point. TGFβ1 ELISA assay Human and mouse TGFβ1 secretion were measured by ELISA assay (R&D system, Minneapolis, MN) per the manufacturers’ recommendations and analyzed with microplate readers (TECAN, Männedorf, Switzerland). Scratch wound-healing assay Cell migration was analyzed by a scratch wound-healing assay. Cells were grown to full confluence, and a scratch wound in the monolayer was performed by dragging a 1 mL pipette tip across the layer. Detached cells were washed away with PBS. The remaining cells were cultured in medium, and closure of the wound was observed under a microscope post inflicting the wound for 18 hours. Trans-well migration assay The assay was performed in 24-well transwells (Corning, NY). A total of 2.5 x 104 cells in 0.2 mL medium were seeded to a membrane in the upper well of a trans-well apparatus and allowed to migrate for 18 hours. Cells were fixed with 4% formaldehyde for 10 min and permeablized by 0.1% Triton X100 for 10 min. After removing the non-migrated cells, hematoxylin staining was performed at room temperature for 20 min to visualize the cells passing through the membrane. The migration activity of cells was determined by counting the cells under a microscope, at ×100 magnification. siRNA transfection siRNA oligos were purchased from Invitrogen (Grand Island, NY). Briefly, 50 nM of target siRNA and control siRNA (Stealth RNAi™ siRNA Negative Control, Med GC) were transfected with RNAimax transfection reagent (Invitrogen, Grand Island, NY) at 37⁰C. Cells were washed with PBS and the medium was replaced with differentiation or de-differentiation medium after 18 hours. For Fig. 4B-D, the siRNA against the DNMTs was transfected at day 7 and day 11 of differentiation, and the samples were collected at day 14 and day 28. For Figure 4E, the siRNA against the DNMTs was transfected at day 28 of differentiation with MSC-maintenance medium. Target sequences: mouse DNMT1 siRNA, CAGAGCAGAUUGAGAAGUAUGAUAA, GAGGACAUCCUGUGGUGCACUGAAA, CAGCACAGAGUAUCAGGAUGAUAAA. Microarray and antibody array analysis

mouse and

DNMT3a mouse

DNMT3b

siRNA, siRNA,

MSCs, un-isolated dHep, ddHeps, isolated CD26high Heps and un-isolated MLCs were used to perform microarray and antibody array. For transcriptome analysis, total RNA were hybridized to the Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA) at the National Research Progress for Genomic Medicine Microarray and Gene Expression Analysis Core Facility, National Yang-Ming University VYM Genome Research Center, Taiwan. For proteomic analysis, total proteins were analyzed on Lseries mouse antibody array L-308 (RayBiotech, Norcross, GA). All data have been uploaded to the Gene Expression Omnibus (GEO) repository (GSE73617 for microarray and GSE73762 for protein array). One-step Tukey’s BiWeight (TBW) method (Hubbell et al., 2002) were applied to protein expression data to adjust the negative signals. Data were preprocessed and analyzed using packages of Bioconductor (Gentleman et al., 2004) on the R programming language (Ihaka and Gentleman, 1996). The ‘affy’ package (version 1.42.3) was used for data preprocessing and normalization; the ‘affyQCReport’ package (1.42.0) was used to assess quality of microarray data; correlations between microarray samples was shown in heatmap created via the ‘gplots’ package (version 2.16.0). Heatmaps and Hierarchical clustering of gene expression profiles were created by TM4:MeV software (Saeed et al., 2003) (http://www.tm4.org/mev.html). Enrichment analysis was carried out via the Database for Annotation, Visualization and Integrated Discovery (DAVID) (Huang da et al., 2009a, b) and Kyoto Encyclopedia of Genes and Genomes (KEGG). For signaling pathway identification, first 299 intersected genes/proteins were selected and the Pearson’s correlation coefficient (PCC) between the microarray data and protein data for each gene/protein was calculated. Next, the candidates were chosen according to 3 criteria: (1) PCC between DNMT1 and each candidate should be greater than 0.6; (2) gene expression and protein expression of each candidate in MSC should be greater than Heps, as well as DNMT1; (3) PCC between gene expression and protein expression of each candidate should be greater than 0.7. Final, all candidates were arranged by PCC between gene expression and protein expression in descending order. 4 out of 9 selected candidates with high PCC (Tgfb1, Thbs2, Thbs1 and Thbs3) were associated with the TGF-beta signaling pathway. Tumorigenicity assays For tumorigenicity assays, 1 x 106 cells suspended in 100 μL culture medium were injected subcutaneously into 6 week-old male BALB/c nude mice with 6 mice in each group. Animals were monitored regularly for tumor occurrence. An approval prior to the commencement of the experiment from IACUC has been obtained regarding the use of animal. Animal Experimentation In the study, we assured that all animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985). All animal studies were approved by the Taipei Veterans General Hospital Institutional Animal Care and Use Committee (IACUC 2014-043).

Statistical analysis All of the quantitative results are shown as the means ± SD from at least three independent measurements. Statistical analyses were performed using Student’s paired t test. Value of P