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Am J Physiol Gastrointest Liver Physiol 293: G1089-G1098, 2007. First published September 20, 2007; doi:10.1152/ajpgi.00187.2007
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LIVER AND BILIARY TRACT

A role of Wnt/beta-catenin signals in hepatic fate specification of human umbilical cord blood-derived mesenchymal stem cells

Yoko Yoshida,1 Takashi Shimomura,1 Tomohiko Sakabe,1 Kyoko Ishii,1 Kazue Gonda,1 Saori Matsuoka,1 Yumi Watanabe,1 Kazuko Takubo,2 Hiroyuki Tsuchiya,1 Yoshiko Hoshikawa,1 Akihiro Kurimasa,1 Ichiro Hisatome,3 Taro Uyama,4 Masanori Terai,4 Akihiro Umezawa,4 and Goshi Shiota1

1Division of Molecular and Genetic Medicine, Department of Genetic Medicine and Regenerative Therapeutics, Graduate School of Medicine, Tottori University; 2Division of Oral and Maxillofacial Biopathological Surgery, Department of Medicine of Sensory and Motor Organs, Faculty of Medicine, Tottori University; 3Division of Regenerative Medicine, Department of Genetic Medicine and Regenerative Therapeutics, Graduate School of Medicine, Tottori University; and 4Department of Reproductive Biology and Pathology, National Research Institute for Child Health and Development, Tokyo, Japan

Submitted 30 April 2007 ; accepted in final form 7 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to be an excellent source of cells for transplantation. In addition, the stem cell plasticity of human UCBMSCs, which can transdifferentiate into hepatocytes, has been reported. However, the mechanisms involved remain to be clarified. To identify the genes and/or signals that are important in specifying the hepatic fate of human UCBMSCs, we analyzed gene expression profiles during the hepatic differentiation of UCBMSCs with human telomerase reverse transcriptase, UCBMSCs immortalized by infection with a retrovirus carrying telomerase reverse transcriptase, but whose differentiation potential remains unchanged. Efficient differentiation was induced by 5-azacytidine (5-aza)/hepatocyte growth factor (HGF)/oncostatin M (OSM)/fibroblast growth factor 2 (FGF2) treatment in terms of function as well as protein expression: 2.5-fold increase in albumin, 4-fold increase in CCAAT enhancer-binding protein {alpha}, 1.5-fold increase in cytochrome p450 1A1/2, and 8-fold increase in periodic acid-Schiff staining. Consequently, we found that the expression of Wnt/beta-catenin-related genes downregulated, and the translocation of beta-catenin was observed along the cell membrane and in the cytoplasm, although some beta-catenin was still in the nucleus. Downregulation of Wnt/beta-catenin signals in the cells by Fz8-small interference RNA treatment, which was analyzed with a Tcf4 promoter-luciferase assay, resulted in similar hepatic differentiation to that observed with 5-azacytidine/HGF/OSM/FGF2. In addition, the subcellular distribution of beta-catenin was similar to that of cells treated with 5-azacytidine/HGF/OSM/FGF2. In conclusion, the suppression of Wnt/beta-catenin signaling induced the hepatic differentiation of UCBMSCs, suggesting that Wnt/beta-catenin signals play an important role in the hepatic fate specification of human UCBMSCs.

mesenchymal stem cell; Wnt/beta-catenin signaling pathway; hepatic differentiation

HUMAN UMBILICAL CORD BLOOD (UCB) has been reported to contain stem/progenitor cells at concentrations greater than or equal to those in bone marrow and adult peripheral blood (28, 30). In addition, the use of UCB entails few ethical problems, and even now most UCB is regarded as medical waste in delivery rooms. Although bone marrow is an important source of stem cells, its use has several disadvantages: aspirating bone marrow from patients is an invasive procedure, and the capacity of stem cells to differentiate decreases with the donor's age (7). UCB-cell transplantation for various blood diseases has recently been successful, with a lower incidence of graft-vs.-host disease than that seen in many conventional treatments (18). Thus UCB is considered useful for therapeutic applications.

It has long been thought that the differentiation potential of adult stem cells is limited to their germ layer of origin, but recent studies have demonstrated that adult stem cells are more plastic than once believed (1, 20–22, 47). Mesenchymal stem cells (MSCs) can differentiate into several lineages including osteoblasts, chondrocytes, and adipocytes. Recently, in vivo transplantation studies showed that MSCs can differentiate into endodermal cell types as well as most mesodermal and neuroectodermal types (15). Human MSCs from bone marrow and UCB-derived MSCs (UCBMSCs) can differentiate into hepatocytes in vitro (41, 24, 25). UCBMSC may become an important alternative source of cells for transplantation; however, the hepatic differentiation of MSCs is not efficient enough for clinical use. Therefore, the molecular mechanism of hepatic differentiation should be clarified. Newly established UCBMSCs with human telomerase reverse transcriptase (UCBTERT) proliferated for >120 population doublings, and the characteristics of the cells including differentiation potential remained unchanged (46). Therefore, UCBTERT cells provide a powerful model for the application of stem cell-based therapies.

Adult stem cells, under certain microenvironmental conditions, give rise to cell types besides the cell type of origin, indicating that they can switch cell fate, a feature termed "stem cell plasticity" (23). An alternative mechanism to induce cell plasticity could be the cell fusion of a bone marrow-derived cell with a nonhematopoietic cell (19). Although use of the Cre/loxP system revealed that some hepatocytes from bone marrow-derived cells are really produced by cell fusion in vivo (2), in vitro hepatic differentiation of MSCs demonstrated that stem cell plasticity really exists (24, 25). In addition, we previously reported that the hepatic differentiation of UCB cells as well as cell fusion occurred simultaneously in vivo (45). Since cells generated from cell fusions may be more likely to undergo transformation, hepatocytes differentiated from UCB cells in vitro, which do not mediate cell fusion, are a reliable cell source for regenerative medicine. We therefore attempted to identify the genes and/or signals that regulate the hepatic differentiation of human UCBMSCs.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and oncostatin M (OSM) were purchased from PeproTech EC (London, UK). Recombinant Wnt-3A was obtained from R&D Systems. Mesenchymal stem cell growth medium (MSCGM, PT-3001) and SYTOX green nucleic acid stain were purchased from Cambrex Bio Science. 5-Azacytidine (5-aza) was obtained from Nacalai Tesque (Kyoto, Japan). Anti-human serum albumin antibody (A 6684), anti-C/EBP{alpha} antibody (sc-61), and anti-CYP1A1/1A2 (AB1255), and Cy3-conjugated secondary antibody (AP132C) were purchased from Sigma Aldrich (St. Louis, MO), Santa Cruz Biotechnology (Santa Cruz, CA), and Chemicon International, respectively.

Culture of UCBTERT-21 cells. Human UCBMSCs, the life span of which was prolonged by infection with a retrovirus encoding human telomerase reverse transcriptase (hTERT), designated UCBTERT-21 cells (46), were used. All cultures were maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO2. To induce the hepatic differentiation of UCBTERT-21 cells in vitro, we added several cytokines to the culture media. UCBTERT-21 cells were plated into six-well plates at 2 x 104 cells/well and cultured overnight to allow cell attachment. To induce hepatic differentiation, UCBTERT-21 cells were cultured in MSCGM containing 1 µM 5-aza for 24 h and then cultured in MSCGM containing 10% FBS, 10 ng/ml FGF2, 20 ng/ml HGF, and 20 ng/ml OSM for 3 wk with special reference to Refs. 25, 29, and 41. The medium was changed weekly (Fig. 1). Hepatic differentiation was assessed by reverse transcription-polymerase chain reaction (RT-PCR), immunostaining, periodic acid-Schiff (PAS) staining, and urea assay.


Figure 1
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  		Fig. 1. Induction of hepatic differentiation of umbilical cord blood-derived mesenchymal stem cells with human telomerase reverse transcriptase (UCBTERT-21) cells. Experimental protocol. UCBTERT-21 cells were transferred into 6-well plates at 2 x 104 cells/well. The cells were cultured in mesenchymal stem cell growth medium (MSCGM) with or without 1 µM 5-azacytidine (5-aza) or not for 24 h and then cultured in MSCGM containing 20 ng/ml hepatocyte growth factor (HGF), 20 ng/ml oncostatin M (OSM), and/or 10 ng/ml fibroblast growth factor 2 (FGF2) for 3 wk. The culture medium was exchanged weekly. Total RNA was extracted from the UCBTERT-21 cells for RT-PCR.

 
RNA extraction and RT-PCR analysis. Total RNA was extracted from UCBTERT-21 cells by using a Total RNA Isolation kit (Promega, Madison, WI). cDNA samples were synthesized from 1 µg of total RNA by using a Super Script II first-strand synthesis system (Invitrogen, Carlsbad, CA) with a-oligo(dT)-adaptor primer according to the manufacturer's instructions. The cDNA preparation template was used for subsequent PCR amplification by using Taq DNA polymerase. The primers for albumin, CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}), CCAAT/enhancer-binding protein beta (C/EBPbeta), cytochrome p4501A1 (CYP1A1), cytochrome p4501A2 (CYP1A2), phosphoenolpyruvate carboxykinase (PEPCK), and GAPDH were as follows: albumin forward primer, 5'-TTGGAAAAATCCCACTGCAT-3'; albumin reverse primer, 5'-CTCCAAGCTGCTCAAAAAGC-3'; C/EBP{alpha} forward primer, 5'-CACGAAGCACGATCAGTCCAT-3'; C/EBP{alpha} reverse primer, 5'-CGCACATTCACATTGCACAAG-3'; C/EBPbeta forward primer, 5'-GCCAAGAAGACCGTGGACA-3'; C/EBPbeta reverse primer, 5'-GCCAAGAAGACCGTGGACA-3'; CYP1A1 forward primer, 5'-ACCATGACCAGAAGCTATGGGT-3'; CYP1A1 reverse primer, 5'-TTAACACCTTGTCGATAGCACCA-3'; CYP1A2 forward primer, 5'-ACCATGACCCAGAGCTGTGG-3'; CYP1A2 reverse primer, 5'-TCACTCAAGGGCTTGTTAAT-3' PEPCK forward primer, 5'-CAGGCAGCTGAAGAAGTATGA-3', PEPCK reverse primer, 5'-AACCGTCTTGCTTTCGATCCT-3', GAPDH forward primer, 5'-GTCTTCTCCACCATGGAGAAGGCT-3'; GAPDH reverse primer, 5'-CATGCCAGTGAGCTTCCCGTTCA-3'. Albumin was amplified at 95°C for 2 min, with denaturing at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s for 35 cycles. GAPDH was amplified at 95°C for 2 min, with denaturing at 95°C 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s for 30 cycles. The PCR products were analyzed by electrophoresis with a 2% agarose gel and stained with ethidium bromide. The intensity of the UV-light illuminated bands was measured by ImageJ (http://rsb.info.nih.gov/ij/) and was expressed after being normalized to GAPDH. Control RNA from normal adult liver was obtained when a patient with colon cancer metastasis underwent partial hepatectomy under informed consent.

Real-time quantitative PCR analysis. The cDNA template was amplified by use of a LightCycler (Roche). beta-Actin was employed as an internal reference gene to normalize cDNA input. The mRNA expression levels of beta-catenin, PP2A, and frizzled 8 (Fz8) were defined as the ratio of the value of each gene product to that of the beta-actin product. The primers were as follows: albumin forward primer, 5'- TGTTGCATGAGAAAACGCCA-3'; albumin reverse primer, 5'- GTCGCCTGTTCACCAAGGAT-3'; Fz8 forward primer, 5'-TCTGGTGGGTGATCTTGTCG-3'; Fz8 reverse primer, 5'-AGCACCGCGATGGACTTGAC-3'; beta-actin forward primer, 5'-CACTCTTCCAGCCTTCCTTCC-3'; beta-actin reverse primer, 5'-CGTACAGGTCTTTGCGGATGTC-3'. The real-time PCR assays were performed with SYBR green (Roche) according to the manufacturer's instructions.

PAS staining for glycogen. Cells were fixed with PBS containing 4% formaldehyde for 20 min, permeabilized with PBS containing 0.1% Triton X-100 for 10 min, and incubated in the presence or absence of 1 mg/ml diastase for 1 h at 37°C. Samples were then oxidized in 1% periodic acid for 5 min, treated with Schiff's reagent for 15 min, rinsed three times in a sodium sulfite solution (0.5% sodium sulfite, 0.05 N HCl), and rinsed another three times in H2O. Sections were assessed under a light microscope.

Urea assay. UCBTERT-21 cells at 1 x 105 cells/well were cultured, and the cell density was adjusted weekly. After 3 wk, 3 x 104 cells were incubated with 5 mM ammonium chloride, and the amount of urea secreted into the medium was measured every 24 h for up to 96 h. Urea concentrations were determined with a QuantiChrom urea assay kit (BioAssay Systems) according to the manufacturer's instructions.

Immunocytochemistry. Cultures were collected by enzymatic methods and were plated onto coverslips. The coverslips were fixed in PBS containing 4% formaldehyde for 20 min and permeabilized with PBS containing 0.2% Triton X-100 for 10 min. Samples were blocked with goat serum for 20 min and then incubated with anti-human serum albumin, anti-human C/EBP{alpha}, anti-CYP1A1/1A2, or anti-beta-catenin antibodies, followed by Vectastain ABC Systems (Vector Laboratories, Burlingame, CA) or Cy3-conjugated secondary antibody. Hematoxylin or SYTOX green was utilized for nuclear counterstaining. The coverslips were mounted with Gel/Mount, and the sections were assessed under a light microscope or fluorescence microscope.

DNA microarrays. RNA was extracted from UCBTERT-21 cells treated with or without 5-aza/HGF/OSM/FGF2 on day 7 by use of an RNeasy mini kit (Qiagen), followed by digestion with RNase-free DNase (Qiagen). DNA microarray analysis was performed using AceGene human oligo chip 30k 1 chip version (Hitachi Software Engineering, Yokohama, Japan), which contains the oligonucleotide probe sets for 30,000 human genes. The intensity of fluorescence of each probe was measured with a Fuji FLA-8000 scanner (FujiPhoto Film, Tokyo, Japan) and quantified using Array Gauge software (FujiPhoto Film). The expression of each gene was compared between UCBTERT-21 cells treated with and without 5-aza/HGF/OSM/FGF2.

Transfection of siRNA. The Fz8 small interference RNA (siRNA; Fzd8-siRNA, cat. SI02646413) and nonsilencing siRNA control (negative control siRNA, cat. 1022563) were purchased from Qiagen. UCBTERT-21 cells were transfected with siRNA in multiwell plates by using RNAiFect transfection reagent (Qiagen). A mixture of 1 µg siRNA, 100 µl Buffer EC-R, and 6 µl RNAiFect transfection reagent was incubated for 15 min at room temperature. One hundred microliters of the mixture and 300 µl of medium containing 10% FBS were incubated with the cells for 6 h. The effect of mRNA silencing was confirmed by real-time quantitative PCR analysis.

Gene reporter assay. The plasmid Tcf4-CMVpro-Luc contains three copies of the optimal Tcf motif CCTTTGATC upstream of the CMV promoter that drives the expression of luciferase (GoGoFig. 4B) (38). The CMV promoter sequence was inserted into pGL3-Basic vector (Promega). The plasmid pRL-TK (Promega) was used as an internal control. Transient transfection was performed using RNAiFect transfection reagent. As a positive control, 100 ng/ml of Wnt-3a was added 24 h after transfection. At 48 h after transfection, the cell lysates were used for gene reporter assays with the Dual-Luciferase reporter assay system (Promega).


Figure 2
Figure 2
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  		Fig. 2. Hepatic differentiation of UCBTERT-21 cells. A: expression of the albumin gene evaluated by quantitative real-time RT-PCR. The level of albumin gene expression is shown as a ratio of that in the control on day 21. bullet, 10% FBS (control); {blacksquare}, 10% FBS with 5-aza/HGF/OSM/FGF2. Data are expressed as means ± SE of 3 experiments. **P < 0.01 compared with the control. B: induction of hepatic specific genes in UCBTERT-21 cells on day 21 was analyzed by cytochemistry. a–d, 10% FBS; e–h, 10% FBS with 5-aza/HGF/OSM/FGF2. Cells were immunostained with anti-albumin (a, e), anti-CCAAT enhancer-binding protein {alpha} (C/EBP{alpha}) (b, f), and anti-cytochrome p450 1A1/2 (CYP1A1/1A2) (c, g) antibodies. Glycogen stored in the cells was stained by periodic acid-Schiff (PAS) stain (d, h). Scale bar, 100 µm. C: efficiency of hepatic differentiation of UCBTERT-21 cells on day 21. Open bars, 10% FBS (control); solid bars, 10% FBS with 5-aza/HGF/OSM/FGF2. Data are expressed as means ± SE of 6 experiments. **P < 0.01, *P < 0.05, compared with control. D: urea production in the culture medium of differentiated UCBTERT-21 cells. bullet, 10% FBS (control); {blacksquare}, 10% FBS with 5-aza/HGF/OSM/FGF2. Data are expressed as means ± SE of 6 experiments. *P < 0.05, compared with control. E: expression of hepatocyte-specific genes in 5-aza/HGF/OSM/FGF2-treated cells compared with human adult normal liver. Hepatocyte-specific genes included albumin, C/EBP{alpha}, C/EBPbeta, CYP1A1, CYP1A2, and phosphoenolpyruvate carboxykinase (PEPCK). PEPCK is the rate-limiting enzyme of glucogenesis. UCBTERT day 0, UCBTERT 21d control, UCBTERT 21d 5-aza/HOF, and Adult liver represent UCBTERT cells before incubation, cells incubated in 10% FBS for 21 days, cells incubated in 5-aza/HGF/OSM/FGF2 for 21 days, and adult liver tissue, respectively. Data are expressed as means ± SE of 3 experiments. *P < 0.05, compared with UCBTERT day 0.

 

Figure 3
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  		Fig. 3. Downregulation of Wnt/beta-catenin genes during hepatic differentiation of UCBTERT-21 cells. A: mRNA levels of Wnt/beta-catenin signaling genes at day 21 of control and 5-aza/HGF/OSM/FGF2 treatment as assessed by real-time RT-PCR. Expression levels of the beta-catenin (a), PP2CA (b), and Fz8 (c) genes are expressed as a ratio of the control treatment. Data are expressed as means ± SE of 3 experiments. **P < 0.01, compared with control. B: expression of Fz8 mRNA on days 0, 7, 14, and 21 was assessed by real-time RT-PCR. The level of Fz8 is expressed as a ratio of that on day 0. bullet, 10% FBS (control); {blacksquare}, 5-aza/HGF/OSM/FGF2. Data are expressed as means ± SE of 3 experiments. **P < 0.01, compared with control. C: localization of beta-catenin in UCBTERT-21 cells during hepatic differentiation. a, b, d, and f, 10% FBS (control); c, e, and g, 10% FBS with 5-aza/HGF/OSM/FGF2. Light micrograph (Light), beta-catenin staining (beta-catenin), nuclear staining (Nuclear), and merged images of beta-catenin and nuclear staining (Merged) are shown. SYTOX green was utilized for nuclear staining. beta-Catenin was located in the cytoplasm (arrows) of several differentiated cells. Scale bar, 100 µm.

 

Figure 4
Figure 4
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  		Fig. 4. Effect of Fz8-small interference RNA (siRNA) on hepatic differentiation of UCBTERT-21 cells. A: time course of Fz8 mRNA expression on transfection of Fz8-siRNA. The mRNA level of Fz8 was measured by real-time RT-PCR. The level of fzd8 was expressed as a ratio of that at 0 h. bullet, negative control siRNA; {blacksquare}, Fz8-siRNA. Data are expressed as means ± SE of 3 experiments. **P < 0.01, compared with the negative control siRNA. B: construction of plasmids for the beta-catenin-Tcf reporter gene assay. The plasmid contained 3 copies of the optimal Tcf motif upstream of the CMV promoter driving luciferase expression (Tcf4-CMVpro-Luc). A plasmid with the thymidine kinase promoter driving Renilla luciferase expression (pRL-TK) was used as an internal control. C: reduction of transactivational properties of beta-catenin-Tcf4 in UCBTERT-21 cells transfected with fz8-siRNA. The activation level of beta-catenin-Tcf4 was expressed as a ratio of that of the negative control siRNA. The level of luciferase activity was examined at 48 h after transfection of pTcf4-CMVpro-luc, pRL-TK, and Fz8-siRNA. The data are expressed as a ratio to Renilla luciferase activity. The data from the addition of 100 ng/ml Wnt-3a at 24 h after transfection served a positive control. Data are expressed as means ± SE of 6 experiments. **P < 0.001, compared with negative control siRNA. D: expression of the albumin gene was induced by Fz8-siRNA transfection. The level of albumin, as evaluated by real-time RT-PCR, was expressed as a ratio of that on day 0. bullet, negative control siRNA; {blacksquare}, Fz8-siRNA. Data are expressed as means ± SE of 3 experiments. **P < 0.01, compared with day 0. E: induction of hepatocyte-specific proteins in UCBTERT-21 cells transfected with Fz8-siRNA on day 21. a–d, negative control siRNA; e–h, Fz8-siRNA. Cells were immunostained with anti-albumin (a, e), anti-C/EBP{alpha} (b, f), and anti-CYP1A1/1A2 (c, g) antibodies. Glycogen stored in cells was stained by PAS stain (d, h). Scale bar, 100 µm. F: efficiency of hepatic differentiation of UCBTERT-21 cells with fzd8-siRNA. Open bars, negative control siRNA; solid bars, Fz8-siRNA. Data are expressed as means ± SE of 6 experiments. **P < 0.001 compared with negative control siRNA. G: localization of beta-catenin in UCBTERT-21 cells transfected with Fz8-siRNA. a: Negative control siRNA on day 21. b: Fz8-siRNA on day 21. Light micrograph (Light), beta-catenin staining (beta-catenin), nuclear staining (Nuclear), and merged images of beta-catenin and nuclear staining (Merged) are shown. SYTOX green was utilized for nuclear staining. beta-Catenin was located in the cytoplasm (arrows) of several differentiated cells. Scale bar, 100 µm.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Optimal conditions for hepatic differentiation. UCBTERT-21 cells are suitable for this study because they proliferate clonally and expand for a prolonged number of passages, and, importantly, introduction of the hTERT gene did not change the characteristics of the cells (46). UCBTERT-21 cells were expanded in six-well plates at 2.0 x 104 cells/well. The cells were cultured first with or without 5-aza for 24 h and then with a combination of HGF, OSM, and FGF2 for 3 wk (Fig. 1). Albumin mRNA expression was evaluated by RT-PCR with seven combinations of these agents: 5-aza/HGF/OSM/FGF2, 5-aza/HGF/OSM, 5-aza/HGF, 5-aza, HGF/OSM/FGF2, HGF/OSM, and HGF compared with 10% FBS. On day 0, UCBTERT-21 cells expressed little albumin mRNA, and MSCGH scarcely expressed albumin mRNA without 5-aza or cytokines. The combination of 5-aza/HGF/OSM/FGF2 for 3 wk was more effective than the other seven combinations (data not shown).

To more precisely examine albumin mRNA expression, real-time PCR was performed using RNA obtained under optimal conditions and from the controls (Fig. 2A). The expression level of albumin mRNA after treatment with 5-aza/HGF/OSM/FGF2 was almost eightfold higher than that in MSCGM containing 10% FBS. We investigated whether UCBTERT-21 cells subjected to theses conditions expressed hepatocyte-specific proteins by immunocytochemistry. The expression of albumin was upregulated in the cells treated with 5-aza/HGF/OSM/FGF2, especially in the cells that proliferated at high density (Fig. 2B, a and e). Staining of C/EBP{alpha} was also increased, and the nuclei of treated cells were stained strongly (Fig. 2B, b and f). CYP1A1/2 staining was weak in untreated cells, but it was more intense in treated cells (Fig. 2B, c and g). The cells positive for CYP1A/2 were larger than those negative for CYP1A/2. The presence of stored glycogen, as determined by PAS staining, was observed in treated cells (Fig. 2B, d and h). Untreated cells did not show the ability to synthesize glycogen. When pretreated with diastase to digest glycogen, treated cells stained negative for glycogen (data not shown).

The level of cells positive for hepatic marker proteins and PAS staining in UCBTERT-21 cells treated with 5-aza/HGF/OSM/FGF2 significantly increased, compared with the control: a 2.5-fold increase in albumin, 4-fold increase in C/EBP{alpha}, 1.5-fold increase in CYP1A1/1A2, and 8-fold increase in PAS staining were induced by this treatment (P < 0.01, P < 0.01, P < 0.05, and P < 0.01, respectively, Fig. 2C).

Secretion of urea by the cells was measured every 24 h after the addition of 5 mM ammonium chloride. Urea in the medium became detectable at 24 h in both treated and untreated cells and increased thereafter, tending to be higher in the treated cells at 48 and 72 h. At 96 h, the production of urea was significantly greater in the treated cells compared with the control (P < 0.05, Fig. 2D).

The expression levels of albumin, C/EBP{alpha}, c/EBPbeta, CYP1A1, Cyp1A2, and PEPCK were examined by RT-PCR (Fig. 2E). The expression levels of albumin, C/EBP{alpha}, C/EBPbeta, CYP1A1, CYP1A2, and PEPCK were strongly induced in UCBTERT cells treated with 5-aza/HGF/OSM/FGF2. However, the expression levels of albumin, C/EBP{alpha}, C/EBPbeta, CYP1A1, CYP1A2, and PEPCK in adult normal liver cells were 1.7-fold, 2.0-fold, 1.1-fold, 0.7-fold, and 3.5-fold greater than that of the treatment with 5-aza/HGF/OSM/FGF2. These findings suggest that UCBTERT cells treated with 5-aza/HGF/OSM/FGF2 were still immature compared with adult mature hepatocytes.

Genes that are associated with hepatic differentiation of human MSCs. We investigated the genes whose expression changed during the treatment with 5-aza/HGF/OSM/FGF2. Total RNA extracted from cells, that were treated with 5-aza/HGF/OSM/FGF2 or control medium for one wk, was examined by microarray analysis. We found that the expression levels of many Wnt signal-related molecules were downregulated in the treated cells, compared with the untreated cells (Table 1). The expression level of CTNNB1 (beta-catenin) and many of the Fz family genes was decreased, including the expression level of Fz8. On the other hand, the expression level of CTNNBIP1 (ICAT), which inhibits Wnt signaling (44), increased by 2.4-fold. The expression of PPP2CA (protein phosphatase 2 catalytic subunit), which has a positive role in Wnt signal transduction (36), decreased. Conversely, the expression level of PPP2R1B (protein phosphatase regulatory subunit), which inhibits Wnt signaling (27), increased. Real-time RT-PCR analysis showed that beta-catenin, PPA2CA, and Fz8 were downregulated in their expression (Fig. 3A); treatment with 5-aza/HGF/OSM/FGF2 reduced the expression of beta-catenin, PPA2CA, and Fz8 to 82, 78, and 25%, respectively, of the control. Fz8 maintained its expression at ~20–40% of the control level during the course of hepatic differentiation of UCBMSC (Fig. 3B).


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  		Table 1. Microarray analysis of Wnt signal-related genes

 
Subcellular distribution of beta-catenin during hepatic localization. We investigated the subcellular localization of beta-catenin in UCBTERT-21 cells by immunocytochemistry. beta-Catenin has dual roles, as an adhesion molecule at the plasma membrane and as a key intermediate in the canonical Wnt signaling pathway. On activation of the Wnt cascade, beta-catenin in the cytosolic soluble pool becomes stabilized and then translocates into the nucleus where it coactivates transcription factors of the TCF/LEF family (4). On day 7 after the start of treatment, beta-catenin was mostly located in the nuclei of the cells (Fig. 3C). On day 14 and day 21, beta-catenin was also observed along the cell membrane and in the cytoplasm, but some was still in the nucleus (Fig. 3C). The translocation of beta-catenin was observed during hepatic cell differentiation (5, 31). Thus the changes in beta-catenin localization may be important during hepatic differentiation of progenitor cells.

Knockdown of the genes of MSC leading to hepatic differentiation. Since the expression levels of beta-catenin, PP2CA, and Fz8 were downregulated during the course of hepatic differentiation of UCBTERT-21 cells, the suppressive effect of Fz8, which is essential for Wnt/beta-catenin signaling (4), on hepatic differentiation was examined by using RNA interference. First, we confirmed that the expression level of Fz8 mRNA in Fz8-siRNA-transfected cells was decreased to 60% of that at 0 h at 48 h after transfection (Fig. 4A). To investigate the effect of Fz8 knockdown on beta-catenin/TCF4 transcriptional activity, we performed a luciferase reporter assay with pTcf4-CMVpro-Luc, using pRL-TK as an internal control (Fig. 4B). Treatment with Wnt-3a enhanced luciferase activity, whereas the luciferase activity in the cells transfected with Fz8-siRNA was 25% of that in the cells transfected with control siRNA, indicating that Wnt/beta-catenin signaling was suppressed by transfection with Fz8-siRNA (Fig. 4C). Because the suppressive effect of transfection with lipofection reagent did not last more than 7 days (data not shown), transfection with Fz8-siRNA was repeated every 7 days. Weekly transfection of Fz8-siRNA caused an increase in albumin mRNA expression in UCBTERT-21 cells on day 14 and day 21, as observed in UCBTERT-21 cells treated with 5-aza and cytokines (Fig. 4D).

UCBTERT-21 cells 3 wk after the beginning of Fz8-siRNA treatment were examined for hepatic marker proteins. Suppression of Fz8 by siRNA transfection induced the expression of albumin, C/EBP{alpha}, and CYP1A1/2 in UCBTERT-21 cells (Fig. 4E). The UCBTERT-21 cells treated with Fz8-siRNA were rounder than those subjected to "hepatic induction treatment." The numbers of albumin-, C/EBP{alpha}-, CYP1A1/2-, and PAS-positive cells increased by 3.2-fold, 3-fold, 1.7-fold, and 6-fold, respectively (each, P < 0.01, Fig. 4F). However, there was no significant change in urea synthesis when the cells were transfected with Fd8-siRNA (data not shown).

The subcellular distribution of beta-catenin in UCBTERT-21 cells transfected with Fz8-siRNA was investigated by immunocytochemistry. beta-Catenin was located along the cell membrane and in the cytoplasm of the cells transfected with Fz8-siRNA (Fig. 4G).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we identified an important signal for hepatic fate specification by using a system to induce hepatocytes to differentiate from human UCBMSCs. The combination of HGF, OSM, and FGF2 was examined, since the receptors for these cytokines were highly expressed on UCBTERT-21 cells (data not shown). In addition, during embryonic development, the production of growth factors such as HGF and FGFs has been associated with endoderm specification (40, 16). Furthermore, the first event that occurs after acute hepatitis in humans is a large increase in the serum level of HGF (42), suggesting that HGF plays an important role in the early stages of hepatogenesis. 5-Aza was employed because its efficiency in terms of cardiomyogenic differentiation of murine bone marrow stromal cells was ~30% (29). There is now evidence that the remodeling of chromatin and the alterations of epigenetics, including histone methylation and acetylation and DNA methylation, can cause committed cells to convert from one fate to another (6, 19). In our preliminary experiment, trichostatin A, a histone deacetylase inhibitor, induced apoptosis, but not differentiation, of UCBTERT-21 cells. Hence trichostatin A was not used in the present study. The positive rates of albumin, C/EBP{alpha}, CYP1A1/2, and PAS staining were not identical. This phenomenon may be explained by differences in the sensitivity of the immunostaining. In addition, the hierarchy of expression of liver-enriched transcription factors and proteins during development may explain this discrepancy (48).

Human UCBMSCs are easy to isolate but difficult to study because of their limited life span. The advantages of using UCBTERT-21 cells in a repopulation study are as follows: the cells have the same expression pattern of surface markers as the parental cells, the cells displayed osteogenic and adipogenic differentiation, the cells do not transform, they do not generate tumors in immunosuppressed mice, they do not form foci in vitro, and they stop dividing when confluent (46). Although important concerns regarding the use of these "artificial" cells, into which the hTERT genes has been introduced, for human liver repopulation studies may be raised, the use of UCBTERT-21 cells meets the purpose of the present study. In addition, the use of these cells enabled us to demonstrate the reversibility of differentiation. Therefore, to establish an efficient induction treatment, tissue engineers might apply this protocol to primary cultured human MSCs, which have not been genetically manipulated.

Recent studies have demonstrated that the Wnt/beta-catenin signal plays a crucial role in the regulation of stem cell functions (17). Wnt signaling maintains the self-renewing properties of hematopoietic stem cells and pluripotency of embryonic stem cells (37, 39). In addition to promoting the proliferation of stem/progenitor cells, Wnt also influences the lineage adopted by stem cells. A requirement for Wnt signaling was observed for neuronal specification in the dorsal spinal cord (33). beta-Catenin-deficient mouse stem cells fail to differentiate into follicular keratinocytes and instead adopt an epidermal fate (12).

The expression of the gene encoding a Wnt antagonist, Secreted frizzled-related protein 5 (sFRP5), in the foregut endoderm gave rise to the liver in mouse and Xenopus (10, 35), suggesting that the Wnt signal is a negative regulator of hepatic development (26). In addition, nuclear beta-catenin staining was not observed in hepatocytes from human autopsied tissues at gestational ages 10, 15, 16, 18, 22, and 35 wk (8). Recently, it has been reported that repression of Wnt/beta-catenin signaling in the anterior endoderm is essential for development (32). Additional reports have shown a requirement for Wnt signaling after liver cells are formed and not at the time of endoderm specification to form the liver (3, 43). These reports support the results of the present study. In addition, a report that the combination of Wnt and other hepatic growth factors plays an important role in early liver development (13) is in agreement with the present study. It has been recently reported that mesodermal Wnt2b signaling positively regulates liver specification although the species used were different from the present study (34). Differences may arise as to how canonical Wnts can fulfill diverse functions in stem cell. The fact that stem cells from different locations interpret Wnt in different ways obviously reflects an activation of distinct genetic programs in response to the same signal. Although cyclin D1 and c-myc are direct target genes of beta-catenin/Lef during cell cycle progression, recent studies revealed that proneural genes, neurogenins, are also targets of beta-catenin/TCF/Lef during neurogenesis (11, 14). Thus the activation of specific sets of Wnt target genes is likely to be mediated by cell type-specific intrinsic properties. Besides the cell-intrinsic cues that influence the biological activity of Wnt in distinct stem and progenitor cell types, the same type of stem cell might respond in different ways to Wnts, depending on its extracellular microenvironment (17). In this model, Wnt signaling interacts with signaling pathways triggered by other cues and is involved in cross talk with other signals at several levels. Taken together, downregulation of the Wnt/beta-catenin pathway plays an important role in the transdifferentiation of MSCs into hepatocytes in response to mesenchymal cell-specific intrinsic properties or cross talk between several cytokines.

In conclusion, we found that the downregulation of Wnt/beta-catenin signals play an important role in hepatic differentiation of human UCBMSCs. During hepatic differentiation, Wnt/beta-catenin signaling was downregulated. Conversely, suppression of the signaling stimulated the hepatic differentiation of UCBTERT-21 cells. These findings provide useful information on stem cell biology, which should contribute to the development of regenerative medicine for liver diseases.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Shiota, Division of Molecular and Genetic Medicine, Dept. of Genetic Medicine and Regenerative Therapeutics, Graduate School of Medicine, Tottori Univ., Yonago 683-8504, Japan (e-mail: gshiota{at}grape.med.tottori-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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