Bio-Medical Materials and Engineering 25 (2015) S145–S157 DOI 10.3233/BME-141249 IOS Press

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Differentiation of human umbilical cord-derived mesenchymal stem cells into hepatocytes in vitro Guan Zheng, Yang Liu, Qian Jing and Lei Zhang ∗ Biomedical Research Center, First People’s Hospital of Kunming (Affiliated Calmette Hospital of Kunming Medical University), Yunnan, China Abstract. OBJECTIVE: The stem cell based therapy is a potential alternative to liver transplantation. The aim of the study is to investigate the hepatocytic differentiation ability of human umbilical cord derived mesenchymal stem cells (HUC-MSCs) in vitro. METHODS: The HUC-MSCs were isolated from Wharton’s jelly of human umbilical cord. The cells were identified by assessing the stem cell markers. The HUC-MSCs were characterized by multipotency of differentiation. We modified the hepatogenic differentiation protocol and examine the function of differentiated cell by Periodic acid-Schiff (PAS) staining and Low-Density Lipoprotein (LDL) uptake. The protein expressions of Total protein (TP), Albumin (ALB), Globulin (GLB), Urea (BUN) and α-fetoprotein (AFP) were also detected. RESULTS: The cells of the hepatogenic differentiation group showed the function of hepatocyte. Protein expressions of TP, ALB, GLB BUN and AFP improve that the HUC-MSCs are able to different into the functional hepatogenic-like cell. CONCLUSION: The above founding indicated that the HUC-MSCs are able to different into the functional hepatogenic-like cell by present method. Keywords: Human umbilical cord-derived mesenchymal stem cells (HUC-MSCs), hepatocytic differentiation, hepatocytes

1. Introduction The hepatocytes damage is usually caused by the progression of liver diseases and eventually leads to liver dysfunction. Liver transplantation is still the only curative treatment for liver failure. The liver transplantation technique is improved, and a variety of medical problems such as rejection can be controlled. However, the shortage of the donor liver is the main problem of the liver transplantation. Tissue engineering brings the new hope for solving this problem. Stem cells play an important role in regenerating new tissue [1]. Because of the ethical and safety consideration, the use of embryonic stem cells is prohibited or restricted [2]. Mesenchymal stem cells (MSC) are mesodermal in origin with self-renewal and multilineage differentiation potential, mainly are found in the connective tissue and organic mesenchyme. Previous studies showed the bone marrow mesenchymal stem cells (BMSCs) derived from mice [3], rats [4,5], and humans [6,7] can be differentiated into hepatic cells in vivo. Hong’s group demonstrated the in vitro hepatocytic differentiation of human umbilical cord blood-derived mesenchymal stem cells (HUCBMSCs) [8]. Researchers also found the adipose tissue-derived stem cells (ADSCs) have a similar *

Address for correspondence: Lei Zhang, Biomedical Research Center, Affiliated Calmette Hospital of Kunming Medical University, Yunnan, China. E-mail: [email protected]. 0959-2989/15/$27.50 © 2015 – IOS Press and the authors. All rights reserved

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hepatogenic differentiation potential to BMSCs [9]. However, there are practical limitations of BMSCs, HUCBMSCs and ADSCs. For example, the bone marrow aspiration may unacceptable for some patients. The separation methods of HUCBMSCs require the high quality of the human umbilical cord blood [10]. The density of MSCs in adipose tissue is lower than in other tissue. Therefore it needs to take a lot of adipose tissue to obtain enough MSCs [11]. Moreover, ADSCs were taken a longer hepatogenic differentiation culture period than BMSCs [9]. Human umbilical cord derived mesenchymal stem cells are low immunogenicity, can suppress allograft immune responses, and reduce graft versus host disease [12]. The human umbilical cord mesenchymal stem cells could be differentiated into hepatocyte-like cells in DMEM with HGF, bFGF and Oncostatin, and the doses of cytokine and the methods for hepatogenic differentiation from different research report are vary [13–15]. The present study was to investigate the hepatocytic differentiation ability of human umbilical cord derived mesenchymal stem cells in vitro by modifying and optimizing the differentiation method from previous studies. 2. Materials and methods 2.1. Isolation and culture of MSCs from human umbilical cord The human umbilical cord derived mesenchymal stem cells were isolated from Wharton’s jelly of human umbilical cord (n = 6, gestational ages, 40 weeks). All samples were collected with informed consent. The cells were isolated by adherence method. Tissue culture method was modified from a previous study [16]. The umbilical cord was collected and stored in a sterile bottle containing 0.9% normal saline at 4°C until processing. The cord was manipulated in a sterile 10 cm culture dish after complete sterilization, and it was cut into 3–5 cm long pieces. Blood vessels were removed from each piece after incising the cord lengthwise. The remaining tissue was washed by PBS (Invitrogen, USA) to remove remain blood, and then minced to 2–3 mm long pieces. The minced tissue was coated on 6-well plate and was incubated at 37°C, 5% CO2 for 30 minutes. Minced tissue was cultured in DMEM (Sigma) supplemented with 10% FBS (GBICO, South America) and 2 mM L-glutamine (GBICO, USA) for 1 week to allow the cells attach. The cell colonies (50 cells) were transferred to T-75 cultural flasks (Corning, USA) and were cultured up to 70–80% confluence, and then they were passaged at 1 : 3 ratios for experiment or storage until cell passage 3–5. 2.2. Flow cytometry analysis Cells at passage 3 were identified by assessing the stem cell markers such as CD34 (Invitrogen, USA), CD45 (Invitrogen, USA), CD 90 (eBioscience, USA), CD105 (Invitrogen, USA) and OCT-4 (Invitrogen, USA). Flow cytometric analysis was performed with flow cytometer (CytomicsTM FC500, Beckman Coulter, USA) after labeling 1 × 106 cells with direct conjugated antibodies of CD34, CD 45, CD90 and CD105. Cell density of 1 × 106 were blocked and stained with RatAnti-Human/Mouse Oct-4, and the isotype-matched controls were set. 2.3. Osteogenic differentiation To induce osteogenic differentiation, the 5th-passage umbilical cord mesenchymal stem cells (n = 6) were seeded at the density of 1 × 105 cells/well in 6-well-plates. Those cells were than cultured with the normal growth media until reaching 80% confluence, after that, DMEM (Sigma) 2 ml supplemented with

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10% FBS, 10 mM β-glycerophosphate (Sigma), 10−7 M dexamethasone (Sigma), 50 µM L-ascorbic acid 2-phosphate (Sigma), 2 mM L-glutamine (GBICO, USA) were added to three wells. The cells within normal media were set as control in other three wells. The plates were cultured at 37°C in an incubator setting in a humidified atmosphere of 5% CO2 . The media were changed twice weekly for 3 weeks. 2.4. Adipogenic differentiation For adipogenic differentiation, the 5th-passage cells (n = 6) were seeded at the density of 1 × 105 cells/well in 6-well-plates. After reaching 80% confluence, the cells were treated with adipogenic medium for 3 weeks. Medium changes were performed twice weekly. Adipogenic medium consists of DMDM-LG supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich), 0.1 mM indomethacin (Sigma-Aldrich), and 10% FBS, 10 µg/ml insulin, 1 µM dexamethasone. The cells within normal media were set as control in other three wells. The plates were cultured at 37°C in an incubator setting in a humidified atmosphere of 5% CO2 . 2.5. Hepatogenic differentiation For hepatogenic differentiation, the 5th-passage cells (n = 12) in density of 1 × 105 cells/well were cultured until reaching 80% confluence, and then those cells were serum deprived for 2 days, in DMEM-LG supplemented with 20 ng/ml EGF (Sigma-Aldrich) and 10 ng/ml bFGF (R&D Systems, Minneapolis, MN), prior to induction by a 2-step protocol. Differentiation was induced by treating the cells with Step-1 differentiation medium, consisting of DMEM-LG supplemented with 20 ng/ml HGF (R&D Systems, Minneapolis, MN) and 10 ng/ml bFGF, nicotinamide (Sigma-Aldrich) 0.61 g/l, for 7 days, followed by treatment with step-2 maturation medium, consisting of DMEM-LG supplemented with 20 ng/ml oncostatin M (PeproTech EC, London, UK), 1 µmol/l dexamethasone, and 1X of ITSpremix (Sigma-Aldrich). Thereafter, medium changes were performed twice weekly. 2.6. Identification of cell differentiation 2.6.1. Osteogenic differentiation After 3 weeks of osteogenic differentiation, nodule formations were examined. The samples were then fixed with 10% formalin and stained for calcium by Alizarin red (Sigma-Aldrich) staining. The mineral phase formed nodules were examined under the microscope for visual inspection. 2.6.2. Adipogenic differentiation After adipogenic differentiation for 21 days, the cells were fixed using 10% formalin and stained with oil red O for 60 min at room temperature followed by thorough wash and microscopic examination. 2.6.3. Hepatogenic differentiation Hepatogenic differentiation was identified by Periodic acid-Schiff staining, Low-Density Lipoprotein (LDL) uptake. The protein expressions of total protein (TP), Albumin (ALB), Globulin (GLB), Urea (BUN) and α-fetoprotein (AFP) were also examined. 2.7. Periodic acid-Schiff treatment for glycogen On days 21 of hepatogenic differentiation, cells were washed with PBS twice and were fixed with 10% formalin for 30 min. Those cells were oxidized in 10 g/l periodic acid for 10 min and rinsed thrice in dH2 O. Afterwards, cells were treated with Schiff’s reagent for 10 min, rinsed in dH2 O for

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10 min, stained with hematoxylin for 2 min, differentiated by 1% alcohol–HCl, and rinsed in dH2 O again, observed under an invert microscope. 2.8. Uptake of Low-Density Lipoprotein (LDL) The Dil-Ac-LDL staining kit was purchased from Biomedical Technologies (Stoughton, MA) and assay will be performed by the manufacturer’s instructions. The concentration of 4 µg/ml Dil-Ac-LDL staining solution with medium will be added into each well of test and control groups 2 days prior to the assay. Dil-Ac-LDL staining was performed by removing the medium and Dil-Ac-LDL staining solution, washing triple with PBS, fixing by 1 ml of 4% paraformaldehyde (room temperature, 1 h) and examining under the fluorescence microscope. We randomly selected 9 visions of both test and control groups. The results were analyzed by LAS AF Lite and Image Pro-Plus 6.0. mean density = (IOD SUM)/(area sum) [17,18]. 2.9. Protein expressions Cell culture supernatants were kept when media were changed during the 14 days, 21 days and 28 days of hepatogenic differentiation. The culture supernatants were stored at −80°C immediately after they were collected. The protein expressions of total protein (TP), Albumin (ALB), Globulin (GLB), Urea (BUN) and α-fetoprotein (AFP) were determined colorimetry, and the assay were performed by Automatic Biochemical Analyzer (Olympus, AU5421). 2.10. Data analysis Statistical analyses were performed by using SPSS software (Version 16.0, Standard Software Package Inc., USA). The data was presented as mean ± SD. Differences among groups or differences among time intervals were analyzed using one-way analysis of variance (ANOVA). When a difference was statistically significant at P < 0.05, a multiple comparison test was performed. If the variances of the data were normal, the Scheffe method was used. If the variances of the data were not normal, the Dunnette T3 method was used. Significant differences were set at 95% confidence. 3. Results 3.1. Characterization of HUC-MSCs We obtained mesenchymal stem cells from human umbilical cord. Those cells presented a spindleshaped morphology. Figure 1(a) was taken after 17 days of primary culture. From Fig. 1(a), cells aggregated in groups of more than 50 cells. It indicated that the cell clones were formed. Figure 1(b) shows cell morphology at cell passage 3. The surface antigen markers of human mesenchymal stem cells CD34, CD45, CD90 and CD105 expression on human umbilical cord-derived cells were analyzed by flow cytometry at cell passage 3. Meanwhile expression of undifferentiated embryonic stem cell marker, OCT-4, was analyzed. HUC-MSCs were positive for the mesenchymal markers CD90 (Fig. 2(a)) and CD105 (Fig. 2(b)), but did not express the hematopoietic marker CD34 (Fig. 2(c)) and CD45 (Fig. 2(d)) and Fig. 2(e) shows the expression of OCT-4. Moreover, all HUC-MSC samples of this experiment were collected from six different donors. CD90 and CD105 expression of those cells were 96.0 ± 2.0% and

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(b) Fig. 1. (a) Cell clones were formed after 17 days of primary culture. (Scale bars, 50 µm. Original magnification, ×100.) (b) Cell morphology at cell passage 3. (Scale bars, 50 µm. Original magnification, ×100.)

98.8±1.0%, respectively (n = 6), while the mean of both CD 34 and CD 45 expression were 0.5±0.2% (Fig. 2(f)). As shown in Fig. 2(f), the mean of OCT-4 was 42.2 ± 5.5%. The human umbilical cord derived mesenchymal stem cells were characterized by multipotency of differentiation. HUC-MSCs were able to differentiate into osteoblasts showing mineral nodules (Fig. 3(a)) in osteogenic condition af-

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Fig. 2. HUC-MSCs were positive for the mesenchymal markers CD90 (Thy-1) PE (a), and CD105 PE (b), but did not express the hematopoietic marker CD34 FITC (c) and CD45 FITC (d). (e) Expression of OCT-4. (f) CD90 and CD105 expression of HUC-MSCs (n = 6) were 96.0 ± 2.0% and 98.8 ± 1.0%, respectively, while the mean of both CD 34 and CD 45 expression were 0.5 ± 0.2%. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-141249.)

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(b) Fig. 3. (a) HUC-MSCs were able to differentiate into osteoblasts showing mineral nodules in osteogenic condition after 3 weeks. (Scale bars, 100 µm. Original magnification, ×100.) (b) Under adipogenic induction conditions for 3 weeks, the intracellular microdroplets were formed. (Scale bars, 100 µm. Original magnification, ×200.) (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-141249.)

ter 3 weeks. Under adipogenic induction conditions for 3 weeks, the intracellular microdroplets were formed (Fig. 3(b)). 3.2. Identification of the hepatogenic differentiation The cell morphology of hepatogenic differentiation group was changed from spindle-shaped (Fig. 4(a)) to oblateness (Fig. 4(b)). The special function of hepatocyte is whether it can synthesis and store glycogen. After the Periodic acid-Schiff staining, the cells will stain if the glycogen synthesis. We observed the cells of the hepatogenic differentiation group were strongly positive for PAS staining on day 14th (Fig. 5(a)), whereas the PAS staining was not show in control group (Fig. 5(b)). Moreover, on

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(b) Fig. 4. (a) HUC-MSC morphology of undifferentiation group was spindle-shaped. (Scale bars, 100 µm. Original magnification, ×200.) (b) HUC-MSC morphology of hepatogenic differentiation group was oblateness. (Scale bars, 100 µm. Original magnification, ×200.) (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-141249.)

day 21st, the DiI-Ac-LDL incorporation could be observed under fluorescence microscopy in both hepatogenic differentiation group and undifferential HUC-MSCs group. However, the fluorescence intensity of hepatogenic differentiation group (Fig. 6(b)) was much stronger than the undifferential HUC-MSCs group (Fig. 6(a)). After analyzed the pictures by LAS AF Lite and Image Pro-Plus 6.0, fluorescence intensity was transferred to the mean density (MD). Mean density of hepatogenic differentiation group (235.1 ± 8.0) was significant higher than MD of control group (163.0 ± 11.0) (Fig. 6(c)). As shown in Fig. 7 and Table 1, the protein expression of TP (Fig. 7(a)), ALB (Fig. 7(b)), GLB (Fig. 7(c)) and BUN

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(b) Fig. 5. (a) HUC-MSCs of the hepatogenic differentiation group were strongly positive for PAS staining on day 14th. (Scale bars, 100 µm. Original magnification, ×200.) (b) PAS staining was not show in control group. (Scale bars, 100 µm. Original magnification, ×200.) (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-141249.)

(Fig. 7(d)) could be detected on day 14th, 21st and 28th. The protein expression of TP, ALB, GLB had significant differences between day 14th, day 21st and day 28th, respectively. The protein expression of BUN was significantly different between day 21st and day 28th, and there was no significant difference between day 14th and day 21st. The protein expression of AFP (0.9 ± 0.2) ng/ml could be found only on day 28th. 4. Discussion Recent years, research of human umbilical cord-derived mesenchymal stem cells attracts more attentions because of its self-renewal property, multiple-lineage differentiation potential, low immunogenicity

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Fig. 6. (a) DiI-Ac-LDL incorporation of undifferential HUC-MSCs group, (b) DiI-Ac-LDL incorporation of hepatogenic differentiation group. On day 21st, the DiI-Ac-LDL incorporation could be observed under fluorescence microscopy in both hepatogenic differentiation group and undifferential HUC-MSCs group. However, the fluorescence intensity of hepatogenic differentiation group (b) was much stronger than the undifferential HUC-MSCs group (a). (c) Data conversion of fluorescence. Mean density of hepatogenic differentiation group (235.1±8.0) was significant higher than MD of control group (163.0±11.0), ∗ P < 0.05 compared with control. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/ BME-141249.)

[1,2], without limited source and ethical issues. From our study, HUC-MSCs possessed multiple-lineage differentiation ability. Those cells highly expressed mesenchymal markers CD90 and CD105. More than forty percent HUC-MSCs expressed OCT-4. The OCT-4 is the undifferentiated embryonic stem cell maker [19], and the cells obtained from umbilical cord could be differentiated into osteoblasts, adipocytes and hepatocytes in our research. These results indicated that the cells are mesenchymal stem cells and they have a high stem cell quality. Previous studies [13–15] reported that HUC-MSCs could be differentiated into hepatogenic-like cells in vitro. The induced cells could be express hepatogenic specific marker CK18, CK19, α-fetoprotein and albumin and could be presented some hepatocyte func-

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Fig. 7. (a) Protein expression of TP, (b) ALB, (c) GLB had significant differences between day 14th, day 21st and day 28th, respectively. (d) BUN expression was significantly different between day 21st and day 28th, and there was no significant difference between day 14th and day 21st. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/ BME-141249.) Table 1 Protein expression of TP, ALB, GLB and BUN on day 14th, 21st and 28th TP ALB GLB BUN

Day 14 4.50 ± 1.10∗ 0.80 ± 0.12∗ 3.60 ± 1.80∗ 0.70 ± 0.20

Day 21 8.30 ± 1.75∗ 1.80 ± 0.55∗ 6.10 ± 2.70∗ 1.10 ± 0.40∗

Day 28 10.40 ± 0.68∗ 2.30 ± 0.77∗ 8.10 ± 1.60∗ 2.30 ± 0.87∗

Notes: The protein expression of TP, ALB, GLB had significant differences between day 14th, day 21st and day 28th, respectively. Protein expression of BUN was significantly different between day 21st and day 28th. (∗ P < 0.05 between each time point.)

tions. Two studies used two steps in long term in vitro culture, and the induction period was 29 days [13,15]. One study induced the cells by a 18-day one step method [14]. All studies used the different cytokine doses. In our experiments, we applied a two step method, however we shorted the culture period of step 1 from 14 days to 7 days and decreased the dose of OSM from 50 ng/ml to 20 ng/ml. Our results showed that the induced cells not only were able to present the functional hepatocyte characteristic but also appeared earlier. The presence of stored glycogen is considered as the hepatocyte function. It can be one evaluation criteria of hepatogenic differentiation. Result from Zhu et al. showed their induced cells were positive for glycogen staining at 4 weeks [13]. The other previous study showed the PAS staining could be found on day 18th [14]. In our study, the cells of the hepatogenic differentiation group were strongly positive for PAS staining on day 14th. Furthermore, the strong fluorescence intensity of hepatogenic differentiation group were observed on day 21 indicated that most cells of test group were hepatogenic differentiated. For the LDL up-taken study, the results of Ren’s study showed that the LDL up-taken were observed on most cells on Day 28 [15]. LDL metabolism is another ability of health hepatocyte whereas the LDL up-taken of undifferentiated MSC is much lower. In our research, both glycogen formation and LDL up-taken appeared earlier compared with previous result, it means the cells is more verge on the hepatocyte with our differentiating methods. What is more, our studies also showed some of those proteins could be found at about 20–21 days [13,15]. The protein studies showed an expression of TP, ALB, GLB and BUN were detected since day 14th. The above results indicate that the human umbilical cord derived mesenchymal stem cells are able to be differentiated into functional hepatogenic-like cell. The methods of the present study improved the process efficiency. Interestingly, unlike the protein expression of AFP appearance were on day 7th in previous studies [13–15], it could be detected only on day 28th in our study. In our protocol, we decreased the dose of the OSM and delayed

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the timing of adding OSM. Report has shown that OSM increased hepatocyte size and enhanced hepatic differentiation [20]. Cheng et al. suggested that oncostatin M could increase the induced efficiency [14]. OSM seems to be the most critical cytokine in hepatogenic differentiation and may be related to the AFP expression. Therefore, we conclude that the hepatogenic differentiation can be more efficiency by using our current method. Oncostatin M (OSM) may be act an important role in hepatogenic differentiation.

Acknowledgements This program is initially supported by grants from the French embassy in China (Xu-Guangqi en 2011 and 2012, 26184WF), and is now supported by grants from Yun Nan province (2012FB105).

References [1] P. Bianco and P.G. Robey, Stem cells in tissue engineering, Nature 414(6859) (2001), 118–121. [2] M. Quattrocelli, M. Cassano, S. Crippa, I. Perini and M. Sampaolesi, Cell therapy strategies and improvements for muscular dystrophy, Cell Death Differ. 17 (2010), 1222–1229. [3] N.D. Theise, S. Badve, R. Saxena, O. Henegariu, S. Sell, J.M. Crawford and D.S. Krause, Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation, Hepatology 31 (2000), 235–240. [4] B.E. Petersen, W.C. Bowen, K.D. Patrene, W.M. Mars, A.K. Sullivan, N. Murase, S.S. Boggs, J.S. Greenberger and J.P. Goff, Bone marrow as a potential source of hepatic oval cells, Science 284 (1999), 1168–1170. [5] S.N. Shu, L. Wei, J.H. Wang, Y.T. Zhan, H.S. Chen and Y. Wang, Hepatic differentiation capability of rat bone marrowderived mesenchymal stem cells and hematopoietic stem cells, World J. Gastroenterol. 10 (2004), 2818–2822. [6] N.D. Theise, M. Nimmakayalu, R. Gardner, P.B. Illei, G. Morgan, L. Teperman, O. Henegariu and D.S. Krause, Liver from bone marrow in humans, Hepatology 32 (2000), 11–16. [7] M.R. Alison, R. Poulsom, R. Jeffery, A.P. Dhillon, A. Quaglia, J. Jacob, M. Novelli, G. Prentice, J. Williamson and N.A. Wright, Hepatocytes from non-hepatic adult stem cells, Nature 406 (2000), 257. [8] S.H. Hong, E.J. Gang, J.A. Jeong, C.Y. Ahn, S.H. Hwang, I.H. Yang, H.K. Park, H. Han and H. Kim, In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells, Biochem. Biophys. Res. Commun. 330 (2005), 1153–1161. [9] R. Taléns-Visconti, A. Bonora, R. Jover, V. Mirabet, F. Carbonell, J.V. Castell and M.J. Gómez-Lechón, Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells, World J. Gastroenterol. 12(36) (2006), 5834–5845. [10] K. Bieback, S. Kern, H. Kluter et al., Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood, Stem Cells 22 (2004), 625–634. [11] A. Graziano, R. d’Aquino, G. Laino and G. Papaccio, Dental pulp stem cells: a promising tool for bone regeneration, Stem Cell Rev. 4 (2008), 21–26. [12] L.Y. Liu, J.K. Chai, Y.F. Han, T.J. Sun, D.J. Li and J.Y. Zhao, Research progress of biological characteristics and advantages of Wharton’s jelly-mesenchymal stem cells, Chinese J. Reparative and Reconstructive Sur. 06 (2011), 745–749. [13] Z.Y. Zhu, J.Q. Yan, T. Han, Z. Du, Y. Luo, P. Wang, Y.T. Gao and T. Liu, Long-term in vitro culture and hepatocytic differentiation of human umbilical cord-derived mesenchymal stem cells, J. R. Soc. Interface 13(49) (2009), 9792–9796. [14] J. Chen, Y.X. Liu, W.H. Jiang, G. Han and W.P. Jiang, Differentiation of human umbilical cord mesenchymal stem cells into hepatocyte – like cells in vitro, J. R. Soc. Interface 14(49) (2010), 9257–9261. [15] H.Y. Ren, Q.J. Zhao, W. Xing, S.G. Yang, S.H. Lu, Q. Ren, L. Zhang and Z.C. Han, Differentiation of human umbilical cord derived mesenchymal stem cells into low immunogenic and functional hepatocyte-like cells in vitro, Acta Acad. Med. Sin. 32(2) (2010), 190–194. [16] K. Seshareddy, D. Troyer and M.L. Weiss, Method to isolate mesenchymal-like cells from Wharton’s jelly of umbilical cord, Methods Cell Biol. 86 (2008), 101–119. [17] J.M. Kim, A. Ogura, M. Nagata and F. Aoki, Analysis of the mechanism for chromatin remodeling in embryos reconstructed by somatic nuclear transfer, Biol. Reprod. 67(3) (2002), 760–766. [18] Y. Liu, B. Ding, X.X. Yang, M.B. Shang, X.H. Lei, R. Wang et al., Dynamic transformation of DNA methylation and chromatin configuration in porcine oocyte during follicular growth, J. Anim. Vet. Adv. 11(10) (2012), 1739–1744.

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[19] L.H. Looijenga, H. Stoop, H.P. de Leeuw et al., POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors, Cancer Res. 63(9) (2003), 2244–2250. [20] C.A. Lázaro, E.J. Croager, C. Mitchell, J.S. Campbell, C. Yu, J. Foraker, J.A. Rhim, G.C. Yeoh and N. Fausto, Establishment, characterization, and long-term maintenance of cultures of human fetal hepatocytes, Hepatology 38 (2003), 1095–1106.

Differentiation of human umbilical cord-derived mesenchymal stem cells into hepatocytes in vitro.

The stem cell based therapy is a potential alternative to liver transplantation. The aim of the study is to investigate the hepatocytic differentiatio...
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