Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10278


Human endometrial stem cells differentiation into functional hepatocyte-like cells Farzaneh Khademi1,2, Masoud Soleimani2,3, Javad Verdi1,4, Seyed Mohammad Tavangar1,5, Esmaeil Sadroddiny6, Mohammad Masumi2,7 and Jafar Ai1,8* 1 2 3 4 5 6 7 8

Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Stem Cells Technology Research Center, Tehran, Iran Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran Department of Applied Cell, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Pathology, Shariaty Hospital, Tehran University of Medical Sciences, Tehran, Iran Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Induced Pluripotent Stem Cell Biotechnology Team, Stem Cells Department, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Brain and Spinal Injury Research Center, Tehran University of Medical Sciences, Tehran, Iran

Abstract In spite of certain clinical limitations, such as teratoma formation, the use of stem cells is considered as an appropriate source in cell therapy and tissue engineering. This study shows human endometrial stem cells (hEnSCs) has exceptional differentiation ability in hepatocyte formation. hEnSCs have high purification rate and immune-tolerance, and can be used as an appropriate substitute for hepatocytes in liver disorders. Differentiation required hepatogenic medium. Quantitative reverse transcription-polymerase chain reaction and immunofluorescent staining of hepatic genes and proteins including cytokeratin 18 (ck18), alpha-fetoprotein (afp), and albumin (alb) were used to assess differentiation. Cells differentiated with a hepatocyte-like morphology and expressed hepatic markers on 30 days of differentiation. The Periodic Acid-Schiff (PAS) reaction showed storage of glycogen, and albumin and afp secretions were also detected. In vitro hEnSCs behave like hepatocyte after differentiation and may be a suitable source of cells in liver regeneration. Keywords: cell therapy; differentiation; growth factors; hepatic repopulation; hepatocyte-like cells; human endometrial stem cells; regenerative medicine

Introduction Liver is one of the vital organs in the body, having metabolic, exocrine, and endocrinal functions in the hepatocytes (Takiguchi, 1998). Chronic hepatic failure usually leads to organ transplantation with inherent problems related to the transplantation, donor shortage, and high mortality rates. To overcome these limitations, many studies have suggested alternative strategies. Tissue engineering and cell therapy have been approved for chronic organ failure as an alternative treatment (Atala, 2004; Caldas et al., 2011). Regeneration and self-repair in various organs by stem cells in the body have been reported (Young et al., 2001). The therapeutic potential of stem cells in liver diseases and regeneration have demonstrated that from in vitro or in vivo they can become mature hepatocyte-like cells with live

functions, for example, albumin production and urea metabolism (Ogawa and Miyagawa, 2009). Colonogenic cells with both self-renewal and multi-lineage differentiation, stem cells, are proving useful for the repair and reconstruction of tissues (Young et al., 2001). Liver biopsy specimens from human recipients may be harmful, of low quality and cannot expand easily in in vitro culture. Liver stem cells have regenerative capacity in a moderate size graft of a damage liver (Sangan and Tosh, 2010). Adult stem cells are an appropriate cell source with plasticity (Theise et al., 2000; Lowes et al., 2003), and has no ethical issues attached. Hence, we introduced endometrial stem cells, a type of adult stem cells, showing high proliferation and differentiation ability, angiogenesis in women menstrual period and immune-modulatory property for fetus, and easy to isolate (Gargett et al., 2007; Ai et al., 2012a; Mobarakeh

Corresponding author: e-mail: [email protected]

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et al., 2012; Ai and Mehrabani, 2010; Ebrahimi-Barough et al., 2013a). Therefore, endometrial stem cells are an appropriate choice for hepatic cell therapy and can be used an alternative for embryonic or mesenchymal stem cells. We report that hepatocyte-like cells differentiating from human endometrial stem cells (hEnSCs) are strong candidates for regeneration of hepatic tissues. We describe isolation of hEnSCs from human uterus and explain their differentiation into functional hepatocyte cells, using molecular, cytochemical, and functional aspects.

100 mm strainers, centrifuged (1,000g for 15 min) and gently overlaid on Ficol–Hypaque (3 mL) to eliminate unwanted cell types. After washing in PBS, the isolated stromal cells were inoculated in media DMEM-F12 supplemented with 15% FBS, 100 antibiotic, Insulin–Transferrin–Selenite (ITS). Inoculated cells were incubated at 37 C in a humidified air atmosphere with 5% CO2 incubator. Medium was changed twice a week. When cells reached 80% confluence, they were collected with 0.25% trypsin–EDTA solution and seeded into three subcultures.

Materials and methods

Flow cytometric analysis Materials Cell culture media and supplements were acquired from Gibco BioCult (Paisley, UK). Antibodies for flow cytometric assay were purchased from Dako (Denmark) and Oxford Biomedical Research (UK). RNA extraction kit, cDNA Synthesis Kit, the materials for PCR amplification and primers for quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) and SYBR Green PCR Master Mix were purchased from Qiagen (USA) and Fermentas (USA). Albumin ELISA Quantitation kit was bought from Bethyl Laboratories (Montgomery, USA). Mouse antihuman monoclonal antibodies for albumin and alphafetoprotein, goat anti-mouse FITC-conjugated immunoglobulin G (IgG) were obtained from Santa Cruz Biotechnology Inc. (USA), hepatocyte growth factor (HGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), dexamethasone (DEX), oncostatin M (OSM), Periodic Acid-Schiff Staining kit and Alizarin red staining kit, Oil red o-staining kit, and other reagents were purchased from Sigma Chemical Co. (St. Louis, USA).

Isolation, culture, and expansion of hEnSCs Endometrial samples were obtained from volunteers under the supervision of the Medical Ethics Committee, Ministry of Health, I.R. Iran. Written informed consent was given by all donors (according to the ethical guidelines of the 1975 Declaration of Helsinki). Endometrial biopsies of volunteers aged 19–28 years was taken with an endometrial sampling device (Endocell; Wallach Surgical Devices, Inc., Orange, CT, USA) at the infertility center, Valiasr Hospital, Tehran, Iran. Samples were obtained from the endometrium in the proliferative phase of the menstrual cycle. Based on the previous reports (Esfandiari et al., 2007), endometrial stem cells were isolated from the uterus using the following techniques (Ebrahimi-Barough et al., 2013b,c). Stem cell were put in Hank’s media containing 100 antibiotic, minced, and treated with collagenase (2 mg/mL) at 37 C for 2 h. The epithelial and stromal cells were separated by 45 and 826

Expression of hEnSCs surface markers was assessed in cells after three passage by staining with the following antihuman antibodies: fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated mouse immunoglobulin G (IgG) isotype control, mouse anti-human CD31, CD34 (hematopoietic markers), CD90, CD105, CD146 (MSC markers), CD44, CD45 (leukocyte common antigen), CD133 (hematopoietic/angioblast marker), Stro1 (BMMSC marker) for 1 h in 4 C. Cells were washed in PBS and centrifuged for 5 min. The cells were analyzed by a FACS-Caliber flow cytometer (Beckman Coulter, Fullerton, CA, USA), and the Win MDI 2.8 software (Scripps Institute, La Jolla, CA, USA). This test was replicated thrice.

Adipogenic and osteogenic differentiation of hEnSCs To assess the potential of hEnSCs for osteogenic and adipogenic differentiation, the cells were incubated in osteogenic medium containing 10–7 M dexamethasone, 50 mg/ml L-ascorbic acid 2-phosphate, and 10 mM b-glycerophosphate (10 ng/mL) for 3 weeks (Kazemnejad et al., 2009). Medium was changed every 3–4 days. To verifying the extracellular matrix mineralization and calcification nodules, cells were stained with 2% alizarin red. For adipogenic differentiation, the cells were incubated in the adipogenic medium consisting of DMEM with 10 g/mL insulin, 1 M dexamethasone, 200 M indomethacin, and 0.5 mM isobutylmethylxanthine (Chan et al., 2004). Medium was changed every 3 days for 3 weeks. The cells were fixed in 4% paraformaldehyde for 10 min and stained with oil red O solution. They were washed and examined by light microscopy (IX50, Olympus, Japan).

Hepatic differentiation A 3-step differentiation protocol (Leelawat et al., 2010) was used, based on liver development. In the pre-differentiation phase, the cells were inoculated in DMEM medium supplemented with 20 ng/mL epidermal growth factor Cell Biol Int 38 (2014) 825–834 © 2014 International Federation for Cell Biology

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(EGF) and 10 ng/ml basic fibroblast growth factors (bFGFs). The next step called the differentiation phase involved medium consisting of DMEM supplemented with 20 ng/mL HGF, 10 ng/mL bFGF, and 4.9 mmol/L nicotinamide, for 7 days, followed by treatment with step-3 maturation medium, consisting of DMEM supplemented with 20 ng/mL OMS, 1 mmol/L dexamethasone, and 10 mL/mL ITS þ premix to achieve cell maturation (up to day 30). Medium was refreshed twice in week.

The fixed cells got permeable with 0.4% Triton X-100/ PBS for 20 min, and were blocked for 30 min at 37 C with 10% goat serum/PBS-Tween 20. Cells were incubated overnight at 4 C with primary antibodies: mouse monoclonal antialbumin (ALB, at a 1:200 dilution), anti-alpha-fetoprotein (AFP, 1:200) and then for 4 h with secondary antibody (rabbit anti-mouse IgG-FITC, 1:200) at 37 C. Nuclei counterstaining was performed using 40 ,6-diamidino-2phenylindole (DAPI) and cells were analyzed by fluorescence microscope (BX51, Olympus).

Cell morphology The morphology of cells on the plastic flask was examined under a phase-contrast microscopy on 1, 15, and 30 days of differentiation to appraise cuboidal cell semblance. Microphotographs were taken with 10 objective (TS-100 Nikon, Japan).

Real-time and RT-PCR analysis To evaluate the expression of differentiated hEnSCs markers into a hepatic cell lineage, trizol reagent (Invitrogen) was used for total cellular RNA of cells extraction in accord with the manufacturer’s instructions from differentiated cells. cDNA was synthesized from 5 g of total RNA using Moloney-murine leukemia virus (MMLV), superscript II reverse transcriptase (Promega), and random hexamer primers. The gene expression was quantified by real-time PCR using SYBR Green PCR Master Mix and Real-Time detection system software. The relative levels of gene mRNAs were normalized to housekeeping gene GAPDH mRNA. HepG2 was used as positive control. Samples were collected from at least three independent experiments. Table 1 shows the primer sequences.

Functional assay Periodic Acid-Schiff reaction At day 30, Periodic Acid-Schiff (PAS) staining determined glycogen storage of hEnSCs-derived hepatocytes. The differentiated cells fixed with 4% paraformaldehyde, then oxidized in 1% periodic acid for 5 min and washed twice. The cells were exposed to the Schiff reagent for 10 min followed by rinsing with deionized water (Baharvand et al., 2007) and observed under a light microscope (IX50, Olympus). Undifferentiated hEnSCs and HepG2 cell lines were used as a negative and a positive control, respectively. Albumin and a-fetoprotein synthesis The cultured media from differentiated cells were collected after 1st, 15th, and 30th day and stored at 70 C for afp and albumin secretion measurement. Quantitative enzymelinked immunusorbent assay kit (ELISA) was used according to instructions of manufacturer. Eventually, afp and albumin concentration was determined from different dilutions of afp and albumin standard.

Statistical analysis Immunocytochemical analysis Immunostaining was performed for albumin, and alphafetoprotein markers. To confirm hepatic differentiation of hEnSCs, the cells were stabilized with 4% paraformaldehyde (Sigma, USA) for 20 min at 4 C followed by cold PBS rinse.

Statistical analyses were conducted with SPSS software (version 19.0). Student’s t-test was used to determined difference between the two groups. The results were given as mean  SEM and values of P < 0.05 were considered to be statistically significant.

Table 1 The human primers used in RT-PCR studies: gene primer sequence, annealing temperature ( C), length (bp), gene bank code.

Gene Albumin (ALB) Alpha-fetoprotein (AFP) Cytokeratin 18 (CK18) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)


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Annealing temperature ( C)

Length (bp)













Gene bank code


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Phenotypic characterization of hEnSCs After three passages, the morphology of hEnSCs relatively became homogene and spindle shapes on the plastic surface (Figure 2). Flow cytometric analysis characterized isolated hEnSCs and capability of hepatic differentiation upon their exposure to suitable medium including adipogenic and osteogenic-specific factors. Flow cytometry confirmed the presence of CD90, CD105, CD146, CD44 and absence of CD34, CD31, CD45, CD133, and Stro1 in undifferentiated hEnSCs (Figure 1). The cells multipotency was confirmed by using alizarin red and oil red O staining for presence of calcium deposits, and lipid droplets (Figures 3a and 3b). Above mentioned

results demonstrated that the isolated hEnSCs ability of differentiation and can be characterized as stem cells (Figures 1 and 3).

Confirmation of hepatic differentiation by morphologic changes in cultured hEnSCs Applying a protocol described earlier to induce hepatic differentiation of human endometrium-derived endometrial stem cells, the spindle-shaped hEnSCs cells were converted to a cuboidal morphology developed similar to hepatocytes by step two–three of differentiation (7 days up to 30 days) (Figure 2).

Hepatic gene expression The expression of human hepatocyte genes, including CK18, AFP, and albumin were measured by qRT-PCR analysis on

Figure 1 Flow cytometric analysis of isolated hEnSCs are positive for CD146, CD90, CD44, and CD105 (mesenchymal markers), and are negative for CD31 (endothelial stem cell marker), CD34, CD133, CD45 (hematopoietic stem cell markers) and Stro1 (bone marrow mesenchymal stem cell marker).


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Figure 2 The photomicrograph of morphological characteristics of hEnSCs in passage three (left), the cells are spindle shape (below, left). Fifteenth day (middle), the differentiating cells are going to cuboidal shape with one nucleus (below, middle) and 30th day, the differentiated cells (right). The accumulations of cells are showed with one or two nuclei (below, right). Scale bar: 100 mm.

15th and 30th day of differentiation. Results indicated that the cells differentiated from hEnSCs show phenotypic properties of human hepatocytes which suggested the hepatic genes, Ck18, afp, and alb were gradually upregulated alike HepG2 cells and peaked at 30 days (Figure 4). These markers were significantly higher than hEnSCs (P < 0.0.001, P < 0.036, P < 0.032, respectively).

reaction with PAS, the purple spots were shown in the cytoplasm of hepatocyte-like cells. Albumin and alpha-fetoprotein was secreted in differentiated cells and HepG2 cell line on 15 and 30 days of differentiation (Figures 6d and 6e). The experimental cells were significantly high compared with hEnSCs (alb ¼ P < 0.001, afp ¼ P < 0.000). There was no significant difference between differentiated cells and HepG2 cell lines.

Immunofluorescence staining To ascertain cells differentiating further toward a hepatic phenotype, immunostaining was performed for afp and alb. On 30th day, the hEnSCs-derived hepatocyte-like cells expressed these hepatic proteins and immunoreactivity was observed. These results were compared with hEnS cells (negative control). But, these markers were not express in hEnSCs. Nucleuses staining with DAPI was done in both differentiate and undifferentiated cells (Figure 5).

Periodic Acid-Schiff staining reaction, albumin and a-fetoprotein synthesis The hEnSCs-derived hepatocyte-like cells were screened for glycogen storage by PAS staining after 30 days of differentiation. Glycogen granules were detected in the differentiated cells cytoplasm shown (Figures 6a and 6b). In Cell Biol Int 38 (2014) 825–834 © 2014 International Federation for Cell Biology

Discussion Stem cells have great advocated in recent years by their potential therapeutic uses. They have been shown liver function improvement and have been presented an interesting alternative to live with more plasticity in differentiation than previous thought (Kuo et al., 2008). Stem cells are an ideal for damaged organ (Ai et al., 2012b) application and recently propose to tissue reconstruction. But, there are controversy in their presence and function of these cells, timely. Hence, the use of extra-hepatic source of stem cells is a considered option in clinic for liver cell therapy. One of the strategies for liver cell transplantation is using appropriate cell sources. In this way, previous studies have used to many stem cells as a non-hepatic origin such as embryonic stem cells (Hay et al., 2008; Kim et al., 2011), cord umbilical blood-derived stromal cells (Hashemi et al., 2009), 829

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bone marrow-derived mesenchymal stem cells (Di Bonzo et al., 2008; Allameh et al., 2009), adipose tissue derived mesenchymal stem cells (Puglisi et al., 2011), induced pluripotent stem (ips) cells (Saramipoor Behbahan et al., 2011) into hepatic differentiation. Previous studies have been demonstrated endometrial stem cells as the mesenchymal stem cells with differential potency to various cells such as osteoblast (Donofrio et al., 2008; Azami et al., 2013), insulin producer cells (Li et al., 2010). By the way, angiogenic property was proved of these cells in three-dimensional culture (Esfandiari et al., 2007a,b, 2008) as well as mesenchymal stem cells to the endometrium layers (Taylor, 2004). Our study was confirmed the Lee and coworkers hypothesis about differentiating ability of mesenchymal stem cells (MSCs) obtained from different sources in body into functional hepatocyte-like cells in vitro (Lee et al., 2004). On top of that hEnSCs compared with MSCs from other sources such as bone marrow are an easy accessible source of cell with a high proliferation capacity which can maintain a normal karyotype normality for 68 doublings confirmed lack of tumorigenicity of these cells (Meng et al., 2007). The allograft-immunity is considerable factor which exists for all organ transplantation and cell therapy using stem cells. The hEnSCs due to the innocence of these aspects as an immunomodulator (Herath et al., 2006) can be used in transplantation without restriction on different people as a heterologous cell sources. Stem cells can proliferate and differentiate by environmental factors such as morphogens and extracellular matrix components to recreate these events in vitro. The comprehension of engaged factors in fetal organogenesis is important because of these agents like HGF and FGFs has been related to endodermal development. In addition, OSM, insulin, and glucocorticoids are involved in the late maturation stage (Hamazaki et al., 2001). Also, these factors play critical role throughout cell cycle. For example, HGF and EGF allow progression of the hepatocyte from G1 to S stage in the cell cycle and insulin acts as a growth promoting and growth factors for supporter hormone (Teratani et al., 2005). Hence, we were applied a three-step protocol for hepatic differentiation from hEnSCs. EGF and bFGF factors were used at a first-stage, predifferentiation, for preparing of differentiation. The second-stage was start using bFGF and HGF factors to induce the endodermal and hepatocyte lineage differentiation on 7 days. Finally, OSM, ITS, and dexamethasone were added at late stage from days 7 to 30. Over the past decade, it has been illustrated that growth factors are pivotal element in liver regeneration. They act as regulators of hepatic proliferation and liver regeneration namely HGF, EGF, OSM, FGF (Baharvand et al., 2007; Cai et al., 2007; Agarwal et al., 2008; Hay et al., 2008). These researchers have successfully generated 830

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cultures where up to 70% of the cells with a hepotocyte-like phenotype using mouse or embryonic stem cells. To confirm this, many investigators were transfused ES cells-derived hepotocyte-like cells into mice with hepatic injury models and were exhibited a modest rescue of hepatic function. However, engraftment of these cells into the host liver was low (Baharvand et al., 2007; Cai et al., 2007; Hay et al., 2008). Okaya et al. (2005) showed hepatocyte induction using oncostatin M (OSM) for rat oval cell line OC15-5 treatment, they were observed by alterations in morphology in which microvilli appeared and a large cytoplasm developed with organelles, and likewise the expression of hepatic cells markers, for example, albumin and tyrosine aminotransferase (TAT). In current study, we established and characterized a hepatocyte-like cells derived from human endometrial stromal stem cells and then analyzed their multipotency. Our results showed that the cultured hEnSCs on twodimensional culture had a typical undifferentiated morphology, spindle-shape (Figure 2), and expressed multipotencyassociated markers of the endometrial stem cells namely CD90, CD105, CD44, and CD146. And negative expression for markers such as CD133, CD34 (known as hematopoietic lineage markers), CD31 (as an endothelial lineage marker), leukocyte common antigen (CD45), and Stro1 (as bone marrow-mesenchymal stem cells) was used (Figure 1) that these consented with Meng and Gargett studies (Meng et al., 2007; Gargett and Masuda, 2010). To approve the nature of hEnSCs, we were also investigated the differentiation aptitude of this stem cells toward osteogenic and adipogenic lineages (Figure 3). The expression of biochemical markers are commonly used to assess the differentiation of hEnSCs into hepatic lineages, particularly albumin and alpha-fetoprotein (Thorgeirsson, 1996). In this study, the results were showed the expression of endodermal markers such as cytokeratin18, alpha-fetoprotein, and albumin that confirms commitment into hepatocyte cells (Figure 4). And also, differentiated hEnSCs were expressed endodermal and hepatic proteins such as alpha-fetoprotein and albumin on day 30 of differentiation (Figure 5). Correspondingly, Herrera et al. (2006) were demonstrated that cells in stimulated medium by HGF and FGF4 factors could secret albumin marker on human hepatic stem cells in vitro condition. Although Meng et al. (2007) have claimed the obtained endometrial stem cells from menstrual blood could differentiate into many different cell lineages, the generated hepatocyte-like cells in their study were not further validated for hepatocyte function. Current study has tested the functionality of differentiated cells both qualitative and quantitative. Therefore, to determine functional properties, glycogen storage was performed (Figure 6b). In hepatogenic condition, the spindle shape of hEnSCs was gradually Cell Biol Int 38 (2014) 825–834 © 2014 International Federation for Cell Biology

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Figure 3 Alizarin Red staining for human endometrial stem cells (hEnSCs) differentiate into osteocytes (a) and Oil Red O staining for adipocytes (b). Scale bar: 100 mm.

Figure 4 Hepatic-related gene expression analysis by quantitative RT-PCR after 15 days (a) and 30 days post treatment (b). Data are expressed as the means  SEM of three independent experiments. *P < 0.05, significantly different from negative control group (hEnSCs).

Figure 5 Immunocytochemistry staining for alpha-fetoprotein (AFP: b, b0 , b00 , b000 ) and albumin (ALB: a, a0 , a00 , a000 ) protein into hepatocytelike cells derived human endometrium stem cells. Differentiated cells were compared with undifferentiated endometrial stem cells as negative control. Scale bar: 100 mm. Cell Biol Int 38 (2014) 825–834 © 2014 International Federation for Cell Biology


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Figure 6 Functional assay of the hepatocyte-like cells derived from hEnSCs: PAS staining for glycogen storage revealed that differentiated cells (b) could produce and store glycogen by hepatogenic induction for 30 days. Undifferentiated cells (c) and HepG2 cell line (a) stained as negative and positive control, respectively. Differentiated cells synthesized afp (d) and alb (e) on days 15 and 30 of differentiation (down). *P < 0.05, significant differences between differentiated and negative control group (hEnSCs). Scale bar: 100 mm.

changed to the cuboidal shape of the mature hepatocytes until day 30 post-induction of differentiation (Figure 2). In vitro functional assays on differentiated cells at the end of induction time were confirmed with the morphological changes. Positive-stained cytoplasm of cells were exhibited glycogen storage as a function by hEnSCs-derived hepatocyte-like cells similar to Hashemi and Chivu (2009) studies that were used accumulate glycogen assay to evaluate liver function. Our study, in serological functional assay, was proved the hEnSCs-derived hepatocyte-like cells synthesized both alpha-fetoprotein and albumin. In a similar manner, some researchers were achieved functional hepatocyte derived from adult stem cells using sequential treatment with factors (Dong et al., 2013; Kazemnejad et al., 2009; Hashemi et al., 2009; No et al., 2012). These differentiated cells had hepatocyte cells feature comprising alpha-fetoprotein, albumin, urea secretion, glycogen storage, indocyanine green uptake. Our findings not only showed hepatocyte cell morphology as it expressed hepatic-specific markers from the differentiated hepatocyte cells, but also possessed the characteristics of mature hepatocyte function which has been makes them an excellent option for hepatic repopulation in the treatment of end-stage liver disease. 832

Acknowledgments and funding We would like to thank Stem Cell Technology Research Centre and Tehran University of Medical Sciences for financial supported (Grants 90-03-87-14665). Conflict of interest None declared. References Ai J, Shahverdi AR, Ebrahimi-Barough S, Kouchesfehani HM, Heidari S, Roozafzoon R, Verdi J, Khoshzaban A (2012a) Derivation of adipocytes from human endometrial stem cells (EnSCs). J Reprod Infertil 13(3): 151–57. Ai J, Ebrahimi S, Khoshzaban A, Jafarzadeh Kashi TS, Mehrabani D (2012b) Tissue engineering using human mineralized bone xenograft and bone marrow mesenchymal stem cells allograft in healing of tibial fracture of experimental rabbit model. IRCM J 14(2): 96–103. Ai J, Mehrabani D (2010) The potential of human endometrial stem cells for osteoblast differentiation. IRCM J 12(5): 585–7. Alison MR, Islam S, Lim S, (2009) Stem cells in liver regeneration, fibrosis and cancer: the good, the bad and the ugly. J Pathol 217: 282–98.

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Cell Biol Int 38 (2014) 825–834 © 2014 International Federation for Cell Biology

Human endometrial stem cells differentiation into functional hepatocyte-like cells.

In spite of certain clinical limitations, such as teratoma formation, the use of stem cells is considered as an appropriate source in cell therapy and...
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