Differentiation 84 (2012) 223–231
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Comparison of in vitro hepatogenic differentiation potential between various placenta-derived stem cells and other adult stem cells as an alternative source of functional hepatocytes$ Hyun-Jung Lee a, Jieun Jung b, Kyung Jin Cho c, Chang Kyou Lee c, Seong-Gyu Hwang d, Gi Jin Kim a,b,n a
CHA Placenta Institute, CHA University, Seoul, Republic of Korea Department of Biomedical Science, CHA University, Seoul, Republic of Korea c Department of Biomedical Science, College of Health Science, Korea University, Seoul, Republic of Korea d Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Republic of Korea b
a r t i c l e i n f o
Article history: Received 20 July 2011 Received in revised form 24 March 2012 Accepted 25 May 2012 Available online 10 August 2012
Mesenchymal stem cells (MSCs) are powerful sources for cell therapy in regenerative medicine. The capability to obtain effective stem cell-derived hepatocytes would improve cell therapy for liver diseases. Recently, various placenta-derived stem cells (PDSCs) depending on the localization of placenta have been suggested as alternative sources of stem cells are similar to bone marrow-derived MSC (BM-MSCs) and adipose-derived MSC (AD-MSCs). However, comparative studies for the potentials of the hepatogenic differentiation among various MSCs largely lacking. Therefore, we investigated to compare the potentials for hepatogenic differentiation of PDSCs with BM-MSCs, AD-MSCs, and UCBMSCs. Several MSCs were isolated from human term placenta, adipose tissue, and umbilical cord blood and characterized isolated MSCs and BM-MSCs was performed by quantitative reverse transcriptionPCR (RT-PCR) and special stains after mesodermal differentiation. The hepatogenic potential of PDSCs was compared with AD-MSCs, UCB-MSCs, and BM-MSCs using RT-PCR, PAS stain, ICG up-take assays, albumin expression, urea production, and cytokine assays. MSCs isolated from different tissues all presented similar characteristics of MSCs. However, the proliferative potential of PDSCs and the expression of hepatogenic markers in differentiated PDSCs were higher than other MSCs. Interestingly, the expression of hepatocyte growth factor (HGF) increased in PDSCs after hepatogenic differentiation. Interestingly, stem cell factor (SCF) expression in chorionic plate-derived MSCs, one of the PDSCs, was signiﬁcantly higher than in the other PDSCs. Taken together, the results of the present study suggest that MSCs isolated from various adult tissues can be induced to undergo hepatogenic differentiation in vitro, and that PDSCs may have the greatest potential for hepatogenic differentiation and proliferation. Therefore, PDSCs could be used as a stem cell source for cell therapy in liver diseases. & 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.
Keywords: Adult stem cells Placenta-derived stem cells Hepatocyte Differentiation
1. Introduction Hepatic failure is one of the major causes of morbidity and mortality worldwide. Although it is the best way to treat liver transplantation for acute and chronic hepatic failure patients, there are several obstacles (e.g., lack of donor organs, invasive procedure) Haydon and Neuberger (2000). Therapy using hepatocyte transplantation has emerged as an attractive treatment for patients with late-stage liver diseases Zaret and Grompe (2008). Mature hepatocytes isolated from the liver have important
’’Join the International Society for Differentiation (www.isdifferentiation.org)’’. Correspondence to: Department of Biomedical Science, CHA University, 606-1 Yeoksam 1-dong Kangnam-ku, Seoul 135-081, Republic of Korea. Tel.: þ 82 2 3468 3687; fax: þ 82 2 538 4102. E-mail address: [email protected]
(G.J. Kim). n
functions when they are transplanted; however, it is hard to expect any effective results from the transplantation of isolated hepatocytes due to the limited number of functional hepatocytes via their dedifferentiation during in vitro cultivation, limited ability to be cryopreserved and short-term survival (Elaut et al., 2006; Terry et al., 2007). Therefore, exploring other sources of cells to replace damaged hepatocytes in hepatogenic diseases is required. Recently, stem cells have been spotlighted as alternative sources of hepatocytes because they have potential for hepatogenic differentiation (Cai et al., 2007; Cantz et al., 2008). Human embryonic stem cells (hESCs) have unlimited proliferation capacity in vitro as well as a potential for differentiation into all cell types under specialized differentiation conditions (Thomson et al., 1998). Many researchers have demonstrated that hESCs are capable of hepatogenic differentiation into hepatocytes or
0301-4681/$ - see front matter & 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. Join the International Society for Differentiation (www.isdifferentiation.org) http://dx.doi.org/10.1016/j.diff.2012.05.007
H.-J. Lee et al. / Differentiation 84 (2012) 223–231
hepatocyte-like cells. However, the efﬁciency of their hepatogenic differentiation is still low and controversial according to the differentiation procedures and conditions (Cai et al., 2007). In particular, there are risks in using partially differentiated hESCs because they can induce teratoma formation due to the heterogenous population of partially differentiated hESCs Gallicano and Mishra (2010). In addition, Activin A is a necessary factor for hepatogenic speciﬁcation of undifferentiated hESCs to induce the hepatogenic differentiation of hESCs in vitro because it can induce endodermal differentiation of ESCs. However, the application of Activin A for hepatogenic differentiation causes the loss of many cells via cell death due to the cytotoxicity of Activin A (Parashurama et al., 2008). In addition, collecting available hepatocytes or hepatocyte-like cells, and differentiating functional hepatocytes from hESCs are time-consuming processes (Cai et al., 2007; Hay et al., 2008). To enhance the potential for this approach, stem cell therapy requires a renewable cell source capable of functional hepatocytes(Hay et al., 2008). Therefore, hepatogenic differentiation using mesenchymal stem cells derived from various sources (e.g., bone marrow, adipose, cord blood, and placenta) has been developed to enhance the efﬁciency of hepatogenic differentiation using simple procedures, as well as to ensure the safety in stem cell application (Lee et al., 2004a; Snykers et al., 2006; Lee et al., 2010; Shin et al., 2010). The plasticity means that MSCs derived from adult tissue can generate differentiated cell types of a different tissue. This ability is variously referred to as ‘‘plasticity’’ or ‘‘transdifferentiation’’ (Anderson et al., 2001; Krause et al., 2001; Barzilay et al., 2009). Although MSCs are capable of differentiating into mesodermal lineages such as osteoblasts, chondocyte and adipocytes, they are also able to give rise to multiple lineages including ectodermal (neurons) and endodermal (hepatocyte) cells (Jiang et al., 2002; Greco et al., 2007; Bianco et al., 2008). The potential for hepatogenic differentiation of various MSCs has been evaluated by measuring the expression of endodermal or hepatocyte markers including a-fetoprotein (AFP), hepatocyte nuclear factors (HNFs), albumin, and tyrosine aminotransferase (TAT), as well as urea production after inducing hepatogenic differentiation (Snykers et al., 2009). In addition, MSCs show therapeutic effects when transplanted into animal models with liver diseases, regardless of whether differentiated or undifferentiated MSCs are used (Banas et al., 2008; Shi et al., 2009; Lee et al., 2010). Furthermore, the hepatogenic differentiation potential of iPS cells, which could provide a source of autologus hepatocytes, has been introduced (Espejel et al., 2010; Sancho-Bru et al., 2011). But, hepatogenic differentiation potential of iPS cells should be studied on the efﬁciency and the safety because the generation of iPS is laborintensive as well as based on virus gene delivery system. Due to the reason, there are no reports comparing the efﬁciency of hepatogenic differentiation among various MSCs derived from adult tissues including bone marrow, adipose tissue, cord blood and placenta. Placenta-derived stem cells (PDSCs) have several advantages for use in cell-based therapy. They have a higher proliferative potential that is associated with short population doubling time, as well as ethical advantages. PDSCs contain several types of stem cells based on placental anatomy: chorionic villi (CV-MSCs), amnion (AE-MSCs), chorionic plate (CP-MSCs) and Wharton’s jelly of the umbilical cord (WJ-MSCs) (Igura et al., 2004; Parolini et al., 2008; Troyer and Weiss, 2008). These placenta-derived MSCs have the ability to differentiate into various types of cells, including adipocytes, chondrocytes, osteocytes, neuronal cells and hepatocytes, given the appropriate induction conditions (Moon et al., 2008). Therefore, we isolated several types of MSCs from human placenta, characterized the differences among several adult stem
cells, and compared the hepatogenic differentiation of PDSCs including AE-MSCs, CP-MSCs, CV-MSCs, WJ-MSCs, AD-MSCs, BM-MSCs and UCB-MSCs.
2. Material and methods 2.1. Cell culture The collection of placenta samples was approved by the Institutional Review Board of CHA General Hospital, Seoul, Korea. All participating women provided written, informed consent prior to the collection of samples. Placentas were collected from women who were free of medical, obstetrical, and surgical complications and who delivered at term (Z37 gestational weeks). PDSCs including AE-MSC, CP-MSC, CV-MSC, and WJ-MSC were isolated from the placentas after term delivery. Various PDSCs were harvested, as described, with some modiﬁcations (Parolini et al., 2008; Kim et al., 2011). Harvested cells were cultured in Ham’s F-12 medium/DMEM medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS; Invitrogen), penicillin (100 U/ml), streptomycin (100 mg/ml) (Invitrogen), 25 ng/mL FGF-4 (Peprotech Inc) and 1 mg/mL heparin (Sigma-Aldrich) at 37 1C in a humidiﬁed atmosphere containing 5% CO2. PDSCs were passaged every 48–72 h at a 1:3 ratio. AD-MSCs were kindly provided by Dr. Jong-Hyuk Seong (CHA University, Seoul Korea) and cultured with medium contained a-MEM (GIBCO-BRL, USA) supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml) and 10% fetal bovine serum (GIBCO-BRL, USA). Bone marrow-derived mesenchymal stem cells (BM-MSCs) were obtained from Cambrex Bioscience Walkersville (Cambrex BioScience Walkersville, Walkersville, MD) and cultured with medium contained a-MEM (GIBCO-BRL, USA) supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), 1 mM sodium pyruvate (GIBCO 11360070) and 10% fetal bovine serum (GIBCO-BRL, USA). UCB-MSCs were kindly provided by Dr. Young Soo Choi (CHA University, Seoul Korea) and were cultured with medium contained a-MEM (GIBCO-BRL, USA) supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml) and 20% fetal bovine serum (GIBCO-BRL, USA). Cells were split every 48–72 h at 1:3 ratio. To measure the growth of adult stem cells with respect to cell types, each cell type was seeded at a density of 2 104 cells into 60 mm dishes. At 0, 24, 48, 72, 96 and 120 h cells were digested with trypsin, stained with 0.2% trypan blue, and counted using a hemocytometer. The viability of all MSCs was determined by trypan blue exclusion. To analyze the growth kinetics of individual stem cells, cells were plated at 2 104 cells/cm2 on culture dishes. Cell number was determined from each dish 24 h after plating and every 24 h until day 5. 2.2. Expression of stemness markers and lineage-speciﬁc markers of various MSCs by reverse transcription-PCR Total RNA was extracted from the naı¨ve stem cells and cells that had differentiated into osteogenic, adipogenic and hepatogenic lineages using RNeasy plus mini kits (QIAgen, Valencia, CA, USA), and 1 mg of RNA was reverse transcribed into cDNA using the Superscript Plus ﬁrst-strand synthesis system (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cDNA samples were subjected to polymerase chain reaction (PCR) ampliﬁcation. The PCR primers and the length of the ampliﬁed products are shown in Table 1. PCR conditions were as follows: initial denaturation (95 1C, 15 min), annealing (95 1C, 20 s; 55 1C, 58 1C, 59 1C or 60 1C, 40 s; 72 1C, 1 min) and ﬁnal extension (72 1C, 5 min). All PCR reactions were performed using 40 cycles. The ampliﬁed PCR products were analyzed by electrophoresis on 1% agarose gels and stained with ethidium bromide for visualization.
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Table 1 RT-PCR primers used in the study. Gene
Oct-4 Nanog Sox-2 NF-68 Cardiac AFP HLA-G TERT OC Adiponectin AFP Albumin TAT Antitrypsin Connexin 32 Connexin 43 CYP2B6 CYP3A4 Actin
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
Primer sequences (50 -30 )
AGT GAG AGG CAA CCT GGA GA GTG AAG TGA GGG CTC CCA TA TTC TTG ACT GGG ACC TTG TC GCT TGC CTT GCT TTG AAG CA AGA ACC CCA AGA TGC ACA AC GGG CAG CGT GTA CTT ATC CT GAG TGA AAT GGC ACG ATA CCT A TTT CCT CTC CTT CTT CTT CAC CTT C GGA GTT ATG GTG GGT ATG GGT C AGT GGT GAC AAA GGA GTA GCC A ATG CTG CAA ACT GAC CAC GC GCT TCG CTT TGC CAA TGC TT GCG GCT ACT ACA ACC AGA GC GCA CAT GGC ACG TGT ATC TC GAG CTG ACG TGG AAG ATG AG CTT CAA GTG CTG TCT GAT TCC AAT G GCA GCG AGG TAG TGA AGA GA CGA TGT GGT CAG CCA ACT GCT GGA GTT CAG TGG TGT GA ACC AAC CTG ACG AAT GTG GT AGC TTG GTG GAT GAA AC TCC AAC AGG CCT GAG AAA TC CCC CAA GTG TCA ACT CCA AC CCA GAA GAC ACC CTC CAA AGG AC AAC GAT GTG GAG TTC ACG G GAC ACA TCC TCA GGA GAA TGG TCG CTA CAG CCT TTG CAA TG TTG AGG GTA CGG AGG AGT TCC ATG AAC TGG ACA GGT TTG TAC ATG TGT TGC TGG TGA GCC A GCG TGA GGA AAG TAC CAA AC GGG CAA CCT TGA GTT CTT CC CTT GAC CTG CTG CTT CTT CC TGC TTC CCG CCT CAG ATT TCT C ATC ATT GCT GTC TCC AAC CTT CAC TGC TTC CCG CCT CAG ATT TCT C TCC TTC TGC ATC CTG TCA GCA CAG GAG ATG GCC ACT GCC GCA
2.3. Differentiation of various mesenchymal stem cells into mesodermal lineages All MSCs were analyzed at the early passages (6–8 passages) for their potentials for hepatogenic differentiation. To induce adipogenic differentiation, seven types of MSCs were plated at a density of 5 103 cells/cm2 in adipogenic induction medium containing 1 mM dexamethasone, 0.5 mM isobutyl methylxanthine (IBMX), 0.2 mM indomethacin, 1.7 mM insulin (all from Sigma-Aldrich), 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) with medium changes twice a week. After 21 days, PDSCs were ﬁxed with 4% paraformaldehyde (PFA) and lipid vesicles were revealed by Oil-Red O (Sigma-Aldrich) staining for 1 h. Cells were counterstained with Mayer’s hematoxylin (SigmaAldrich) for 1 min. To induce osteogenic differentiation, all MSCs were plated at a density of 5 103 cells/cm2 in osteogenic induction medium containing 1 mM dexamethasone, 10 mM glycerol-2phosphate (Sigma-Aldrich), 50 mM L-ascorbic acid 2-phosphate (all from Sigma-Aldrich), 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) with medium changes twice a week. After 21 days, calcium deposits in adult stem cells were evaluated by von Kossa staining using 5% silver nitrate (Sigma-Aldrich) under light for 1 h. 2.4. Hepatogenic differentiation of various MSCs To direct the differentiation of AE-MSC, CP-MSC, CV-MSC, WJ-MSC, AD-MSC, BM-MSC, and UCB-MSC into hepatocytes in vitro, the cells were plated at a density of 2 104 cells/cm2
on 0.1% collagen-coated dishes in culture medium (60% DMEM-LG, 40% MCDB201, 2% FBS, and 1% P/S) containing 20 ng/ml EGF and 10 ng/ml bFGF. Cells were grown to 60% conﬂuence and then incubated for 7 days in basal medium supplemented with 2% FBS, 20 ng/ml HGF, 10 ng/ml bFGF, and 0.61 g/L nicotinamide. To induce maturation, the cells were treated with maturation medium (basal medium supplemented with 2% FBS, 1 mM dexamethasone, 50 mg/ ml ITS þ premix, and 20 ng/ml oncostatin M) for a further 2 weeks. The medium was replaced every 3 days. After hepatogenic differentiation, the hepatogenic induced cells were uptaken of CM-Dil for identiﬁcation of hepatogenic differentiation. Subsequently, the induced cells were harvested for analysis of hepatocyte-speciﬁc gene expression using reverse transcription PCR. 2.5. CM-Dil uptake assay After washing the cultured cells with PBS, a Vybrant CM-Dil cell labeling solution (Invitrogen cat.no V22888) was added to the plates at a ﬁnal concentration of 5 mM CM-Dil, incubated at 37 1C for 20 min, rinsed three times with PBS, and then reﬁlled with DMEM containing 10% FBS (GIBCO-BRL, USA). 2.6. ELISA assay All adult stem cells were cultured to differentiate into hepatocytes using induction media. The cell culture supernatant was collected after hepatogenic differentiation. The supernatant of normal cell cultures was also collected and used as a negative control. Using the supernatant, an HGF ELISA assay (Peprotech cat.no 100-39-10),
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urea assay (Bioassay systems DIUR-500) and SCF ELISA assay (Multiplex detection kit, Bio-rad, Hercules, CA, USA) were performed, following the manufacturer’s protocol. 2.7. Immunoﬂuorescence staining analysis Cells were ﬁxed in 4% PFA and stained with an anti-albumin polyclonal antibody (1:50 dilution; Sigma-Aldrich, St Louis, MO) at 4 1C overnight. An Alexa 488-conjugated polyclonal antibody (Invitrogen) was used as a secondary antibody and DAPI staining was performed (Vector Laboratories, Burlingame, CA, USA). 2.8. Analysis of glycogen storage using periodic acid-schiff (PAS) staining Culture dishes containing cells were ﬁxed in 4% formaldehyde and oxidized in 1% periodic acid for 5 min, rinsed three times in dH2O, treated with Schiff’s reagent (SIGMA, St. Louis, MO, USA) for 15 min, and rinsed in dH2O for 5–10 min. Samples were counterstained with Mayer’s hematoxylin (DAKO, Glostrup, Denmark) for 1 min, rinsed in dH2O, and assessed using a light microscope.
was centrifuged at 13,000 g for 10 min at 4 1C. The supernatant was harvested, and its protein concentration measured using a BCA protein assay kit (Pierce, Rockford, IL, USA). For electrophoresis, 30 mg of protein was dissolved in sample buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 1% b-mercaptoethanol, 12.5 mM EDTA, 0.02% Bromophenol blue), boiled for 5 min, and separated on an 8% SDS-PAGE reducing gel. Separated proteins were transferred onto polyvinylidene diﬂuoride (PVDF) membranes (Bio-rad, Hercules, CA, USA) using a trans-blot system (Bio-rad). Blots were blocked for 1 h in phosphate-buffered saline (PBS) containing 8% non-fat dry milk (BD, Sparks,MD,USA) and 0.05% Tween 20 at room temperature, washed three times with PBST, and incubated at 4 1C overnight with a primary antibody speciﬁc for human albumin (1:1000 dilution; SIGMA, St. Louis, MO, USA) in PBST containing 2% non-fat dry milk. The next day, blots were washed six times with PBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution; Bio-rad, Hercules, CA, USA) in PBST containing 2% non-fat dry milk at room temperature. After being washed six times with PBST, the proteins were visualized with an ECL detection system (Amersham, Piscataway, NJ, USA).
2.9. Western blotting
2.10. Statistical analysis
Cells were washed with PBSs and lysed in 100 ml of cold Cell lysis buffer (Fermentas, Glen Burnie, Maryland, USA) with a protease inhibitor cocktail (Roche, Mannheim, Germany). Cell lysate
Results are presented as the means7SD. Statistical signiﬁcance measured multiple comparisons were performed using the t-test with a signiﬁcance level of Po0.05.
Fig. 1. Characterization of adult MSCs derived from different tissues. (A) Morphologies of human adult MSCs derived from different tissues in undifferentiated culture conditions ( 100). (B) Cell proliferation of various stem cells. To conﬁrm the proliferation potential of adult stem cells, the cell number was determined from each dish at 24 h after plating and at every 24 h until day 5. (C) RT-PCR analysis for stem cell markers in several MSCs. Scale bars: 25 mm. a: AE-MSC, b: CP-MSCs, c: CV-MSC, d: WJ-MSC, e: AD-MSC, f: BM-MSC, g: UCB-MSC.
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3. Results 3.1. Growth kinetics of MSCs derived from placenta, adipose, bone marrow, and umbilical cord blood MSCs derived from placenta (amnion, chorionic plate, chorionic villi, Wharton jelly), adipose, bone marrow, and umbilical cord blood were plated at 2 104 cells/cm2 on culture dishes and cultured for 3, 4 days. In general, their morphologies were similar to the ﬁbroblast phenotype without differences between the types of stem cells (Fig. 1a). The MSCs were assayed for cell proliferation activity. The growth curves of MSCs were derived as described in the Materials and Methods section from the ﬁrst 5 day after plating. There were no differences among AE-MSC, CVMSC, AD-MSC, BM-MSC, and UCB-MSCs. However, the CP-MSCs and WJ-MSCs exhibited a higher growth rate by 4th and 5th day after cell seeding (Fig. 1b). Growth rates of CP-MSCs and WJ-MSCs were over 2-fold greater than others MSCs at day 5. These ﬁndings
indicate that CP-MSCs and WJ-MSCs derived from placenta have a strong proliferation activity. 3.2. Characterization of various MSCs derived from placenta, adipose tissue, bone marrow, and umbilical cord blood The expressions of markers of stemness and the three germ lineage in seven types of MSCs were analyzed by RT-PCR. There were differences in the expression of self-renewal markers (e.g., Oct4, Nanog, Sox2) and germ lineage-speciﬁc markers (e.g., NF68, cardiac, AFP) among MSCs (Fig. 1c). Oct4 expression in WJ-MSCs, AD-MSCs, and BM-MSCs was strongly expressed. Furthermore, the three germ lineage-speciﬁc markers were all expressed in CPMSCs, WJ-MSCs, and AD-MSCs. After inducing mesodermal lineage differentiation, lineage-speciﬁc markers were analyzed by RTPCR and chemical staining. As shown in Fig. 2, the expression of osteocalcin (OC), which is an osteocyte marker, increased in CP-MSCs, WJ-MSCs, BM-MSCs, and UCB-MSCs. In addition, the
Fig. 2. Differentiation potential of several MSCs into mesodermal lineages. (A) Expression of various mesodermal-speciﬁc genes during osteogenic and adipogenic differentiation by RT-PCR analysis. (B) Osteogenic differentiation was conﬁrmed by von Kossa staining. (C) Adiopgenic differentiation was conﬁrmed by Oil-Red O staining. Scale bars: 25 mm. a: AE-MSC, b: CP-MSCs, c: CV-MSC, d: WJ-MSC, e: AD-MSC, f: BM-MSC, g: UCB-MSC, h: CP-MSC as a negative control.
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expression of adiponectin, which is an adipose marker, was enhanced in CP-MSCs, WJ-MSCs, AD-MSC, and BM-MSCs (Fig. 2a). The differentiation potentials for osteogenic and adipogenic lineages were conﬁrmed by von Kossa and Oil-Red O staining, respectively (Figs. 2b and c). These ﬁndings indicate that the characteristics of MSCs derived from various tissues are similar. 3.3. In vitro hepatogenic differentiation of various MSCs derived from placenta, adipose, bone marrow, and umbilical cord blood Each type of adult stem cell was plated at 2 104 cells/cm2 in culture dishes and cultured in culture medium with 2% FBS, after a pre-culture for 2 days in serum-free medium supplement with EGF, basic FGF4 and BMP-4 to inhibit cell proliferation. Cell morphologies of all adult stem cells did not change during the initiation step, when cultures were treated with HGF, although the ﬁbroblastic morphology was lost and the cells developed a broadened ﬂattened shape. A polygonal shape developed during
the maturation step when cells were exposed to medium containing oncostatin M and insulin, transferrin, and selenium (Fig. 3a). To determine whether differentiated cells expressed hepatogenic phenotype markers, total RNA was isolated from all MSCs after hepatogenic differentiation and the mRNA levels of several hepatogenic genes were examined by RT-PCR. Interestingly, the expression of AFP, which is expressed in the early stages of hepatogenesis, decreased in all MSCs with the exception of UCB-MSCs. The expression of albumin increased in hepatogenic differentiated cells. There was no difference in the expression of anti-trypsin and connexin 43 among MSCs. However, alterations of several markers related to hepatogenic function exhibited different patterns depending on the type of MSC (Fig. 2b). These ﬁndings suggest that the protocol for hepatogenic differentiation used in this study proved to be an effective method to induce the hepatogenic differentiation of adult stem cells, and that the potential for hepatogenic differentiation of MSCs derived from tissues could be different, although several markers correlated with functional hepatocytes were expressed.
Fig. 3. Hepatogenic differentiation of several MSCs. (A) The morphological changes of several MSCs between undifferentiated (h) and differentiated (a–g) conditions ( 200). (B) Expression of hepatogenic-speciﬁc genes depends on hepatogenic differentiation by RT-PCR analysis. Scale bars: 25 mm. a: AE-MSC, b: CP-MSCs, c: CV-MSC, d: WJ-MSC, e: AD-MSC, f: BM-MSC, g: UCB-MSC, h: CP-MSC as a negative control.
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3.4. Functional analysis of hepatocyte-like cells derived from placenta, adipose tissue, bone marrow, and umbilical cord blood after hepatogenic differentiation To analyze hepatocyte-speciﬁc functions in the differentiated MSCs, we performed PAS staining, Western blots and ELISA assays. As shown on Fig. 4, the glycogen stores in the cytoplasm of hepatocyte differentiated MSCs were observed in almost all types of MSCs by PAS staining with the exception of UCB-MSCs (Fig. 4a). In addition, in vitro differentiation of MSCs into hepatocyte-like cells was identiﬁed by cellular uptake of indocyanine green (ICG) (Fig.4b). The expression of albumin in all MSCs increased after hepatogenic differentiation and was especially higher in AE-MSCs, CP-MSCs, AD-MSCs, and UCB-MSCs (Fig. 4c). There were no differences in the expression pattern of albumin in
WJ-MSCs and BM-MSCs. However, urea production was highly expressed in BM-MSCs after hepatogenic differentiation (Fig. 4d). These results indicate that the efﬁciency for hepatogenic differentiation of MSCs depends on the tissue origin could be different although they have a potential for hepatogenic differentiation. 3.5. Expression of HGF and SCF in MSCs derived from placenta, adipose tissue, bone marrow, and umbilical cord blood Because several cytokines effectively induce hepatogenic differentiation through their alteration during differentiation, the expression of HGF and SCF was analyzed by ELISA. The expression of HGF was dramatically increased in all MSCs derived from placenta after inducing hepatogenic differentiation (Fig. 5a) and was higher than in other MSCs. Additionally, the expression of SCF increased in
Fig. 4. Functional assay for hepatogenic differentiation of several MSCs. (A) Glycogen storage using PAS staining of MSCs between undifferentiated (h) and differentiated (a–g) conditions. (B) ICG uptake by MSCs between undifferentiated (h) and differentiated (a–g) conditions ( 200). (C) Albumin expression of MSCs according to hepatogenic differentiation detected by Western blot. GAPDH was used as an internal control. (D) Urea production in MSCs according to hepatogenic differentiation using an ELISA assay. Scale bars: 5 mm, 25 mm. a: AE-MSC, b: CP-MSCs, c: CV-MSC, d: WJ-MSC, e: AD-MSC, f: BM-MSC, g: UCB-MSC, h: CP-MSC as a negative control.
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Fig. 5. Expression of HGF and SCF by MSCs according to hepatogenic differentiation using an ELISA assay. (A) HGF expression of MSCs according to hepatogenic differentiation. (B) SCF expression of MSCs according to hepatogenic differentiation.
CP-MSCs, AD-MSCs, and BM-MSCs. Interestingly, the expression of SCF was dramatically increased only in CP-MSCs, among the placentaderived MSCs (Fig. 5b). These ﬁndings strongly indicate that there are differences in the potential for hepatogenic differentiation among MSCs derived from different tissues, and show that these differences depend on the cell origin in placenta tissues. Furthermore, MSCs derived from the placenta actively express cytokines.
4. Discussion MSCs are a powerful source for clinical applications in regenerative medicine and tissue/organ replacement because they can be easily isolated from various tissues, expanded in quantities relevant to clinical applications, and induced into multiple lineages including mesodermal lineage cells (Bobis et al., 2006). Among the types of MSCs derived from various tissues BM-MSCs have been studied as a representative source of MSCs for a long time (Pittenger et al., 1999). AD-MSCs have become more of a concern given the increasing problem posed by obesity in recent decades. AD-MSCs can be sourced from excess discarded fat tissues (Gimble et al., 2007). In addition, AD-MSCs have a similar proliferative ability and differentiation potential to BM-MSCs Parker and Katz (2006). Human UCB, also a source of MSCs, have the ability to differentiate into multiple lineages, given the appropriate conditions (Lee et al., 2004b). Therefore, adipose tissue, bone marrow and UCB may be readily accessible and good candidates as a source of stem cells with therapeutic potential for use in regenerative medicine. However, there are still considerations, including the invasive procedures and limitations in collecting cells from tissues, and the decreased stem cell content in elderly patients, that limit the application of adult stem cells in clinical stem cell-based therapies. Therefore, many researchers have tried to evaluate the potential of MSCs derived from various tissues to select the best MSC source for stem cell therapy Rao and Mattson (2001).
Recently, progenitor cells or stem cells could be isolated from human term placenta. These cells, referred to as PDSCs, can be expanded beyond 20 cell generations and can generate mesodermal cells, as well as neural and hepatogenic cells. PDSCs express CD90, SH105, SH3, and SH4, as representative markers of MSCs, and are negative for CD34, CD45, and CD117 (c-kit), which are markers of hematopoietic stem cells isolated from bone marrow or UCB. The properties of PDSCs are similar to other MSCs isolated from some adult tissues. In particular, PDSCs have several advantages such as the earliest stage of adult stem cells, easiness to harvest cells without invasive procedures, their activities for self-renewal and multi-potent, and immunomodulatory properties (Li et al., 2007). In addition, PDSCs contains a variety of localization-speciﬁc MSCs (e.g., AE-MSCs, CV-MSCs and CP-MSCs) in placental tissues (Parolini et al., 2008). We reported previously that CP-MSCs isolated from human term placenta have the potential for self-renewal and hepatogenic differentiation, and CP-MSCs have therapeutic effects in CCl4-injured liver rat models through an anti-ﬁbrotic effect due to the expression of MMP-9 (Lee et al., 2010). Since CP-MSCs and other MSCs share several stem cell properties, despite their different origins, these progenitor cells may possess an equivalent hepatogenic differentiation potential. However, their potential for hepatogenic differentiation was controversial because hepatocytes originated from an endodermal lineage and hepatogenic development progresses through a tightly regulated program of expression of transcriptional factors and cytokines in the stages of hepatocyte differentiation (Snykers et al., 2009). Therefore, it is important to use hepatocyte lineage-speciﬁc markers in representing stages in the potential sequence of molecular events of hepatocyte differentiation (Schwartz et al., 2002; Lee et al., 2004a). We used a two-step differentiation protocol with sequential addition of bFGF, the cytokines OSM and HGF, and hormones (dexamethasone and insulin), which have been reported to be involved in the development and differentiation of hepatocytes (Talens-Visconti et al., 2006). As previously mentioned, HGF plays an essential role in the development and regeneration of the liver, bFGF is required to induce a hepatogenic fate in the foregut endoderm, and OSM increases hepatocyte size and hepatocyte maturation. The morphologic and phenotypic features and gene expression changes in all types of MSCs were compared (Fig. 3a). Before hepatogenic differentiation, MSCs have their distinct morphology, but the morphology of the cells induced to become hepatocytes is polygonal in shape. To identify the efﬁciency of hepatogenic differentiation, the levels of gene expression of hepatogenic markers were determined by RT-PCR because their expression is primarily regulated at the transcriptional level. The expression of albumin, the abundant protein synthesized by mature hepatocytes, starts in early fetal hepatocytes and reaches the maximal level in adult hepatocytes. CYP2B6 (Cytochrome P450 2B6) is also considered speciﬁc to functional hepatocytes. TAT (tyrosine aminotransferase) is known to be a late marker of the hepatocyte lineage, but the level of TAT expression was only detected in PDSCs, not other stem cells (Fig. 3b). To conﬁrm the functional activity of hepatogenic differentiated MSCs, HGF secretion was measured at the ﬁnal step of differentiation. HGF highly secretes in PDSCs including AE-MSC, CP-MSC, CV-MSC, and WJ-MSC after hepatogenic differentiation, otherwise, AD-MSC, BM-MSC and UCB-MSC did not secrete HGF (Fig. 5a). These results indirectly suggest that PDSCs have a greater potential than other stem cells to differentiate into hepatocytes. Interestingly, the expression of SCF in CP-MSCs differentiated into hepatocytes dramatically increased (Fig. 5b). SCF is a hematopoietic factor, inducing leukocyte maturation and differentiation (Galli et al., 1994). In a previous report, SCF was shown to play a role in the liver’s recovery from a toxic injury, speciﬁcally an acetaminophen
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(APAP)-induced hepatogenic injury (Simpson et al., 2003). Administration of exogenous SCF reduced mortality in APAP-treated mice, increased hepatocyte proliferation, and prevented hepatocyte apoptosis induced by APAP (Hu and Colletti (2008)). On the basis of these previous reports, we thought that SCF might play a role hepatogenic differentiation as an important factor for hepatogenesis. In particular, increased SCF in CP-MSCs after hepatogenic differentiation indicates that CP-MSCs, out of several types of adult stem cells, have the best capacity for hepatogenic differentiation. In summary, various MSCs derived from adult tissues are able to differentiate into functional hepatocyte-like cells in vitro regardless of their different origins. PDSCs have hepatogenic differentiation potential, and have a longer culture period and a higher proliferation capacity than other stem cells. Therefore, PDSCs could be used as a stem cell source for cell therapy in liver diseases.
Acknowledgments We specially thank Professor Jonh-Hyul Seong (CHA University, Seoul, Korea) and Professor Young Soo Choi (CHA University, Seoul, Korea) for donating adipose-derived mesenchymal stem cells (AD-MSCs) and umbilical cord blood-mesenchymal stem cells (UCB-MSCs), respectively. This work was supported by a grant from Korea Food & Drug Administration in 2012 (10172KFDA993) and the Korea Research Foundation Grant funded by the Korean Government (MEST) (KRF-2008-313-E00247). References Anderson, D.J., Gage, F.H, Weissman, I.L., 2001. Can stem cells cross lineage boundaries? Nature Medicine 7, 393–395. Banas, A., Teratani, T., Yamamoto, Y., Tokuhara, M., Takeshita, F., Osaki, M., Kawamata, M., Kato, T., Okochi, H., Ochiya, T., 2008. IFATS collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury. Stem Cells 26, 2705–2712. Barzilay, R., Sadan, O., Melamed, E., Offen, D., 2009. Comparative characterization of bone marrow-derived mesenchymal stromal cells from four different rat strains. Cytotherapy 11, 435–442. Bianco, P., Robey, P.G., Simmons, P.J., 2008. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2, 313–319. Bobis, S., Jarocha, D., Majka, M., 2006. Mesenchymal stem cells: characteristics and clinical applications. Folia Histochemica et Cytobiologica 44, 215–230. Cai, J., Zhao, Y., Liu, Y., Ye, F., Song, Z., Qin, H., Meng, S., Chen, Y., Zhou, R., Song, X., et al., 2007. Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology 45, 1229–1239. Cantz, T., Manns, M.P., Ott, M., 2008. Stem cells in liver regeneration and therapy. Cell and Tissue Research 331, 271–282. Elaut, G., Henkens, T., Papeleu, P., Snykers, S., Vinken, M., Vanhaecke, T., Rogiers, V., 2006. Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. Current Drug Metabolism 7, 629–660. Espejel, S., Roll, G.R., McLaughlin, K.J., Lee, A.Y., Zhang, J.Y., Laird, D.J., Okita, K., Yamanaka, S., Willenbring, H., 2010. Induced pluripotent stem cell-derived hepatocytes have the functional and proliferative capabilities needed for liver regeneration in mice. Journal of Clinical Investigation 120, 3120–3126. Galli, S.J., Zsebo, K.M., Geissler, E.N., 1994. The kit ligand, stem cell factor. Advances in Immunology 55, 1–96. Gallicano, G.I., Mishra, L., 2010. Hepatocytes from induced pluripotent stem cells: a giant leap forward for hepatology. Hepatology 51, 20–22. Gimble, J.M., Katz, A.J., Bunnell, B.A., 2007. Adipose-derived stem cells for regenerative medicine. Circulation Research 100, 1249–1260. Greco, S.J., Liu, K., Rameshwar, P., 2007. Functional similarities among genes regulated by OCT4 in human mesenchymal and embryonic stem cells. Stem Cells 25, 3143–3154. Hay, D.C., Fletcher, J., Payne, C., Terrace, J.D., Gallagher, R.C., Snoeys, J., Black, J.R., Wojtacha, D., Samuel, K., Hannoun, Z., et al., 2008. Highly efﬁcient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proceedings of the National Academy of Sciences USA 105, 12301–12306. Haydon, G.H., Neuberger, J., 2000. Liver transplantation of patients in end-stage cirrhosis. Baillieres Best Practice and Research Clinical Gastroenterology 14, 1049–1073. Hu, B., Colletti, L.M., 2008. Stem cell factor and c-kit are involved in hepatic recovery after acetaminophen-induced liver injury in mice. American Journal of Physiology Gastrointestinal and Liver Physiology 295, G45–G53.
Igura, K., Zhang, X., Takahashi, K., Mitsuru, A., Yamaguchi, S., Takashi, T.A., 2004. Isolation and characterization of mesenchymal progenitor cells from chorionic villi of human placenta. Cytotherapy 6, 543–553. Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz-Gonzalez, X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., et al., 2002. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41–49. Kim, M.J., Shin, K.S., Jeon, J.H., Lee, D.R., Shim, S.H., Kim, J.K., Cha, D.H., Yoon, T.K, Kim, G.J., 2011. Human chorionic-plate-derived mesenchymal stem cells and Wharton’s jelly-derived mesenchymal stem cells: a comparative analysis of their potential as placenta-derived stem cells. Cell and Tissue Research 346, 53–64. Krause, D.S., Theise, N.D., Collector, M.I., Henegariu, O., Hwang, S., Gardner, R., Neutzel, S., Sharkis, S.J., 2001. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 105, 369–377. Lee, K.D., Kuo, T.K., Whang-Peng, J., Chung, Y.F., Lin, C.T., Chou, S.H., Chen, J.R., Chen, Y.P, Lee, O.K., 2004a. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 40, 1275–1284. Lee, M.J., Jung, J., Na, K.H., Moon, J.S., Lee, H.J., Kim, J.H., Kim, G.I., Kwon, S.W., Hwang, S.G, Kim, G.J., 2010. Anti-ﬁbrotic effect of chorionic plate-derived mesenchymal stem cells isolated from human placenta in a rat model of CCl(4)-injured liver: potential application to the treatment of hepatic diseases. Journal of Cellular Biochemistry 111, 1453–1463. Lee, O.K., Kuo, T.K., Chen, W.M., Lee, K.D., Hsieh, S.L., Chen, T.H., 2004b. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 103, 1669–1675. Li, C., Zhang, W., Jiang, X., Mao, N., 2007. Human-placenta-derived mesenchymal stem cells inhibit proliferation and function of allogeneic immune cells. Cell and Tissue Research 330, 437–446. Moon, Y.J., Lee, M.W., Yoon, H.H., Yang, M.S., Jang, I.K., Lee, J.E., Kim, H.E., Eom, Y.W., Park, J.S., Kim, H.C., et al., 2008. Hepatic differentiation of cord bloodderived multipotent progenitor cells (MPCs) in vitro. Cell Biology International 32, 1293–1301. Parashurama, N., Nahmias, Y., Cho, C.H., van Poll, D., Tilles, A.W., Berthiaume, F., Yarmush, M.L., 2008. Activin alters the kinetics of endoderm induction in embryonic stem cells cultured on collagen gels. Stem Cells 26, 474–484. Parker, A.M., Katz, A.J., 2006. Adipose-derived stem cells for the regeneration of damaged tissues. Expert Opinion on Biological Therapy 6, 567–578. Parolini, O., Alviano, F., Bagnara, G.P., Bilic, G., Buhring, H.J., Evangelista, M., Hennerbichler, S., Liu, B., Magatti, M., Mao, N., et al., 2008. Concise review: isolation and characterization of cells from human term placenta: outcome of the ﬁrst international workshop on placenta derived stem cells. Stem Cells 26, 300–311. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147. Rao, M.S., Mattson, M.P., 2001. Stem cells and aging: expanding the possibilities. Mechanisms of Ageing and Development 122, 713–734. Sancho-Bru, P., Roelandt, P., Narain, N., Pauwelyn, K., Notelaers, T., Shimizu, T., Ott, M., Verfaillie, C., 2011. Directed differentiation of murine-induced pluripotent stem cells to functional hepatocyte-like cells. Journal of Hepatology 54, 98–107. Schwartz, R.E., Reyes, M., Koodie, L., Jiang, Y., Blackstad, M., Lund, T., Lenvik, T., Johnson, S., Hu, W.S., Verfaillie, C.M., 2002. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. Journal of Clinical Investigation 109, 1291–1302. Shi, X.L., Zhang, Y., Gu, J.Y., Ding, Y.T., 2009. Coencapsulation of hepatocytes with bone marrow mesenchymal stem cells improves hepatocyte-speciﬁc functions. Transplantation 88, 1178–1185. Shin, K.S., Lee, H.J., Jung, J., Cha, D.H., Kim, G.J., 2010. Culture and in vitro hepatogenic differentiation of placenta-derived stem cells, using placental extract as an alternative to serum. Cell Proliferation 43, 435–444. Simpson, K., Hogaboam, C.M., Kunkel, S.L., Harrison, D.J., Bone-Larson, C., Lukacs, N.W., 2003. Stem cell factor attenuates liver damage in a murine model of acetaminophen-induced hepatic injury. Laboratory Investigation 83, 199–206. Snykers, S., De Kock, J., Rogiers, V., Vanhaecke, T., 2009. In vitro differentiation of embryonic and adult stem cells into hepatocytes: state of the art. Stem Cells 27, 577–605. Snykers, S., Vanhaecke, T., Papeleu, P., Luttun, A., Jiang, Y., Vander Heyden, Y., Verfaillie, C., Rogiers, V., 2006. Sequential exposure to cytokines reﬂecting embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells. Toxicological Sciences 94, 330–341, discussion 235–339. Talens-Visconti, R., Bonora, A., Jover, R., Mirabet, V., Carbonell, F., Castell, J.V., Gomez-Lechon, M.J., 2006. Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells. World Journal of Gastroenterology 12, 5834–5845. Terry, C., Hughes, R.D., Mitry, R.R., Lehec, S.C., Dhawan, A., 2007. Cryopreservationinduced nonattachment of human hepatocytes: role of adhesion molecules. Cell Transplantation 16, 639–647. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., Jones, J.M., 1998. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147. Troyer, D.L., Weiss, M.L., 2008. Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells 26, 591–599. Zaret, K.S., Grompe, M., 2008. Generation and regeneration of cells of the liver and pancreas. Science 322, 1490–1494.