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Hepatic differentiation of cord blood-derived multipotent progenitor cells (MPCs) in vitro
Cell Biology International 32 (2008) 1293e1301 www.elsevier.com/locate/cellbi
Hepatic differentiation of cord blood-derived multipotent progenitor cells (MPCs) in vitro Young Joon Moon a,b, Myoung Woo Lee b, Hee Hoon Yoon c, Mal Sook Yang b, In Keun Jang b, Jong Eun Lee b, Hyo Eun Kim b, Young-woo Eom b, Joon Seong Park d, Hugh C. Kim d, Young Jin Kim b, Kwang-Ho Lee a,* a
Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, Republic of Korea b Biomedical Research Institute, LifeCord Inc., Suwon, Republic of Korea c Department of Chemical and Biochemical Engineering, College of Engineering, Dongguk University, Seoul, Republic of Korea d Department of Hematology-Oncology, Ajou University School of Medicine, Suwon, Republic of Korea Received 4 March 2008; revised 22 April 2008; accepted 15 July 2008
Abstract Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells that possesses practical and ethical advantages. We previously reported a novel UCB-derived adult stem cells which we termed umbilical cord blood-derived multipotent progenitor cells’ (MPCs). MPCs were capable of differentiating into functional neuronal cells. Under appropriate conditions lasting several days or weeks, we now show that the MPCs differentiate into hepatocyte-like cells in vitro; their properties were verified using reverse transcription-polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, periodic acid-Schiff (PAS) staining of accumulated glycogen and an enzyme-linked immunosorbent assay (ELISA). We also found that hepatic differentiated cells expressed hepatocyte specific markers, such as albumin, hepatocyte nuclear factor (HNF)-1a, HNF4, cytokeratin (CK)-8, CK-18, tyrosine amino transferase (TAT), and CYP2B6. Moreover, albumin was secreted, which suggests that MPCs from UCB possess multi-differentiation potential and have the capacity to differentiate into functional cells of hepatic lineage in vitro. Ó 2008 Published by Elsevier Ltd on behalf of International Federation for Cell Biology. Keywords: Umbilical cord blood; Multipotent progenitor cells; Hepatic differentiation; Adult stem cells; Hepatocytes
1. Introduction Stem cells have the capacity to self-replicate and produce progeny that can differentiate into one or several specific cell types. Two types of stem cells have been identified: embryonic stem (ES) cells, found in the inner cell mass of the early embryo; and adult tissue specific stem cells. Adult stem cells have been observed in various adult tissues, including bone marrow (BM) (Bianco et al., 1999), umbilical cord blood (UCB) (Erices et al., 2000), peripheral blood (PB) (Domen * Corresponding author. Department of Life Science, College of Natural Science, Chung-Ang University, 221 Heuksuk-Dong, Dongjak-Ku, Seoul 156756, Republic of Korea. Tel.: þ82 2 820 5213; fax: þ82 2 824 9368. E-mail address: [email protected] (K.-H. Lee).
and Weissman, 1999), skeletal muscles (Seale and Rudnicki, 2000), liver (Sell, 1990), and the brain (Davis and Temple, 1994). These cells, which can propagate in large quantities and differentiate into various tissue cell types, could serve as a highly valuable resource for the development of cellular therapies (Kuehnle and Goodell, 2002). Moreover, since the first transplantation was performed in 1988 for a child with Fanconi’s anemia, UCB transplantation has become a safe and accepted mode of transplantation for recipients due to the low incidence of severe graft-versus-host-disease (GVHD), whereas adult BM transplantation requires exact histocompatibility between donors and recipients (Li et al., 2004). UCB is widely accepted as a rich source of hematopoietic stem cells (HSCs) possessing many practical and ethical advantages; they also have a higher proliferative potential that is associated with an
1065-6995/$ - see front matter Ó 2008 Published by Elsevier Ltd on behalf of International Federation for Cell Biology. doi:10.1016/j.cellbi.2008.07.017
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extended life span and longer telomeres. UCB also contains other types of stem cells, such as mesenchymal stem cells (MSCs), which have the ability to differentiate into various types of cells including osteocytes, chondrocytes, adipocytes, muscle cells and neuronal cells, given appropriate conditions; in consequence, UCB could be used to repair and maintain damaged cells in the body (Erices et al., 2000; Lee et al., 2004b). This restorative phenomenon has been described as adult stem cell plasticity and, hence UCB may play a key role in the development of novel stem cell therapies applicable in various organ failures. At present, liver transplantation is the main therapeutic procedure for patients with acute or chronic end stage liver disease. However, there is limited availability of livers for transplantation and heavy the requirement for immunosuppression. Both bioartificial liver and hepatocyte transplantation are means of temporary liver support that can provide some of the necessary functional support (Malhi and Gupta, 2001; Suh et al., 1999). Hepatocyte transplantation has been the alternative option for bridging patients to whole organ transplantation in cases of acute liver failure and for the treatment of metabolic liver disease. Functional hepatocytes are at the core of temporary support, but their low availability restricts their use in transplantation. Thus, stem cells could be ideal resources for functional hepatocytes addressing this problem. Several types of adult stem cells from BM, UCB and adipose tissue can differentiate into hepatocyte-like cells in vitro and/or in vivo (Table 1). We previously reported novel UCB-derived adult stem cells, termed ‘umbilical cord blood-derived multipotent progenitor cells’ (MPCs), which are capable of differentiating into functional neuronal cells (Lee et al., 2007). Here, we will show that cryopreserved UCB-derived MPCs have the intrinsic and direct potential to differentiate into a hepatic cell lineage under appropriate induction conditions. The results indicate that MPCs have trans-differentiation capacity, and moreover, that cryopreserved UCB can be an alternative source of cells capable of hepatic differentiation, potentially extending the
Table 1 Adult stem cells from various sources Cell types
Sources
References
MNCs
BM, CB
HSCs
BM, CB
MSCs
BM, CB
CBEs PCMO MAPCs ASCs
CB Peripheral blood BM Adipose tissue
BM: Petersen et al., 1998 CB: Tang et al., 2006; Sharma et al., 2005 BM: Fiegel et al., 2003; Wang et al., 2003 CB: Kakinuma et al., 2003 BM: Lee et al., 2004 CB: Kang et al., 2006; Hong et al., 2005 McGuckin et al., 2005 Ruhnke et al., 2005 Schwartz et al., 2002 Seo et al., 2005
Abbreviations: ASCs: adipose-tissue derived stem cells; AT: adipose tissue; BM: bone marrow; UCB: umbilical cord blood; CBEs: cord blood-derived embryonic-like stem cells; HSCs: hematopoietic stem cells; MAPCs: multipotent adult progenitor cells; MNCs: mononuclear cells; MSCs: mesenchymal stem cells; PB: peripheral blood; PCMO: programmable cells of monocytic origin.
use of UCB to the treatment of hepatic diseases as well as motivation for studies on the differentiation of cells into the hepatocyte lineage. 2. Materials and methods 2.1. Cell culture and hepatic differentiation The study was approved by the Institutional Review Board of Ajou University Hospital, and all samples were obtained with informed consent. The MPCs were prepared from cryopreserved UCB units as described in Lee et al. (2007). Briefly, thawed UCB-derived mononuclear cells (MNCs), used for culture with no further separation steps, were seeded on noncoated T25 flasks (Nalge Nunc, USA) at 3 105 cells/cm2 in a high glucose Dulbecco’s modified Eagles medium (HGDMEM; Gibco BRL, USA) containing 10% fetal bovine serum (FBS; Gibco BRL, USA), 100 U penicillin/streptomycin (Gibco BRL, USA), 100 ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF; R&D systems, USA), and 2 mM L-glutamine (Gibco BRL, USA). Cells were incubated in a humidified atmosphere at 37 C with 5% CO2 in air, the medium being changed every 7 days for 2e4 weeks. Next, adherent cells were gently resuspended with 0.05% trypsinEDTA (Gibco BRL, USA) and reseeded at 1 106 cells per flask. For hepatogenic differentiation, 1 106 cells of MPCs were plated in a 35 mm MatrigelÔ (BD biosciences, USA) coated culture dish and incubated for 3 days. Subsequently, culture medium was replaced with hepatic differentiation medium. The medium used for hepatic differentiation were HG-DMEM (Gibco BRL, USA) containing 10 ng/ml fibroblast growth factor-4 (FGF-4; R&D systems, USA), 25 ng/ml hepatocyte growth factor (HGF; R&D systems, USA), 10 ng/ ml oncostatin M (OSM; R&D systems, USA), 10 ng/ml epidermal growth factor (EGF; R&D systems, USA) and 10 ng/ml leukemia inhibitory factor (LIF; R&D systems, USA) without FBS. The cells were cultured at 37 C under 5% CO2 in air in a hepatic differentiation medium for 1e3 weeks. All experiments were performed with >5 units of UCB samples. 2.2. RT-PCR analysis Total RNA was extracted from cells using the TRIzol Reagent (Gibco BRL, USA). A total of 2 mg of RNA was reverse-transcribed with AMV reverse transcriptase XL (TaKaRa Shuzo, Japan) for 90 min at 42 C in the presence of oligo-dT primer. PCR was performed using Taq polymerase (BioQuest, Korea) and the primer sequences are given in Table 2. The following conditions were employed for amplification: 35 cycles consisting of a 4-min presoak at 94 C, denaturation for 30 s at 94 C, annealing for 30 s at 55e60 C, and extension for 1 min at 72 C, with an additional 7-min incubation at 72 C after completion of the cycle. Amplified cDNA fragments were electrophoresed on a 2% agarose gel, stained with
Y.J. Moon et al. / Cell Biology International 32 (2008) 1293e1301 Table 2 RT-PCR primers for the detection of hepatic lineage specific mRNA Primer HNF1a (Tokiwa et al., 2006) CK8 (Okumoto et al., 2003) Albumin (Schwartz et al., 2002) TAT (Lee et al., 2004c) HNF4 (Lee et al., 2004a) CYP2B6 (Lee et al., 2004a) GAPDH
Sequences
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Forward; 5 -ttctaagctgagccagctgcagacg-3 Reverse; 50 -gctgaggttctccggctctttcaga-30 Forward; 50 -caatgccaagctggaggatc-30 Reverse; 50 -acctcaggctggcaatgact-30 Forward; 50 -tgcttgaatgtgctgatgacaggg-30 Reverse; 50 -aaggcaagtcagcaggcatctcatc-30 Forward; 50 -tgagcagtctgtccactgcct-30 Reverse; 50 -atgtgaatgaggaggatctgag-30 Forward; 50 -ccaagtacatcccagctttc-30 Reverse; 50 -ttggcatctgggtcaaag-30 Forward; 50 -gacgctacgtttcagtctttc-30 Reverse; 50 -gctgaataccacgccatag-30 Forward: 50 -atcaccatcttccaggagcg-30 Reverse: 50 -gttcttcc-accacttcgtcc-30
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temperature for 10 min, and blocked with 10% normal goat or rabbit serum (Zymed, USA) at room temperature for 30 min. They were incubated with primary antibody specific for human albumin (1:100; Bethyl, USA), alpha-1-antitrypsin (AAT; 1:100; DAKO cytomation, Denmark) and human hepatocyte marker (HepPar1; 1:50; DAKO cytomation, Denmark) at 37 C for 1 h. For fluorescence labeling, cells were incubated with FITC-conjugated secondary antibodies (1:100, Santa Cruz Biotech) at 37 C for 1 h after primary incubation. The cells were also stained with 1 mg/ml 40 , 6-diamino-2phenylindole (DAPI, Sigma, USA) to visualize their nuclei. The slides were observed and photographed under a fluorescent microscope (IX-71, Olympus, Japan).
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ethidium bromide, and photographed under an ultraviolet light transilluminator (BioRad, USA). 2.3. Western blotting Cells were washed with cold DPBS and lysed in 300 mL of cold RIPA buffer (50 mM TriseHCl, pH 7.5, containing 1% Triton X-100, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), and 1% sodium deoxycholate) with a protease inhibitor cocktail (Sigma, USA). Cell lysate was centrifuged at 13,000 g for 10 min at 4 C. The supernatant was harvested, and its protein concentration measured using a protein assay kit (Bio-Rad, USA). For electrophoresis, 50 mg protein was dissolved in sample buffer (60 mM TriseHCl, pH 6.8, containing 14.4 mM b-mercaptoethanol, 25% glycerol, 2% SDS, and 0.1% bromophenol blue), boiled for 5 min, and separated on a 10% SDS reducing gel. Separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, UK) using a trans-blot system (Bio-Rad, USA). Blots were blocked for 1 h in Tris-buffered saline (TBS) (10 mM TriseHCl, pH 7.5, with 150 mM NaCl) containing 5% non-fat dry milk (Bio-Rad, USA) at room temperature, washed three times with TBS, and incubated at 4 C overnight with primary antibody specific for human albumin (1:1000 dilution; Bethyl, USA) in TBST (10 mM Tris, pH 7.5, containing 150 mM NaCl and 0.02% Tween 20) containing 3% non-fat dry milk. The next day, blots were washed three times with TBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution; Santa Cruz Biotech, USA) in TBST containing 3% non-fat dry milk at room temperature. After being washed three times with TBST, the proteins were visualized with an ECL detection system (Amersham Pharmacia Biotech, UK).
2.5. Periodic acid-Schiff (PAS) staining for glycogen 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, USA) for 15 min, and rinsed in dH2O for 5e10 min. Samples were counterstained with Mayer’s hematoxylin for 1 min and rinsed in dH2O and assessed under a light microscope. 2.6. Enzyme-linked immunosorbent assay (ELISA) Differentiation media at various time-points in hepatic differentiation, and the media from various differentiation conditions, were sampled for albumin ELISA. The concentrations of albumin in supernatant were determined in plasma samples by ELISA kit according to the manufacturer’s instructions (Koma biotech, Korea). Briefly, these assays employed the quantitative sandwich enzyme immunoassay technique with monoclonal antibodies specific for albumin precoated on a microplate. Standard controls and samples (100 mL of supernatant) were pipetted into the wells in duplicate. After albumin binding and washing, an enzyme-linked polyclonal antibody specific for human albumin was added to each well. After thorough washing, a substrate solution was added to the wells and color developed in proportion to the amount of albumin bound in the first step. The optical density of each well was determined by a microplate reader at 450 nm. ELISA was performed with the samples from 5 units of umbilical cord blood. 2.7. Statistical analysis All results are expressed as means standard deviation (SD). P < 0.05 was considered significant. Statistical data were calculated with SigmaPlotÔ. 3. Results
2.4. Immunofluorescence analysis Cells were cultured in 2-chamber Lab-Tek slides (Nalge Nunc International, USA) for 7 days. For immunofluorescence staining, cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS at room
3.1. Morphological change of MPCs’ in vitro differentiation in conditioned medium MPCs were plated at 3 104 cells/cm2 on culture dishes and cultured for 3 days. Subsequently, the culture medium was
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replaced with hepatic differentiation medium containing FGF4, HGF, OSM, EGF and LIF. After treatment with hepatocyteconditioned medium, the spindle-shaped morphology of MPCs was lost and most cells displayed oval and round morphology, resembling hepatoblasts and hepatic oval cells 2 weeks post-induction (Fig. 1A).
3.2. Detection of hepatocyte specific marker expressions in MPCs The time-dependence of hepatocyte specific marker expression was examined (Fig. 1B). Hepatic differentiation of MPCs was confirmed in a time-dependent manner by RT-PCR
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Fig. 1. Differentiation of MPCs into hepatic lineage and expression of their hepatocyte specific markers. (A) MPCs were grown for 0, 3 and 14 days in induction medium. Images were viewed using an inverted microscope at 200 magnification. Scale bar ¼ 100 mm. (B) Total RNAs were analyzed by RT-PCR for mRNA expression of human albumin (ALB), TAT, HNF1a, CK-8, HNF4 and GAPDH. cDNA prepared from the total RNAs of hepaticinduced (lanes 3e8) and non-induced (lane 2) MPCs served as the input in PCR reactions containing primers corresponding to hepatic lineage specific differentiation markers. M: size marker; lane 1: HepG2; lane 2: non-induced; lane 3: 1 day; lane 4: 3 days; lane 5: 7 days; lane 6: 14 days; lane 7: 21 days; lane 8: 28 days; lane 9: negative control. Expression of cytochrome P450 was also examined. M: size marker, lane 1: hepatic-induced for 2 weeks; lane 2: non-induced; lane 3: negative control; (C) Expression levels of the important hepatocyte specific marker albumin, were detected by western blotting. The expression levels of b-actin protein were used as the internal control. The lanes contain proteins that have been extracted from cells before induction (lane 1: day 0), after 1 (lane 2), 3 (lane 3), 7 (lane 4), 14 (lane 5) and 21 (lane 6) days in induction medium, and a positive control (lane C: a hepatoma cell line, HepG2).
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for common markers of hepatocyte differentiation. The expression of albumin, cytokeratin-8 (CK-8) and hepatocyte nuclear factor 1a (HNF1a) was readily detectable in hepatic differentiated MPCs with conditioned differentiation medium for 1 day, while hepatocyte nuclear factor 4 (HNF4) and tyrosineamino transferase (TAT) were detected at 3 and 7 postinduction. Albumin expression was detected at all time-points except in the undifferentiated cells, and was maintained throughout the differentiation process. CK-8 expression was gradually enhanced from day 1 to week 1, but rapidly decreased thereafter. HNF1 a expression was detected only from day 1 to week 1, after which it gradually disappeared. HNF4, a transcription factor largely expressed in the adult liver, was detected from day 3 to 2 weeks post-induction. In addition, RT-PCR analysis showed that the expression of the P450 gene (CYP2B6), known as a functional protein related to detoxification activity of mature hepatocyte, was detectable by 2 weeks post-induction. The protein expression of albumin in the differentiated cells was also analyzed through Western blot (WB) and immunofluorescence (IF) staining with the use of appropriate monoclonal antibody. The albumin band was detected on day 1 of the differentiation period and during the maturation step; the band intensity became further enhanced until 3 weeks postdifferentiation (Fig. 1C). To further confirm the expression of hepatocyte specific proteins in hepatic differentiated MPCs, cells were fixed and IF-stained for hepatic lineage specific markers. They expressed hepatic lineage specific markers such as human albumin, human hepatocyte marker (HepPar1) and alpha-1antitrypsin (AAT) (Fig. 2). The results of the protein analysis were in agreement with the RT-PCR results. We attempted to select exclusively MPC colonies that had differentiated into hepatic lineages, which were later confirmed to possess hepatic differentiation capacity. The MPC colonies were produced by the low density seeding of UCB-MNCs (2 105 cells/cm2) into culture chamber slides, followed by the culture method of UCB-MPCs. After their formation, colonies of MPCs were hepatic-induced 1 week and IF stained for hepatocyte specific markers. Differentiated colonies of MPCs were positively stained for hepatocyte specific markers, including albumin, HepPar1 and AAT (Fig. 3). 3.3. Function of hepatic differentiated MPCs and FGF-4, key-cytokine of hepatogenesis Intracellular glycogen accumulation is a feature of adult liver cells. Hepatic differentiation of MPCs was analyzed by staining them with the periodic acid-Schiff (PAS) reagent. Glycogen accumulation was detected in the hepatic differentiated cells, whereas it did not do so in the undifferentiated control (Fig. 4). Albumin is the major plasma protein secreted by hepatocytes (Dufour et al., 2000). Albumin production of hepatic differentiated MPCs was measured at various time-points throughout the differentiating process by ELISA. Hepatic
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differentiated MPCs produced albumin in a time-dependent manner from day 1 (120.14 33.28 ng/ml) and the secretion was maintained until day 28 (239.53 31.26 ng/ml), whereas undifferentiated MPCs did not secrete albumin (Fig. 5A). We also examined the influence of each cytokine to determine their function in MPC hepatic differentiation. In all, 11 combinations of cytokines were tested with MPCs isolated from five donors. Combination of cytokines lacking FGF-4 did not induce secretion of albumin in MPCs (Fig. 5B), whereas only the combination of cytokines and FGF-4 induced the secretion of albumin in MPCs. In addition, Matrigel coating of the culture dishes gave no significant difference in the secretion of albumin compared with uncoated culture dishes. These findings indicate that FGF-4 is essential for the differentiation of MPCs into albumin-secreting cells. 4. Discussion Potential hepatocyte progenitor cells have been identified from BM, peripheral blood, UCB, fetal liver, adult liver and ES cells (Sharma et al., 2005). Recently, classical MSCs derived from BM could differentiate into hepatocyte-like cells under in vitro conditions, with comprehensive phenotype and functional characteristics of liver cells (Lee et al., 2004a). Although the approach shares similarities with that for the isolation of MSCs and other adult stem cells, cell phenotypes and characteristics of hepatic differentiated cells established for novel MPCs are different for those of other cells. The expression profiles of HNF1a, HNF4, CK-8, TAT and albumin mRNA show that MPCs had differentiated into cells of hepatocyte lineage (Fig. 1). On day 0, no hepatocyte markers were detected; however on day 1, the mRNAs of albumin and CK 8 were detected and gradually enhanced for 1 week. The mRNA of HNF1a was detected from day 1 but disappeared 2 weeks after differentiation and CK-8 had decreased after 1 week. But, the mRNA of albumin maintained for 4 weeks of differentiation, and this result coincided with that from the western blot, immunostaining and ELISA. The mRNA of HNF4 and TAT, which are late markers of hepatocyte lineage, were detected on day 3 and at 1 week, respectively, and maintained for 4 weeks. Albumin, the abundant protein synthesized by mature hepatocytes, starts in early fetal hepatocytes and reaches the maximal level in adult hepatocytes (Pan et al., 1998). HNF4 is a transcription factor largely expressed in adult liver (Sladek et al., 1990). TAT is known to be a late marker of hepatocyte lineage and excellent enzymatic marker for peri- or postnatal hepatocyte specific differentiation (Shelly et al., 1989). The CYP2B6 in man is considered specific for functional hepatocytes (Schwartz et al., 2002). Immunofluorescence (IF) analysis also showed that hepatic differentiated MPCs expressed hepatic lineage specific markers. Anti-human hepatocyte monoclonal antibody (HepPar1) reacts with only human hepatocytes and human bile duct cells; alpha-1-antitrypsin (AAT) is a glycoprotein mainly synthesized in the liver (Mercer et al., 2001; Perlmutter, 2002). To confirm the functional activity of hepatic differentiated MPCs, albumin secretion was measured at various times after
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Fig. 2. Immunofluorescence staining of human hepatocyte specific markers in differentiated MPCs. After 1 week of hepatic induction, cells were immunofluorescently stained for human albumin (ALB; panel A), hepatocyte marker (HepPar1: panel C) and alpha-1-antitrypsin (AAT: panel E). The cells were positively stained for those markers. Nuclei were stained with DAPI and merged images are indicated in each panel. Images were viewed using a fluorescence microscope at 200 magnification. Panels (A, C, E): hepatic differentiated cells; panels (B, D, F): undifferentiated MPCs. Scale ¼ 400 mm.
the initiation of cell differentiation. Non-induced MPCs did not secrete albumin, whereas differentiated cells in the induction medium did in a time-dependent manner beginning after the first day of differentiation. It took just 1 day of differentiation for the secretion of albumin of hepatic differentiated MPCs, and the phenomenon appeared more rapidly than any other adult stem cell-derived hepatocyte-like cells
reported by other investigators ((Ruhnke et al., 2005); 10 days, (Shi et al., 2005); 3e6 days, (Kang et al., 2006); 8e12 days, (Schwartz et al., 2002); 5 days). This means that the process of MPC hepatic differentiation would be more rapid than that of other previously reported stem cells. But, further investigation is necessary to understand the distinct mechanism of the MPC phenomenon. In addition, the hepatic differentiated MPCs
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Fig. 3. Immunofluorescence staining of human hepatocyte specific markers in differentiated MPC colonies. Colonies of MPCs were produced by seeding a low density of UCB-MNCs (2 105 cells/cm2) into a culture chamber slide, followed by 10 days of culturing. After colony formation, those permitted to differentiate under induction conditions for 1 week and differentiated cells were immunofluorescently stained for human albumin, HepPar1 and AAT. The colonies were positively stained for these markers assuming that the examined colonies differentiated into hepatic lineages. Nuclei were stained with DAPI and merged images are indicated in each panel. Images were viewed using fluorescence microscope at 200 magnification. Scale bar ¼ 100 mm.
were shown to increase the intracellular accumulation of glycogen and the genuine function of mature hepatocytes. Previous reports have shown that FGF, LIF, EGF, HGF and OSM contributed to the proliferation and/or differentiation of hepatic progenitor cells in distinct ways. Interestingly, it became obvious from to our ELISA analysis that FGF-4 was the most important cytokine for the differentiation of MPCs into functional differentiated cells that produce and secrete albumin in vitro. Albumin secretion is considered one of the most important criteria in assessing stem cell differentiation into functional hepatocytes. FGFs secreted by the cardiac mesoderm induce the expression of liver genes such as albumin in the ventral foregut endoderm (Jung et al., 1999), and the FGF response pathway mediates hepatic gene induction in embryonic endodermal cells (Calmont et al., 2006;
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Wells and Melton, 1999). The other growth factors were influenced positively by albumin secretion of UCB-MPCs, though not at a significant level, and further study is needed on this issue. Stem cell populations, which include UCB-MSCs, BMMSCs and MNCs, may be heterogeneous and consist of several subpopulations (Conget et al., 2001; Moon et al., 2005). Thus, MPCs could also comprise a mixture of different cells. We generated colonies of MPCs which differentiated into hepatic lineages in order to avoid possible problems with cell heterogeneity and to confirm the hepatic differentiation capacity of single-cell-derived MPCs colonies. Differentiated colonies of MPCs were positively stained for hepatocyte specific markers such as albumin, HepPar1 and AAT, which suggests that the characteristics of hepatic lineages of differentiated MPCs
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Fig. 4. Periodic acid-Schiff (PAS) staining of hepatic differentiated MPCs. MPCs that differentiated in induction medium for 1 week stained with PAS. (A) The undifferentiated control; (B) hepatic differentiated MPCs. Scale bar ¼ 200 mm.
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Induction conditions Fig. 5. Albumin secretion of hepatic differentiated MPCs in a time-dependent (A) and condition-dependent (B) manner. Quantification of secreted albumin in induction medium was performed by ELISA. (A) Secretion of albumin was detected on day 1 of differentiation; afterwards, the amount of albumin in the culture medium gradually increased over time until day 28 of induction; (B) MPCs were grown for 1 week in induction media containing various mixtures of cytokines to screen for cytokines essential to the hepatic differentiation of the MPCs. Control: undifferentiated MPCs; H: HGF; O: OSM; F: FGF-4; E: EGF; L: LIF; no coat: cells induced without MatrigelÒ coating. The results are expressed as means SD of the measured amount of albumin from five separate experiments (n ¼ 5).
originate from the MPCs themselves, rather than from cell heterogeneity or contamination of other lineages (Fig. 3). In summary, MPCs derived from UCB are able to differentiate into functional hepatocyte-like cells in vitro. The cells were characterized at the gene and protein levels and exhibited hepatocellular synthesis and metabolic functions specific to hepatocytes. Thus, UCB-MPCs can potentially serve as a new cell source for tissue engineering or hepatic cell therapy. References Bianco P, Riminucci M, Kuznetsov S, Robey PG. Multipotential cells in the bone marrow stroma: regulation in the context of organ physiology. Crit Rev Eukaryot Gene Expr 1999;9:159e73. Calmont A, Wandzioch E, Tremblay KD, Minowada G, Kaestner KH, Martin GR, et al. An FGF response pathway that mediates hepatic gene induction in embryonic endoderm cells. Dev Cell 2006;11:339e48. Conget PA, Allers C, Minguell JJ. Identification of a discrete population of human bone marrow-derived mesenchymal cells exhibiting properties of uncommitted progenitors. J Hematother Stem Cell Res 2001;10:749e58. Davis AA, Temple S. A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 1994;372:263e6. Domen J, Weissman IL. Self-renewal, differentiation or death: regulation and manipulation of hematopoietic stem cell fate. Mol Med Today 1999;5:201e8. Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff LB. Diagnosis and monitoring of hepatic injury. I. Performance characteristics of laboratory tests. Clin Chem 2000;46:2027e49. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 2000;109:235e42. Fiegel HC, Lioznov MV, Cortes-Dericks L, Lange C, Kluth D, Fehse B, et al. Liver-specific gene expression in cultured human hematopoietic stem cells. Stem cells 2003;21:98e104. Hong SH, Gang EJ, Jeong JA, Ahn C, Hwang SH, Yang IH, et al. In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells. Biochem Biophys Res Commun 2005;330: 1153e61. Kakinuma S, Tanaka Y, Chinzei R, Watanabe M, Shimizu-Saito K, Hara Y, et al. Human umbilical cord blood as a source of transplantable hepatic progenitor cells. Stem Cells 2003;21:217e27.
Jung J, Zheng M, Goldfarb M, Zaret KS. Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science 1999; 284:1998e2003. Kang XQ, Zang WJ, Bao LJ, Li DL, Xu XL, Yu XJ. Differentiating characterization of human umbilical cord blood-derived mesenchymal stem cells in vitro. Cell Biol Int 2006;30:569e75. Kuehnle I, Goodell MA. The therapeutic potential of stem cells from adults. BMJ 2002;325:372e6. Lee KD, Kuo TK, Whang-Peng J, Chung YF, Lin CT, Chou SH, et al. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 2004a;40:1275e84. Lee MW, Choi J, Yang MS, Moon YJ, Park JS, Kim HC, et al. Mesenchymal stem cells from cryopreserved human umbilical cord blood. Biochem Biophys Res Commun 2004b;320:273e8. Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 2004c;103:1669e75. Lee MW, Moon YJ, Yang MS, Kim SK, Jang IK, Eom YW, et al. Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood. Biochem Biophys Res Commun 2007;358:637e43. Li CK, Shing MM, Chik KW, Tsang KS, Tang NL, Chan PK, et al. Unrelated umbilical cord blood transplantation in children: experience of the Hong Kong Red Cross Blood Transfusion Service. Hong Kong Med J 2004;10: 89e95. Malhi H, Gupta S. Hepatocyte transplantation: new horizons and challenges. J Hepatobiliary Pancreat Surg 2001;8:40e50. McGuckin CP, Forraz N, Baradez MO, Navran S, Zhao J, Urban R, et al. Production of stem cells with embryonic characteristics from human umbilical cord blood. Cell Prolif 2005;38:245e55. Mercer DF, Schiller DE, Elliott JF, Douglas DN, Hao C, Rinfret A, et al. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 2001;7:927e33. Moon YJ, Lee MW, Yang MS, Kim SK, Park JS, Kim HC, et al. Expression profile of genes representing varied spectra of cell lineages in human umbilical cord blood-derived mesenchymal stem cells. Acta Haematol 2005;114:117e20. Okumoto K, Saito T, Hattori E, Ito JI, Adachi T, Takeda T, et al. Differentiation of bone marrow cells into cells that express liver-specific genes in vitro: implication of the Notch signals in differentiation. Biochem Biophys Res Commun 2003;304:691e5. Pan CJ, Lei KJ, Chen H, Ward JM, Chou JY. Ontogeny of the murine glucose6-phosphatase system. Arch Biochem Biophys 1998;358:17e24.
Y.J. Moon et al. / Cell Biology International 32 (2008) 1293e1301 Perlmutter DH. Liver injury in alpha1-antitrypsin deficiency: an aggregated protein induces mitochondrial injury. J Clin Invest 2002;110:1579e83. Petersen BE, Goff JP, Greenberger JS. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 1998;27:433e45. Ruhnke M, Nussler AK, Ungefroren H, Hengstler JG, Kremer B, Hoeckh W, et al. Human monocyte-derived neohepatocytes: a promising alternative to primary human hepatocytes for autologous cell therapy. Transplantation 2005;79:1097e103. Schwartz RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002;109:1291e302. Seale P, Rudnicki MA. A new look at the origin, function, and ‘‘stem-cell’’ status of muscle satellite cells. Dev Biol 2000;218:115e24. Sell S. Is There a liver stem cell? Cancer Res 1990;50:3811e5. Seo MJ, Suh SY, Bae YC, Jung JS. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem Biophys Res Commun 2005;328:258e64. Sharma AD, Cantz T, Richter R, Eckert K, Henschler R, Wilkens L, et al. Human cord blood stem cells generate human cytokeratin 18-negative hepatocyte-like cells in injured mouse liver. Am J Pathol 2005;167:555e64. Shelly LL, Tynan W, Schmid W, Schutz G, Yeoh GC. Hepatocyte differentiation in vitro: initiation of tyrosine aminotransferase expression in cultured fetal rat hepatocytes. J Cell Biol 1989;109:3403e10.
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Shi XL, Qiu YD, Li Q, Xie T, Zhu ZH, Chen LL, et al. Hepatocyte-like cells from directed differentiation of mouse bone marrow cells in vitro. Acta Pharmacol Sin 2005;26:469e76. Sladek FM, Zhong WM, Lai E, Darnell Jr JE. Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev 1990;4:2353e65. Suh KS, Lilja H, Kamohara Y, Eguchi S, Arkadopoulos N, Neuman T, et al. Bioartificial liver treatment in rats with fulminant hepatic failure: effect on DNA-binding activity of liver-enriched and growth-associated transcription factors. J Surg Res 1999;85:243e50. Tang XP, Zhang M, Yang X, Chen LM, Zeng Y. Differentiation of human umbilical cord blood stem cells into hepatocytes in vivo and in vitro. World J Gastroenterol 2006;12:4014e9. Tokiwa T, Yamazaki T, Xin W, Sugae N, Noguchi M, Enosawa S, et al. Differentiation potential of an immortalized non-tumorigenic human liver epithelial cell line as liver progenitor cells. Cell Biol Int 2006;30: 992e8. Wang X, Ge S, McNamara G, Hao QL, Crooks GM, Nolta JA. Albuminexpressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells. Blood 2003;101:4201e8. Wells JM, Melton DA. Vertebrate endoderm development. Annu Rev Cell Dev Biol 1999;15:393e410.
Hepatic differentiation of cord blood-derived multipotent progenitor cells (MPCs) in vitro Young Joon Moon a,b, Myoung Woo Lee b, Hee Hoon Yoon c, Mal Sook Yang b, In Keun Jang b, Jong Eun Lee b, Hyo Eun Kim b, Young-woo Eom b, Joon Seong Park d, Hugh C. Kim d, Young Jin Kim b, Kwang-Ho Lee a,* a
Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, Republic of Korea b Biomedical Research Institute, LifeCord Inc., Suwon, Republic of Korea c Department of Chemical and Biochemical Engineering, College of Engineering, Dongguk University, Seoul, Republic of Korea d Department of Hematology-Oncology, Ajou University School of Medicine, Suwon, Republic of Korea Received 4 March 2008; revised 22 April 2008; accepted 15 July 2008
Abstract Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells that possesses practical and ethical advantages. We previously reported a novel UCB-derived adult stem cells which we termed umbilical cord blood-derived multipotent progenitor cells’ (MPCs). MPCs were capable of differentiating into functional neuronal cells. Under appropriate conditions lasting several days or weeks, we now show that the MPCs differentiate into hepatocyte-like cells in vitro; their properties were verified using reverse transcription-polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, periodic acid-Schiff (PAS) staining of accumulated glycogen and an enzyme-linked immunosorbent assay (ELISA). We also found that hepatic differentiated cells expressed hepatocyte specific markers, such as albumin, hepatocyte nuclear factor (HNF)-1a, HNF4, cytokeratin (CK)-8, CK-18, tyrosine amino transferase (TAT), and CYP2B6. Moreover, albumin was secreted, which suggests that MPCs from UCB possess multi-differentiation potential and have the capacity to differentiate into functional cells of hepatic lineage in vitro. Ó 2008 Published by Elsevier Ltd on behalf of International Federation for Cell Biology. Keywords: Umbilical cord blood; Multipotent progenitor cells; Hepatic differentiation; Adult stem cells; Hepatocytes
1. Introduction Stem cells have the capacity to self-replicate and produce progeny that can differentiate into one or several specific cell types. Two types of stem cells have been identified: embryonic stem (ES) cells, found in the inner cell mass of the early embryo; and adult tissue specific stem cells. Adult stem cells have been observed in various adult tissues, including bone marrow (BM) (Bianco et al., 1999), umbilical cord blood (UCB) (Erices et al., 2000), peripheral blood (PB) (Domen * Corresponding author. Department of Life Science, College of Natural Science, Chung-Ang University, 221 Heuksuk-Dong, Dongjak-Ku, Seoul 156756, Republic of Korea. Tel.: þ82 2 820 5213; fax: þ82 2 824 9368. E-mail address: [email protected] (K.-H. Lee).
and Weissman, 1999), skeletal muscles (Seale and Rudnicki, 2000), liver (Sell, 1990), and the brain (Davis and Temple, 1994). These cells, which can propagate in large quantities and differentiate into various tissue cell types, could serve as a highly valuable resource for the development of cellular therapies (Kuehnle and Goodell, 2002). Moreover, since the first transplantation was performed in 1988 for a child with Fanconi’s anemia, UCB transplantation has become a safe and accepted mode of transplantation for recipients due to the low incidence of severe graft-versus-host-disease (GVHD), whereas adult BM transplantation requires exact histocompatibility between donors and recipients (Li et al., 2004). UCB is widely accepted as a rich source of hematopoietic stem cells (HSCs) possessing many practical and ethical advantages; they also have a higher proliferative potential that is associated with an
1065-6995/$ - see front matter Ó 2008 Published by Elsevier Ltd on behalf of International Federation for Cell Biology. doi:10.1016/j.cellbi.2008.07.017
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extended life span and longer telomeres. UCB also contains other types of stem cells, such as mesenchymal stem cells (MSCs), which have the ability to differentiate into various types of cells including osteocytes, chondrocytes, adipocytes, muscle cells and neuronal cells, given appropriate conditions; in consequence, UCB could be used to repair and maintain damaged cells in the body (Erices et al., 2000; Lee et al., 2004b). This restorative phenomenon has been described as adult stem cell plasticity and, hence UCB may play a key role in the development of novel stem cell therapies applicable in various organ failures. At present, liver transplantation is the main therapeutic procedure for patients with acute or chronic end stage liver disease. However, there is limited availability of livers for transplantation and heavy the requirement for immunosuppression. Both bioartificial liver and hepatocyte transplantation are means of temporary liver support that can provide some of the necessary functional support (Malhi and Gupta, 2001; Suh et al., 1999). Hepatocyte transplantation has been the alternative option for bridging patients to whole organ transplantation in cases of acute liver failure and for the treatment of metabolic liver disease. Functional hepatocytes are at the core of temporary support, but their low availability restricts their use in transplantation. Thus, stem cells could be ideal resources for functional hepatocytes addressing this problem. Several types of adult stem cells from BM, UCB and adipose tissue can differentiate into hepatocyte-like cells in vitro and/or in vivo (Table 1). We previously reported novel UCB-derived adult stem cells, termed ‘umbilical cord blood-derived multipotent progenitor cells’ (MPCs), which are capable of differentiating into functional neuronal cells (Lee et al., 2007). Here, we will show that cryopreserved UCB-derived MPCs have the intrinsic and direct potential to differentiate into a hepatic cell lineage under appropriate induction conditions. The results indicate that MPCs have trans-differentiation capacity, and moreover, that cryopreserved UCB can be an alternative source of cells capable of hepatic differentiation, potentially extending the
Table 1 Adult stem cells from various sources Cell types
Sources
References
MNCs
BM, CB
HSCs
BM, CB
MSCs
BM, CB
CBEs PCMO MAPCs ASCs
CB Peripheral blood BM Adipose tissue
BM: Petersen et al., 1998 CB: Tang et al., 2006; Sharma et al., 2005 BM: Fiegel et al., 2003; Wang et al., 2003 CB: Kakinuma et al., 2003 BM: Lee et al., 2004 CB: Kang et al., 2006; Hong et al., 2005 McGuckin et al., 2005 Ruhnke et al., 2005 Schwartz et al., 2002 Seo et al., 2005
Abbreviations: ASCs: adipose-tissue derived stem cells; AT: adipose tissue; BM: bone marrow; UCB: umbilical cord blood; CBEs: cord blood-derived embryonic-like stem cells; HSCs: hematopoietic stem cells; MAPCs: multipotent adult progenitor cells; MNCs: mononuclear cells; MSCs: mesenchymal stem cells; PB: peripheral blood; PCMO: programmable cells of monocytic origin.
use of UCB to the treatment of hepatic diseases as well as motivation for studies on the differentiation of cells into the hepatocyte lineage. 2. Materials and methods 2.1. Cell culture and hepatic differentiation The study was approved by the Institutional Review Board of Ajou University Hospital, and all samples were obtained with informed consent. The MPCs were prepared from cryopreserved UCB units as described in Lee et al. (2007). Briefly, thawed UCB-derived mononuclear cells (MNCs), used for culture with no further separation steps, were seeded on noncoated T25 flasks (Nalge Nunc, USA) at 3 105 cells/cm2 in a high glucose Dulbecco’s modified Eagles medium (HGDMEM; Gibco BRL, USA) containing 10% fetal bovine serum (FBS; Gibco BRL, USA), 100 U penicillin/streptomycin (Gibco BRL, USA), 100 ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF; R&D systems, USA), and 2 mM L-glutamine (Gibco BRL, USA). Cells were incubated in a humidified atmosphere at 37 C with 5% CO2 in air, the medium being changed every 7 days for 2e4 weeks. Next, adherent cells were gently resuspended with 0.05% trypsinEDTA (Gibco BRL, USA) and reseeded at 1 106 cells per flask. For hepatogenic differentiation, 1 106 cells of MPCs were plated in a 35 mm MatrigelÔ (BD biosciences, USA) coated culture dish and incubated for 3 days. Subsequently, culture medium was replaced with hepatic differentiation medium. The medium used for hepatic differentiation were HG-DMEM (Gibco BRL, USA) containing 10 ng/ml fibroblast growth factor-4 (FGF-4; R&D systems, USA), 25 ng/ml hepatocyte growth factor (HGF; R&D systems, USA), 10 ng/ ml oncostatin M (OSM; R&D systems, USA), 10 ng/ml epidermal growth factor (EGF; R&D systems, USA) and 10 ng/ml leukemia inhibitory factor (LIF; R&D systems, USA) without FBS. The cells were cultured at 37 C under 5% CO2 in air in a hepatic differentiation medium for 1e3 weeks. All experiments were performed with >5 units of UCB samples. 2.2. RT-PCR analysis Total RNA was extracted from cells using the TRIzol Reagent (Gibco BRL, USA). A total of 2 mg of RNA was reverse-transcribed with AMV reverse transcriptase XL (TaKaRa Shuzo, Japan) for 90 min at 42 C in the presence of oligo-dT primer. PCR was performed using Taq polymerase (BioQuest, Korea) and the primer sequences are given in Table 2. The following conditions were employed for amplification: 35 cycles consisting of a 4-min presoak at 94 C, denaturation for 30 s at 94 C, annealing for 30 s at 55e60 C, and extension for 1 min at 72 C, with an additional 7-min incubation at 72 C after completion of the cycle. Amplified cDNA fragments were electrophoresed on a 2% agarose gel, stained with
Y.J. Moon et al. / Cell Biology International 32 (2008) 1293e1301 Table 2 RT-PCR primers for the detection of hepatic lineage specific mRNA Primer HNF1a (Tokiwa et al., 2006) CK8 (Okumoto et al., 2003) Albumin (Schwartz et al., 2002) TAT (Lee et al., 2004c) HNF4 (Lee et al., 2004a) CYP2B6 (Lee et al., 2004a) GAPDH
Sequences
Size (bp) 0
0
Forward; 5 -ttctaagctgagccagctgcagacg-3 Reverse; 50 -gctgaggttctccggctctttcaga-30 Forward; 50 -caatgccaagctggaggatc-30 Reverse; 50 -acctcaggctggcaatgact-30 Forward; 50 -tgcttgaatgtgctgatgacaggg-30 Reverse; 50 -aaggcaagtcagcaggcatctcatc-30 Forward; 50 -tgagcagtctgtccactgcct-30 Reverse; 50 -atgtgaatgaggaggatctgag-30 Forward; 50 -ccaagtacatcccagctttc-30 Reverse; 50 -ttggcatctgggtcaaag-30 Forward; 50 -gacgctacgtttcagtctttc-30 Reverse; 50 -gctgaataccacgccatag-30 Forward: 50 -atcaccatcttccaggagcg-30 Reverse: 50 -gttcttcc-accacttcgtcc-30
274 437 161 358 295 204
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temperature for 10 min, and blocked with 10% normal goat or rabbit serum (Zymed, USA) at room temperature for 30 min. They were incubated with primary antibody specific for human albumin (1:100; Bethyl, USA), alpha-1-antitrypsin (AAT; 1:100; DAKO cytomation, Denmark) and human hepatocyte marker (HepPar1; 1:50; DAKO cytomation, Denmark) at 37 C for 1 h. For fluorescence labeling, cells were incubated with FITC-conjugated secondary antibodies (1:100, Santa Cruz Biotech) at 37 C for 1 h after primary incubation. The cells were also stained with 1 mg/ml 40 , 6-diamino-2phenylindole (DAPI, Sigma, USA) to visualize their nuclei. The slides were observed and photographed under a fluorescent microscope (IX-71, Olympus, Japan).
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ethidium bromide, and photographed under an ultraviolet light transilluminator (BioRad, USA). 2.3. Western blotting Cells were washed with cold DPBS and lysed in 300 mL of cold RIPA buffer (50 mM TriseHCl, pH 7.5, containing 1% Triton X-100, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), and 1% sodium deoxycholate) with a protease inhibitor cocktail (Sigma, USA). Cell lysate was centrifuged at 13,000 g for 10 min at 4 C. The supernatant was harvested, and its protein concentration measured using a protein assay kit (Bio-Rad, USA). For electrophoresis, 50 mg protein was dissolved in sample buffer (60 mM TriseHCl, pH 6.8, containing 14.4 mM b-mercaptoethanol, 25% glycerol, 2% SDS, and 0.1% bromophenol blue), boiled for 5 min, and separated on a 10% SDS reducing gel. Separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, UK) using a trans-blot system (Bio-Rad, USA). Blots were blocked for 1 h in Tris-buffered saline (TBS) (10 mM TriseHCl, pH 7.5, with 150 mM NaCl) containing 5% non-fat dry milk (Bio-Rad, USA) at room temperature, washed three times with TBS, and incubated at 4 C overnight with primary antibody specific for human albumin (1:1000 dilution; Bethyl, USA) in TBST (10 mM Tris, pH 7.5, containing 150 mM NaCl and 0.02% Tween 20) containing 3% non-fat dry milk. The next day, blots were washed three times with TBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution; Santa Cruz Biotech, USA) in TBST containing 3% non-fat dry milk at room temperature. After being washed three times with TBST, the proteins were visualized with an ECL detection system (Amersham Pharmacia Biotech, UK).
2.5. Periodic acid-Schiff (PAS) staining for glycogen 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, USA) for 15 min, and rinsed in dH2O for 5e10 min. Samples were counterstained with Mayer’s hematoxylin for 1 min and rinsed in dH2O and assessed under a light microscope. 2.6. Enzyme-linked immunosorbent assay (ELISA) Differentiation media at various time-points in hepatic differentiation, and the media from various differentiation conditions, were sampled for albumin ELISA. The concentrations of albumin in supernatant were determined in plasma samples by ELISA kit according to the manufacturer’s instructions (Koma biotech, Korea). Briefly, these assays employed the quantitative sandwich enzyme immunoassay technique with monoclonal antibodies specific for albumin precoated on a microplate. Standard controls and samples (100 mL of supernatant) were pipetted into the wells in duplicate. After albumin binding and washing, an enzyme-linked polyclonal antibody specific for human albumin was added to each well. After thorough washing, a substrate solution was added to the wells and color developed in proportion to the amount of albumin bound in the first step. The optical density of each well was determined by a microplate reader at 450 nm. ELISA was performed with the samples from 5 units of umbilical cord blood. 2.7. Statistical analysis All results are expressed as means standard deviation (SD). P < 0.05 was considered significant. Statistical data were calculated with SigmaPlotÔ. 3. Results
2.4. Immunofluorescence analysis Cells were cultured in 2-chamber Lab-Tek slides (Nalge Nunc International, USA) for 7 days. For immunofluorescence staining, cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS at room
3.1. Morphological change of MPCs’ in vitro differentiation in conditioned medium MPCs were plated at 3 104 cells/cm2 on culture dishes and cultured for 3 days. Subsequently, the culture medium was
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replaced with hepatic differentiation medium containing FGF4, HGF, OSM, EGF and LIF. After treatment with hepatocyteconditioned medium, the spindle-shaped morphology of MPCs was lost and most cells displayed oval and round morphology, resembling hepatoblasts and hepatic oval cells 2 weeks post-induction (Fig. 1A).
3.2. Detection of hepatocyte specific marker expressions in MPCs The time-dependence of hepatocyte specific marker expression was examined (Fig. 1B). Hepatic differentiation of MPCs was confirmed in a time-dependent manner by RT-PCR
A
3 days
0 day
B
M
1
2
2 weeks
3
4
5
6
7
8
9
ALB
TAT
HNF1
CK-8
HNF4
GAPDH M
1
2
3
CYP2B6
C
C ALB
1
2
3
4
5
6
66kD
Actin
Fig. 1. Differentiation of MPCs into hepatic lineage and expression of their hepatocyte specific markers. (A) MPCs were grown for 0, 3 and 14 days in induction medium. Images were viewed using an inverted microscope at 200 magnification. Scale bar ¼ 100 mm. (B) Total RNAs were analyzed by RT-PCR for mRNA expression of human albumin (ALB), TAT, HNF1a, CK-8, HNF4 and GAPDH. cDNA prepared from the total RNAs of hepaticinduced (lanes 3e8) and non-induced (lane 2) MPCs served as the input in PCR reactions containing primers corresponding to hepatic lineage specific differentiation markers. M: size marker; lane 1: HepG2; lane 2: non-induced; lane 3: 1 day; lane 4: 3 days; lane 5: 7 days; lane 6: 14 days; lane 7: 21 days; lane 8: 28 days; lane 9: negative control. Expression of cytochrome P450 was also examined. M: size marker, lane 1: hepatic-induced for 2 weeks; lane 2: non-induced; lane 3: negative control; (C) Expression levels of the important hepatocyte specific marker albumin, were detected by western blotting. The expression levels of b-actin protein were used as the internal control. The lanes contain proteins that have been extracted from cells before induction (lane 1: day 0), after 1 (lane 2), 3 (lane 3), 7 (lane 4), 14 (lane 5) and 21 (lane 6) days in induction medium, and a positive control (lane C: a hepatoma cell line, HepG2).
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for common markers of hepatocyte differentiation. The expression of albumin, cytokeratin-8 (CK-8) and hepatocyte nuclear factor 1a (HNF1a) was readily detectable in hepatic differentiated MPCs with conditioned differentiation medium for 1 day, while hepatocyte nuclear factor 4 (HNF4) and tyrosineamino transferase (TAT) were detected at 3 and 7 postinduction. Albumin expression was detected at all time-points except in the undifferentiated cells, and was maintained throughout the differentiation process. CK-8 expression was gradually enhanced from day 1 to week 1, but rapidly decreased thereafter. HNF1 a expression was detected only from day 1 to week 1, after which it gradually disappeared. HNF4, a transcription factor largely expressed in the adult liver, was detected from day 3 to 2 weeks post-induction. In addition, RT-PCR analysis showed that the expression of the P450 gene (CYP2B6), known as a functional protein related to detoxification activity of mature hepatocyte, was detectable by 2 weeks post-induction. The protein expression of albumin in the differentiated cells was also analyzed through Western blot (WB) and immunofluorescence (IF) staining with the use of appropriate monoclonal antibody. The albumin band was detected on day 1 of the differentiation period and during the maturation step; the band intensity became further enhanced until 3 weeks postdifferentiation (Fig. 1C). To further confirm the expression of hepatocyte specific proteins in hepatic differentiated MPCs, cells were fixed and IF-stained for hepatic lineage specific markers. They expressed hepatic lineage specific markers such as human albumin, human hepatocyte marker (HepPar1) and alpha-1antitrypsin (AAT) (Fig. 2). The results of the protein analysis were in agreement with the RT-PCR results. We attempted to select exclusively MPC colonies that had differentiated into hepatic lineages, which were later confirmed to possess hepatic differentiation capacity. The MPC colonies were produced by the low density seeding of UCB-MNCs (2 105 cells/cm2) into culture chamber slides, followed by the culture method of UCB-MPCs. After their formation, colonies of MPCs were hepatic-induced 1 week and IF stained for hepatocyte specific markers. Differentiated colonies of MPCs were positively stained for hepatocyte specific markers, including albumin, HepPar1 and AAT (Fig. 3). 3.3. Function of hepatic differentiated MPCs and FGF-4, key-cytokine of hepatogenesis Intracellular glycogen accumulation is a feature of adult liver cells. Hepatic differentiation of MPCs was analyzed by staining them with the periodic acid-Schiff (PAS) reagent. Glycogen accumulation was detected in the hepatic differentiated cells, whereas it did not do so in the undifferentiated control (Fig. 4). Albumin is the major plasma protein secreted by hepatocytes (Dufour et al., 2000). Albumin production of hepatic differentiated MPCs was measured at various time-points throughout the differentiating process by ELISA. Hepatic
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differentiated MPCs produced albumin in a time-dependent manner from day 1 (120.14 33.28 ng/ml) and the secretion was maintained until day 28 (239.53 31.26 ng/ml), whereas undifferentiated MPCs did not secrete albumin (Fig. 5A). We also examined the influence of each cytokine to determine their function in MPC hepatic differentiation. In all, 11 combinations of cytokines were tested with MPCs isolated from five donors. Combination of cytokines lacking FGF-4 did not induce secretion of albumin in MPCs (Fig. 5B), whereas only the combination of cytokines and FGF-4 induced the secretion of albumin in MPCs. In addition, Matrigel coating of the culture dishes gave no significant difference in the secretion of albumin compared with uncoated culture dishes. These findings indicate that FGF-4 is essential for the differentiation of MPCs into albumin-secreting cells. 4. Discussion Potential hepatocyte progenitor cells have been identified from BM, peripheral blood, UCB, fetal liver, adult liver and ES cells (Sharma et al., 2005). Recently, classical MSCs derived from BM could differentiate into hepatocyte-like cells under in vitro conditions, with comprehensive phenotype and functional characteristics of liver cells (Lee et al., 2004a). Although the approach shares similarities with that for the isolation of MSCs and other adult stem cells, cell phenotypes and characteristics of hepatic differentiated cells established for novel MPCs are different for those of other cells. The expression profiles of HNF1a, HNF4, CK-8, TAT and albumin mRNA show that MPCs had differentiated into cells of hepatocyte lineage (Fig. 1). On day 0, no hepatocyte markers were detected; however on day 1, the mRNAs of albumin and CK 8 were detected and gradually enhanced for 1 week. The mRNA of HNF1a was detected from day 1 but disappeared 2 weeks after differentiation and CK-8 had decreased after 1 week. But, the mRNA of albumin maintained for 4 weeks of differentiation, and this result coincided with that from the western blot, immunostaining and ELISA. The mRNA of HNF4 and TAT, which are late markers of hepatocyte lineage, were detected on day 3 and at 1 week, respectively, and maintained for 4 weeks. Albumin, the abundant protein synthesized by mature hepatocytes, starts in early fetal hepatocytes and reaches the maximal level in adult hepatocytes (Pan et al., 1998). HNF4 is a transcription factor largely expressed in adult liver (Sladek et al., 1990). TAT is known to be a late marker of hepatocyte lineage and excellent enzymatic marker for peri- or postnatal hepatocyte specific differentiation (Shelly et al., 1989). The CYP2B6 in man is considered specific for functional hepatocytes (Schwartz et al., 2002). Immunofluorescence (IF) analysis also showed that hepatic differentiated MPCs expressed hepatic lineage specific markers. Anti-human hepatocyte monoclonal antibody (HepPar1) reacts with only human hepatocytes and human bile duct cells; alpha-1-antitrypsin (AAT) is a glycoprotein mainly synthesized in the liver (Mercer et al., 2001; Perlmutter, 2002). To confirm the functional activity of hepatic differentiated MPCs, albumin secretion was measured at various times after
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A
ALB
ALB
DAPI
Merged
ALB
DAPI
Merged
HepPar1
DAPI
Merged
HepPar1
DAPI
Merged
AAT
DAPI
Merged
AAT
DAPI
Merged
B
C
HepPar1
D
E
AAT
F
Fig. 2. Immunofluorescence staining of human hepatocyte specific markers in differentiated MPCs. After 1 week of hepatic induction, cells were immunofluorescently stained for human albumin (ALB; panel A), hepatocyte marker (HepPar1: panel C) and alpha-1-antitrypsin (AAT: panel E). The cells were positively stained for those markers. Nuclei were stained with DAPI and merged images are indicated in each panel. Images were viewed using a fluorescence microscope at 200 magnification. Panels (A, C, E): hepatic differentiated cells; panels (B, D, F): undifferentiated MPCs. Scale ¼ 400 mm.
the initiation of cell differentiation. Non-induced MPCs did not secrete albumin, whereas differentiated cells in the induction medium did in a time-dependent manner beginning after the first day of differentiation. It took just 1 day of differentiation for the secretion of albumin of hepatic differentiated MPCs, and the phenomenon appeared more rapidly than any other adult stem cell-derived hepatocyte-like cells
reported by other investigators ((Ruhnke et al., 2005); 10 days, (Shi et al., 2005); 3e6 days, (Kang et al., 2006); 8e12 days, (Schwartz et al., 2002); 5 days). This means that the process of MPC hepatic differentiation would be more rapid than that of other previously reported stem cells. But, further investigation is necessary to understand the distinct mechanism of the MPC phenomenon. In addition, the hepatic differentiated MPCs
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Fig. 3. Immunofluorescence staining of human hepatocyte specific markers in differentiated MPC colonies. Colonies of MPCs were produced by seeding a low density of UCB-MNCs (2 105 cells/cm2) into a culture chamber slide, followed by 10 days of culturing. After colony formation, those permitted to differentiate under induction conditions for 1 week and differentiated cells were immunofluorescently stained for human albumin, HepPar1 and AAT. The colonies were positively stained for these markers assuming that the examined colonies differentiated into hepatic lineages. Nuclei were stained with DAPI and merged images are indicated in each panel. Images were viewed using fluorescence microscope at 200 magnification. Scale bar ¼ 100 mm.
were shown to increase the intracellular accumulation of glycogen and the genuine function of mature hepatocytes. Previous reports have shown that FGF, LIF, EGF, HGF and OSM contributed to the proliferation and/or differentiation of hepatic progenitor cells in distinct ways. Interestingly, it became obvious from to our ELISA analysis that FGF-4 was the most important cytokine for the differentiation of MPCs into functional differentiated cells that produce and secrete albumin in vitro. Albumin secretion is considered one of the most important criteria in assessing stem cell differentiation into functional hepatocytes. FGFs secreted by the cardiac mesoderm induce the expression of liver genes such as albumin in the ventral foregut endoderm (Jung et al., 1999), and the FGF response pathway mediates hepatic gene induction in embryonic endodermal cells (Calmont et al., 2006;
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Wells and Melton, 1999). The other growth factors were influenced positively by albumin secretion of UCB-MPCs, though not at a significant level, and further study is needed on this issue. Stem cell populations, which include UCB-MSCs, BMMSCs and MNCs, may be heterogeneous and consist of several subpopulations (Conget et al., 2001; Moon et al., 2005). Thus, MPCs could also comprise a mixture of different cells. We generated colonies of MPCs which differentiated into hepatic lineages in order to avoid possible problems with cell heterogeneity and to confirm the hepatic differentiation capacity of single-cell-derived MPCs colonies. Differentiated colonies of MPCs were positively stained for hepatocyte specific markers such as albumin, HepPar1 and AAT, which suggests that the characteristics of hepatic lineages of differentiated MPCs
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Fig. 4. Periodic acid-Schiff (PAS) staining of hepatic differentiated MPCs. MPCs that differentiated in induction medium for 1 week stained with PAS. (A) The undifferentiated control; (B) hepatic differentiated MPCs. Scale bar ¼ 200 mm.
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Induction conditions Fig. 5. Albumin secretion of hepatic differentiated MPCs in a time-dependent (A) and condition-dependent (B) manner. Quantification of secreted albumin in induction medium was performed by ELISA. (A) Secretion of albumin was detected on day 1 of differentiation; afterwards, the amount of albumin in the culture medium gradually increased over time until day 28 of induction; (B) MPCs were grown for 1 week in induction media containing various mixtures of cytokines to screen for cytokines essential to the hepatic differentiation of the MPCs. Control: undifferentiated MPCs; H: HGF; O: OSM; F: FGF-4; E: EGF; L: LIF; no coat: cells induced without MatrigelÒ coating. The results are expressed as means SD of the measured amount of albumin from five separate experiments (n ¼ 5).
originate from the MPCs themselves, rather than from cell heterogeneity or contamination of other lineages (Fig. 3). In summary, MPCs derived from UCB are able to differentiate into functional hepatocyte-like cells in vitro. The cells were characterized at the gene and protein levels and exhibited hepatocellular synthesis and metabolic functions specific to hepatocytes. Thus, UCB-MPCs can potentially serve as a new cell source for tissue engineering or hepatic cell therapy. References Bianco P, Riminucci M, Kuznetsov S, Robey PG. Multipotential cells in the bone marrow stroma: regulation in the context of organ physiology. Crit Rev Eukaryot Gene Expr 1999;9:159e73. Calmont A, Wandzioch E, Tremblay KD, Minowada G, Kaestner KH, Martin GR, et al. An FGF response pathway that mediates hepatic gene induction in embryonic endoderm cells. Dev Cell 2006;11:339e48. Conget PA, Allers C, Minguell JJ. Identification of a discrete population of human bone marrow-derived mesenchymal cells exhibiting properties of uncommitted progenitors. J Hematother Stem Cell Res 2001;10:749e58. Davis AA, Temple S. A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 1994;372:263e6. Domen J, Weissman IL. Self-renewal, differentiation or death: regulation and manipulation of hematopoietic stem cell fate. Mol Med Today 1999;5:201e8. Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff LB. Diagnosis and monitoring of hepatic injury. I. Performance characteristics of laboratory tests. Clin Chem 2000;46:2027e49. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 2000;109:235e42. Fiegel HC, Lioznov MV, Cortes-Dericks L, Lange C, Kluth D, Fehse B, et al. Liver-specific gene expression in cultured human hematopoietic stem cells. Stem cells 2003;21:98e104. Hong SH, Gang EJ, Jeong JA, Ahn C, Hwang SH, Yang IH, et al. In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells. Biochem Biophys Res Commun 2005;330: 1153e61. Kakinuma S, Tanaka Y, Chinzei R, Watanabe M, Shimizu-Saito K, Hara Y, et al. Human umbilical cord blood as a source of transplantable hepatic progenitor cells. Stem Cells 2003;21:217e27.
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