In Vitro Differentiation of Human Liver-derived Stem Cells with Mesenchymal Characteristics into Immature Hepatocyte-like Cells J.-H. Leea, H.-J. Parka, I.K. Jangb, H.-E. Kimb, D.-H. Leeb, J.-K. Parkc, S.-K. Leed,*, and H.-H. Yoone,* a Samsung Biomedical Research Institute, Seoul, Republic of Korea; bBiomedical Research Institute, Lifeliver Co Ltd, Yongin, Republic of Korea; cDepartment of Medical Biotechnology, Dongguk University, Seoul, Republic of Korea; dDepartment of Surgery, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul, Republic of Korea; and eDongguk University Research Institute of Biotechnology, Dongguk University, Seoul, Republic of Korea
ABSTRACT Liver transplantation is severely limited by donor shortage although it is the only effective treatment for end-stage liver disease. So the best alternative is hepatocyte transplantation. For obtaining human hepatocytes, some stem cells originating from extrahepatic or intraheptic tissues have been isolated and characterized. Previously we have reported that human liver-derived stem cells (HLSCs) could be isolated and expanded from donated livers unsuitable for transplantation; they expressed some markers of mesenchymal stem cells but neither hematopoietic nor oval cells. In this study, we isolated and expanded HLSCs with mesenchymal characteristics from another adult human liver. They showed mesenchymal morphology and grew well under serum condition similar to our previous reports. Also, they expressed some markers of mesenchymal stem cells, such as CD44, CD73, CD90, and CD105, through fluorescence-activated cell sorting analysis. When HLSCs were sequentially exposed to fibroblast growth factor 1 (FGF-1), FGF-4, and hepatocyte growth factor (HGF) followed by FGF-4, HGF, oncostatin M, and dexamethasone, they became round or polygonal, and expressed some hepatic markers such as albumin and a1-antitrypsin in the gene or protein level. Also, they showed urea synthesis activity 7 days after treatment of FGF-4, HGF, oncostatin M, and dexamethasone. These results provided that HLSCs would be a useful cell source in the field of regenerative medicine as well as liver cell biology.
T
HE BEST treatment for hepatic dysfunction is orthotopic liver transplantation including whole liver transplantation and living donor liver transplantation. But orthotopic liver transplantation is severely limited by the shortage of available donor livers [1,2]. The parenchymal cells in the liver are called hepatocytes, which perform a lot of important catabolic and anabolic functions. The alternative to OLT is transplantation of hepatocytes from graftincompatible cadaveric livers because of steatosis, fibrosis, etc [3,4]. However, use of these organs is also severely limited by the shortage despite technical advances. Therefore, it is necessary to obtain human hepatocytes through lineage-restricted differentiation of stem cells. Stem cells for hepatocytes are categorized into extrahepatic and intrahepatic stem cells with respect to their origin. It has been reported that hematopoietic stem cells [5e7] and mesenchymal stem cells (MSCs) [8e11] as extrahepatic
stem cell can differentiate into hepatocyte-like cells in vitro. There are some types of intrahepatic stem cells, which are regarded having more hepatic potential than extrahepatic stem cells because of their pre-existence in the liver microenvironment. These intrahepatic stem cells include oval cells, clonogenic epithelial cells, and mesenchymal stem-like cells. This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A101758). *Address correspondence to Suk-Koo Lee, MD, Department of Surgery, Samsung Medical Center, 50 Ilwon-dong, Kangnam-Ku, Seoul 135-710, Republic of Korea. E-mail: [email protected]; and Hee-Hoon Yoon, PhD, Dongguk University Research Institute of Biotechnology, Dongguk University, Seuol 100-715, Republic of Korea. E-mail: [email protected]
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.12.070
Transplantation Proceedings, 46, 1633e1637 (2014)
1633
1634
LEE, PARK, JANG ET AL
Previously we have reported that human liver-derived stem cells (HLSCs) could be isolated and expanded, and they showed mesenchymal morphology expressing some MSC markers. Also, they could differentiate osteoblasts and chondrocytes but not adipocytes [12,13]. Beside oval or clonogenic epithelial cells, some mixed types of cells with epithelial and mesenchymal characteristics have been isolated, expanded, and differentiated to functional hepatocytes. They are regarded as mesodermal precursor cells which give rise to endodermal cells and mesenchymal cells during development. They have an attractive benefit in easy cell expansion to large quantity under serum due to their mesenchymal characteristics [14e16]. In this study, we isolated and expanded HLSCs with mesenchymal characteristics from an adult human liver and differentiated to immature hepatocyte-like cells in vitro by using sequential differentiation. MATERIALS AND METHODS Primary and Subculture of HLSCs This study was approved by the Institutional Review Board of Samsung Medical Center, and all samples were obtained with informed consent. Human liver tissue was obtained from a nontumor part of liver from a patient who received hepatic resection for hepatic hemangioendothelioma. Isolation of HLSCs was performed using the two-step collagenase perfusion technique as previously described [12,13].
every 2 days. HepG2 cells, a human hepatoma cell population, were expanded in high-glucose DMEM/10% fetal bovine serum (FBS) and were used as positive control for reverse transcriptase polymerase chain reaction (RT-PCR) analysis.
Flow Cytometry for Surface Antigen Expression Analysis A total of 5 105 cells were resuspended in 200 mL phosphatebuffered saline (PBS) and incubated with fluorescein isothiocyanate- or phycoerythrin- conjugated antibodies for 20 minutes at room temperature. Conjugated antibodies against CD29, CD34, CD38, CD44, CD45, CD54, CD62L, CD73, CD144, CD166, CD326 (BD Bioscience, San Jose, Calif, United States), CD9, CD31, CD90, CD105, CD117, HLA-ABC, and HLA-DR (Beckman Coulter, Fullerton, Calif, United States) were used. The fluorescence intensity of the cells was evaluated by a flow cytometer (FC 500; Beckman Coulter), and the data were analyzed with the CXP software (Beckman Coulter).
RT-PCR Total RNA was isolated from cells using Trizol Reagent (Invitorgen-Gibco); 2 mg RNA was reverse-transcribed with the AMV reverse transcriptase XL (TaKaRaShuzo, Shiga, Japan) for 90 minutes at 42 C in the presence of oligo(dT) primer. PCR was performed using Taq polymerase (KomaBiotech, Seoul, Korea). Hepatic markers for RT-PCR in this study were albumin, a1-antitrypsin, tryptophan 2,3-dioxygenase, and glutamine synthetase. Primer sequences and reaction conditions are shown in Table 1.
Immunofluorescence Hepatic Differentiation of HLSCs HLSCs at fifth passage were seeded into a fibronectin-coated 6-well plate at a density of 1 104 cells/cm2 and differentiated using the two-step protocol. First, the cells were treated with Dulbecco’s Modified Eagle Medium (DMEM; low glucose) containing 1% insulin-transferrin-selenium, 10 ng/mL fibroblast growth factor 1 (FGF-1), 10 ng/mL FGF-4, and 20 ng/mL hepatocyte growth factor (HGF; all from R&D Systems, Minneapolis, MN, United States) for 5 days. Then they were sequentially treated with the same basal medium containing 100 nM dexamethasone (Sigma-Aldrich, St. Louis, Mo), 10 ng/ mL FGF-4, 20 ng/mL HGF (both from R&D Systems), 10 ng/mL oncostatin M (OSM), and 0.5% dimethyl sulfoxide (both from Sigma-Aldrich) for designated days. Medium was exchanged
Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes, then were blocked in PBS containing 1.5% bovine serum albumin (Sigma-Aldrich) and 0.5% Triton X-100 (Sigma-Aldrich) for permeabilization, followed by incubation with appropriate primary antibody, including anti-human albumin (1:200, Bethyl Laboratories Inc., Montgomery, TX, United States) or antihuman a1-antitrypsin antibody (1:100, DakoCytomation, Glostrub, Denmark) for 1 hour at room temperature. After washing with PBS for 10 minutes, the cells were incubated with bovine anti-rabbit immunoglobulin G Rhodamine-conjugated secondary antibody (1:100, Santa Cruz Biotechnology Inc., Delaware, Calif, United States) for 1 hour at room temperature. The glass coverslips were counterstained with 2-(4-amidinophenyl)-1H -indole-6-carboxamidine (Sigma-Aldrich) and mounted onto
Table 1. Primer Sequences and Reaction Conditions for RT-PCR Gene
Albumin
a1-antitrypsin Tryptophan 2,3-dioxygenase Glutamine synthetase GAPDH
Primer
Annealing Temperature ( C)
Product (bp)
F: 5- tgc ttg aat gtg ctg atg aca ggg -3 R: 5- aag gca agt cag cag gca tct cat c -3 F: 5- cca tgt ttg tca aag agc aac -3 R: 5- gga agt aag gta tag tca ggt -3 F: 5- ata cag agc act tca ggg agc -3 R: 5- gtt ggg ttc atc ttc ggt atc -3 F: 5- gtc aag att gcg ggg act aa -3 R: 5- tac gat tgg cta cac cac ca -3 F: 5- atc acc atc ttc cag gag cg -3 R: 5- cct gct tca cca cct tct tg -3
60
161
60
345
60
297
55
396
62
573
Abbreviations: RT-PCR, reverse transcriptase polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
HUMAN LIVER-DERIVED STEM CELLS
1635
Fig 1. Morphology (A) and surface antigen expression (B) of HLSCs. They had mesenchymal elongated and spindle shape in culture. This morphology was maintained at least from P 0 to P 5. Scale bar indicates 500 mm. HLSCs expressed some of the mesenchymal stem cell markers, but not hematopoietic lineage and endothelial cell markers.
microscope slides. The cells were examined using a fluorescent microscope (IX-71, Olympus, Japan).
and reagent B) for 50 minutes at room temperature; optical density was read at 430 nm.
Urea Synthesis Assay To determine urea synthesis activity, Quantichrome urea assay kit (Bioassay Systems, Hayward, Calif, United States) was used. According to manufacturer’s instruction, each 50 mL of sample was reacted with 200 mL of working reagent (1:1 mixture of reagent A
Fig 2. Morphology (A) and gene expression (B) of HLSCs during sequential hepatic differentiation. Their morphology was dramatically changed to highly round or polygonal shape as they were exposed to OSM, dexamethasone. Scale bar indicates 500 mm. Some hepatic markers were upregulated with time of differentiation, especially after treatment of OSM and dexamethasone.
Statistical Analysis Data were given as means standard deviation and were statistically analyzed using SigmaPlot paired t-test. The difference between the means was considered significant when P < .05.
1636
LEE, PARK, JANG ET AL
Also, the expression of HLA-ABC but not HLA-DR, as shown in MSCs, would be used beneficially for cell transplantation of HLSCs in the future. Hepatic Differentiation of HLSCs
Fig 3. Urea production (A) and immunofluorescence (B) of HLSCs during sequential hepatic differentiation. Urea was produced very low on day 4 but was substantially produced after OSM and dexamethasone treatment. However, undifferentiated HLSCs did not produce urea. *P < .01. Albumin and a1-antirypsin, two hepatic markers, were expressed by sequentially differentiated HLSCs for 20 days. Scale bar indicates 200 mm.
RESULTS Primary and Subculture of HLSCs
We isolated and expanded a cell population with mesenchymal characteristics, so-called HLSCs, from a nonparenchymal fraction of adult human liver similar to our previous reports. The origin of HLSCs is thought to be mainly from the nonparenchymal fraction because they were initially collected from the surpernatant after low g force and short centrifugation (50 g for 3 minutes) for removing parenchymal cells. As shown in Fig 1A, HLSCs had an elongated, spindle morphology similar to fibroblasts. They well proliferated over 10 passages under 10% FBS conditions different from epithelial cells. Fluorescence-activated cell sorting analysis showed that HLSCs expressed several MSC markers such as CD44, CD73, CD90, CD105, etc. But they did not express CD34, CD45, CD38, CD62L, CD117, and CD144. This result revealed that HLSCs originated from mesenchymal cells not hematopoietic cells (Fig 1B).
The morphology of HLSCs slightly shortened after exposure to FGF-1, FGF-4, and HGF, and it was subsequently changed to a highly round or polygonal shape according to FGF-4, HGF, OSM, and dexamethasone treatment (Fig 2A). It is postulated that this dramatic change is induced mainly by OSM and dexamethasone. The results from RT-PCR showed that some hepatic markers, such as albumin, a1-antitrypsin, tryptophan 2,3dioxygenase, and glutamine synthetase were upregulated with time of differentiation. Especially, these markers were highly expressed after OSM and dexamethasone treatment (Fig 2B). This result corresponded to that of urea production. Urea produced by HLSCs was undetectable on day 4, but was substantially detected after OSM and dexamethasone treatment. The amount of produced urea was consistently maintained. Therefore, OSM and dexamethasone are two of the major hepatic maturation inducers (Fig 3A). Also, two hepatic markers, albumin and a1-antitrypsin, were expressed in HLSCs 20 days after sequential hepatic differentiation by immunofluorescence (Fig 3B). Although some hepatic markers were efficiently expressed by HLSCs, the most important CYP450 enzymatic functions in relation to detoxification were not detected after treatment of 3-methylcholanthrene or rapamycin (data not shown). Therefore, it was revealed that the full maturation of HLSCs into hepatocytes was not achieved yet. A more sophisticated differentiation protocol will be necessary for the expression of CYP450 function. DISCUSSION
It was reported that a stem cell population derived from human fetal liver, so-called human fetal liver multipotent progenitor cells, had characteristics of mesenchymalepithelial transitional cells, which could differentiate into hepatocytes and biliary epithelial cells as well as mesenchymal lineage cells, including osteoblasts, chondrocytes, adipocytes, and endothelial cells. These cells are a unique cell population and are unlikely to arise from dedifferentiating hepatocytes or transdifferentiating MSCs. It has been hypothesized that mesoderm and endoderm arise from a common bipotential mesendoderm precursor [14]. More similar to our results, Herrera et al reported that a stem cell population isolated from human liver expressed several MSC surface antigens (CD73, CD90, CD29, and CD44) and had differentiation capacity into osteogenic, endothelial cells similar to bone marrow derived MSCs. In contrast to MSCs, these cells did not proliferate in a conventional media for MSC and differentiated into functional hepatocytes but not adipocytes. They suggested that these stem cells may represent a mesenchymal population modified by the influence of the local environment, reflecting the
HUMAN LIVER-DERIVED STEM CELLS
importance of the niche in establishing the phenotype of MSCs [15]. HLSCs were successfully isolated and expanded from an adult human liver. They also had some mesenchymal characteristics with respect to their morphology, proliferation under serum, and surface antigen expression. These cells efficiently differentiated into immature hepatocyte-like cells expressing some hepatic markers, such as albumin, and a1-antitrypsin, as shown using RT-PCR or immunofluorescence. Also, they showed urea synthesis activity, one of the unique functions of hepatocytes. But the degree of differentiation was placed on an immature state because they did not express CYP450 activities yet. Therefore, a more sophisticated differentiation protocol must be developed. Also, it will be necessary to determine what the origin of HLSCs is in the basic research point of view.
REFERENCES [1] Fox IJ, Chowdhury JR. Hepatocyte transplantation. Am J Transplant 2004;4:7. [2] Goldstein MJ, Salame E, Kapur S, et al. Analysis of failure in living donor liver transplantation: differential outcomes in children and adults. World J Surg 2003;27:356. [3] Strom SC, Fisher RA, Thompson MT, et al. Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation 1997;63:559. [4] Strom SC, Chowdhury JR, Fox LJ. Hepatocyte transplantation for the treatment of human disease. Semin Liver Dis 1999;19:39. [5] Avital I, Inderbitzin D, Aoki T, et al. Isolation, characterization, and transplantation of bone marrow-derived
1637 hepatocyte stem cells. Biochem Biophys Res Commun 2001;288:156. [6] Fiegel HC, Lioznov MV, Cortes-Dericks L, et al. Liverspecific gene expression in cultured human hematopoietic stem cells. Stem Cells 2003;21:98. [7] McGuckin CP, Forraz N, Baradez MO, et al. Production of stem cells with embryonic characteristics from human umbilical cord blood. Cell Prolif 2005;38:245. [8] Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002;109:1291. [9] Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41. [10] Lee KD, Kuo TK, Whang-Peng J, et al. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 2004;40:1275. [11] Snykers S, Vanhaecke T, De Becker A, et al. Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow. BMC Dev Biol 2007;7:24. [12] Lee JH, Park HJ, Kim YA, et al. The phenotypic characteristic of liver-derived stem cells from adult human deceased donor liver. Transplant Proc 2012;44:1110. [13] Lee JH, Park HJ, Kim YA, et al. Differentiation and major histocompatibility complex antigen expression in human liverderived stem cells. Transplant Proc 2012;44:1113. [14] Dan YY, Riehle KJ, Lazaro C, et al. Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages. Proc Natl Acad Sci U S A 2006;103:9912. [15] Herrera MB, Bruno S, Buttiglieri S, et al. Isolation and characterization of a stem cell population from adult human liver. Stem Cells 2006;24:2840. [16] Inada M, Follenzi A, Cheng K, et al. Phenotype reversion in fetal human liver epithelial cells identifies the role of an intermediate meso-endodermal stage before hepatic maturation. J Cell Sci 2008;121:1002.
ABSTRACT Liver transplantation is severely limited by donor shortage although it is the only effective treatment for end-stage liver disease. So the best alternative is hepatocyte transplantation. For obtaining human hepatocytes, some stem cells originating from extrahepatic or intraheptic tissues have been isolated and characterized. Previously we have reported that human liver-derived stem cells (HLSCs) could be isolated and expanded from donated livers unsuitable for transplantation; they expressed some markers of mesenchymal stem cells but neither hematopoietic nor oval cells. In this study, we isolated and expanded HLSCs with mesenchymal characteristics from another adult human liver. They showed mesenchymal morphology and grew well under serum condition similar to our previous reports. Also, they expressed some markers of mesenchymal stem cells, such as CD44, CD73, CD90, and CD105, through fluorescence-activated cell sorting analysis. When HLSCs were sequentially exposed to fibroblast growth factor 1 (FGF-1), FGF-4, and hepatocyte growth factor (HGF) followed by FGF-4, HGF, oncostatin M, and dexamethasone, they became round or polygonal, and expressed some hepatic markers such as albumin and a1-antitrypsin in the gene or protein level. Also, they showed urea synthesis activity 7 days after treatment of FGF-4, HGF, oncostatin M, and dexamethasone. These results provided that HLSCs would be a useful cell source in the field of regenerative medicine as well as liver cell biology.
T
HE BEST treatment for hepatic dysfunction is orthotopic liver transplantation including whole liver transplantation and living donor liver transplantation. But orthotopic liver transplantation is severely limited by the shortage of available donor livers [1,2]. The parenchymal cells in the liver are called hepatocytes, which perform a lot of important catabolic and anabolic functions. The alternative to OLT is transplantation of hepatocytes from graftincompatible cadaveric livers because of steatosis, fibrosis, etc [3,4]. However, use of these organs is also severely limited by the shortage despite technical advances. Therefore, it is necessary to obtain human hepatocytes through lineage-restricted differentiation of stem cells. Stem cells for hepatocytes are categorized into extrahepatic and intrahepatic stem cells with respect to their origin. It has been reported that hematopoietic stem cells [5e7] and mesenchymal stem cells (MSCs) [8e11] as extrahepatic
stem cell can differentiate into hepatocyte-like cells in vitro. There are some types of intrahepatic stem cells, which are regarded having more hepatic potential than extrahepatic stem cells because of their pre-existence in the liver microenvironment. These intrahepatic stem cells include oval cells, clonogenic epithelial cells, and mesenchymal stem-like cells. This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A101758). *Address correspondence to Suk-Koo Lee, MD, Department of Surgery, Samsung Medical Center, 50 Ilwon-dong, Kangnam-Ku, Seoul 135-710, Republic of Korea. E-mail: [email protected]; and Hee-Hoon Yoon, PhD, Dongguk University Research Institute of Biotechnology, Dongguk University, Seuol 100-715, Republic of Korea. E-mail: [email protected]
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.12.070
Transplantation Proceedings, 46, 1633e1637 (2014)
1633
1634
LEE, PARK, JANG ET AL
Previously we have reported that human liver-derived stem cells (HLSCs) could be isolated and expanded, and they showed mesenchymal morphology expressing some MSC markers. Also, they could differentiate osteoblasts and chondrocytes but not adipocytes [12,13]. Beside oval or clonogenic epithelial cells, some mixed types of cells with epithelial and mesenchymal characteristics have been isolated, expanded, and differentiated to functional hepatocytes. They are regarded as mesodermal precursor cells which give rise to endodermal cells and mesenchymal cells during development. They have an attractive benefit in easy cell expansion to large quantity under serum due to their mesenchymal characteristics [14e16]. In this study, we isolated and expanded HLSCs with mesenchymal characteristics from an adult human liver and differentiated to immature hepatocyte-like cells in vitro by using sequential differentiation. MATERIALS AND METHODS Primary and Subculture of HLSCs This study was approved by the Institutional Review Board of Samsung Medical Center, and all samples were obtained with informed consent. Human liver tissue was obtained from a nontumor part of liver from a patient who received hepatic resection for hepatic hemangioendothelioma. Isolation of HLSCs was performed using the two-step collagenase perfusion technique as previously described [12,13].
every 2 days. HepG2 cells, a human hepatoma cell population, were expanded in high-glucose DMEM/10% fetal bovine serum (FBS) and were used as positive control for reverse transcriptase polymerase chain reaction (RT-PCR) analysis.
Flow Cytometry for Surface Antigen Expression Analysis A total of 5 105 cells were resuspended in 200 mL phosphatebuffered saline (PBS) and incubated with fluorescein isothiocyanate- or phycoerythrin- conjugated antibodies for 20 minutes at room temperature. Conjugated antibodies against CD29, CD34, CD38, CD44, CD45, CD54, CD62L, CD73, CD144, CD166, CD326 (BD Bioscience, San Jose, Calif, United States), CD9, CD31, CD90, CD105, CD117, HLA-ABC, and HLA-DR (Beckman Coulter, Fullerton, Calif, United States) were used. The fluorescence intensity of the cells was evaluated by a flow cytometer (FC 500; Beckman Coulter), and the data were analyzed with the CXP software (Beckman Coulter).
RT-PCR Total RNA was isolated from cells using Trizol Reagent (Invitorgen-Gibco); 2 mg RNA was reverse-transcribed with the AMV reverse transcriptase XL (TaKaRaShuzo, Shiga, Japan) for 90 minutes at 42 C in the presence of oligo(dT) primer. PCR was performed using Taq polymerase (KomaBiotech, Seoul, Korea). Hepatic markers for RT-PCR in this study were albumin, a1-antitrypsin, tryptophan 2,3-dioxygenase, and glutamine synthetase. Primer sequences and reaction conditions are shown in Table 1.
Immunofluorescence Hepatic Differentiation of HLSCs HLSCs at fifth passage were seeded into a fibronectin-coated 6-well plate at a density of 1 104 cells/cm2 and differentiated using the two-step protocol. First, the cells were treated with Dulbecco’s Modified Eagle Medium (DMEM; low glucose) containing 1% insulin-transferrin-selenium, 10 ng/mL fibroblast growth factor 1 (FGF-1), 10 ng/mL FGF-4, and 20 ng/mL hepatocyte growth factor (HGF; all from R&D Systems, Minneapolis, MN, United States) for 5 days. Then they were sequentially treated with the same basal medium containing 100 nM dexamethasone (Sigma-Aldrich, St. Louis, Mo), 10 ng/ mL FGF-4, 20 ng/mL HGF (both from R&D Systems), 10 ng/mL oncostatin M (OSM), and 0.5% dimethyl sulfoxide (both from Sigma-Aldrich) for designated days. Medium was exchanged
Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes, then were blocked in PBS containing 1.5% bovine serum albumin (Sigma-Aldrich) and 0.5% Triton X-100 (Sigma-Aldrich) for permeabilization, followed by incubation with appropriate primary antibody, including anti-human albumin (1:200, Bethyl Laboratories Inc., Montgomery, TX, United States) or antihuman a1-antitrypsin antibody (1:100, DakoCytomation, Glostrub, Denmark) for 1 hour at room temperature. After washing with PBS for 10 minutes, the cells were incubated with bovine anti-rabbit immunoglobulin G Rhodamine-conjugated secondary antibody (1:100, Santa Cruz Biotechnology Inc., Delaware, Calif, United States) for 1 hour at room temperature. The glass coverslips were counterstained with 2-(4-amidinophenyl)-1H -indole-6-carboxamidine (Sigma-Aldrich) and mounted onto
Table 1. Primer Sequences and Reaction Conditions for RT-PCR Gene
Albumin
a1-antitrypsin Tryptophan 2,3-dioxygenase Glutamine synthetase GAPDH
Primer
Annealing Temperature ( C)
Product (bp)
F: 5- tgc ttg aat gtg ctg atg aca ggg -3 R: 5- aag gca agt cag cag gca tct cat c -3 F: 5- cca tgt ttg tca aag agc aac -3 R: 5- gga agt aag gta tag tca ggt -3 F: 5- ata cag agc act tca ggg agc -3 R: 5- gtt ggg ttc atc ttc ggt atc -3 F: 5- gtc aag att gcg ggg act aa -3 R: 5- tac gat tgg cta cac cac ca -3 F: 5- atc acc atc ttc cag gag cg -3 R: 5- cct gct tca cca cct tct tg -3
60
161
60
345
60
297
55
396
62
573
Abbreviations: RT-PCR, reverse transcriptase polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
HUMAN LIVER-DERIVED STEM CELLS
1635
Fig 1. Morphology (A) and surface antigen expression (B) of HLSCs. They had mesenchymal elongated and spindle shape in culture. This morphology was maintained at least from P 0 to P 5. Scale bar indicates 500 mm. HLSCs expressed some of the mesenchymal stem cell markers, but not hematopoietic lineage and endothelial cell markers.
microscope slides. The cells were examined using a fluorescent microscope (IX-71, Olympus, Japan).
and reagent B) for 50 minutes at room temperature; optical density was read at 430 nm.
Urea Synthesis Assay To determine urea synthesis activity, Quantichrome urea assay kit (Bioassay Systems, Hayward, Calif, United States) was used. According to manufacturer’s instruction, each 50 mL of sample was reacted with 200 mL of working reagent (1:1 mixture of reagent A
Fig 2. Morphology (A) and gene expression (B) of HLSCs during sequential hepatic differentiation. Their morphology was dramatically changed to highly round or polygonal shape as they were exposed to OSM, dexamethasone. Scale bar indicates 500 mm. Some hepatic markers were upregulated with time of differentiation, especially after treatment of OSM and dexamethasone.
Statistical Analysis Data were given as means standard deviation and were statistically analyzed using SigmaPlot paired t-test. The difference between the means was considered significant when P < .05.
1636
LEE, PARK, JANG ET AL
Also, the expression of HLA-ABC but not HLA-DR, as shown in MSCs, would be used beneficially for cell transplantation of HLSCs in the future. Hepatic Differentiation of HLSCs
Fig 3. Urea production (A) and immunofluorescence (B) of HLSCs during sequential hepatic differentiation. Urea was produced very low on day 4 but was substantially produced after OSM and dexamethasone treatment. However, undifferentiated HLSCs did not produce urea. *P < .01. Albumin and a1-antirypsin, two hepatic markers, were expressed by sequentially differentiated HLSCs for 20 days. Scale bar indicates 200 mm.
RESULTS Primary and Subculture of HLSCs
We isolated and expanded a cell population with mesenchymal characteristics, so-called HLSCs, from a nonparenchymal fraction of adult human liver similar to our previous reports. The origin of HLSCs is thought to be mainly from the nonparenchymal fraction because they were initially collected from the surpernatant after low g force and short centrifugation (50 g for 3 minutes) for removing parenchymal cells. As shown in Fig 1A, HLSCs had an elongated, spindle morphology similar to fibroblasts. They well proliferated over 10 passages under 10% FBS conditions different from epithelial cells. Fluorescence-activated cell sorting analysis showed that HLSCs expressed several MSC markers such as CD44, CD73, CD90, CD105, etc. But they did not express CD34, CD45, CD38, CD62L, CD117, and CD144. This result revealed that HLSCs originated from mesenchymal cells not hematopoietic cells (Fig 1B).
The morphology of HLSCs slightly shortened after exposure to FGF-1, FGF-4, and HGF, and it was subsequently changed to a highly round or polygonal shape according to FGF-4, HGF, OSM, and dexamethasone treatment (Fig 2A). It is postulated that this dramatic change is induced mainly by OSM and dexamethasone. The results from RT-PCR showed that some hepatic markers, such as albumin, a1-antitrypsin, tryptophan 2,3dioxygenase, and glutamine synthetase were upregulated with time of differentiation. Especially, these markers were highly expressed after OSM and dexamethasone treatment (Fig 2B). This result corresponded to that of urea production. Urea produced by HLSCs was undetectable on day 4, but was substantially detected after OSM and dexamethasone treatment. The amount of produced urea was consistently maintained. Therefore, OSM and dexamethasone are two of the major hepatic maturation inducers (Fig 3A). Also, two hepatic markers, albumin and a1-antitrypsin, were expressed in HLSCs 20 days after sequential hepatic differentiation by immunofluorescence (Fig 3B). Although some hepatic markers were efficiently expressed by HLSCs, the most important CYP450 enzymatic functions in relation to detoxification were not detected after treatment of 3-methylcholanthrene or rapamycin (data not shown). Therefore, it was revealed that the full maturation of HLSCs into hepatocytes was not achieved yet. A more sophisticated differentiation protocol will be necessary for the expression of CYP450 function. DISCUSSION
It was reported that a stem cell population derived from human fetal liver, so-called human fetal liver multipotent progenitor cells, had characteristics of mesenchymalepithelial transitional cells, which could differentiate into hepatocytes and biliary epithelial cells as well as mesenchymal lineage cells, including osteoblasts, chondrocytes, adipocytes, and endothelial cells. These cells are a unique cell population and are unlikely to arise from dedifferentiating hepatocytes or transdifferentiating MSCs. It has been hypothesized that mesoderm and endoderm arise from a common bipotential mesendoderm precursor [14]. More similar to our results, Herrera et al reported that a stem cell population isolated from human liver expressed several MSC surface antigens (CD73, CD90, CD29, and CD44) and had differentiation capacity into osteogenic, endothelial cells similar to bone marrow derived MSCs. In contrast to MSCs, these cells did not proliferate in a conventional media for MSC and differentiated into functional hepatocytes but not adipocytes. They suggested that these stem cells may represent a mesenchymal population modified by the influence of the local environment, reflecting the
HUMAN LIVER-DERIVED STEM CELLS
importance of the niche in establishing the phenotype of MSCs [15]. HLSCs were successfully isolated and expanded from an adult human liver. They also had some mesenchymal characteristics with respect to their morphology, proliferation under serum, and surface antigen expression. These cells efficiently differentiated into immature hepatocyte-like cells expressing some hepatic markers, such as albumin, and a1-antitrypsin, as shown using RT-PCR or immunofluorescence. Also, they showed urea synthesis activity, one of the unique functions of hepatocytes. But the degree of differentiation was placed on an immature state because they did not express CYP450 activities yet. Therefore, a more sophisticated differentiation protocol must be developed. Also, it will be necessary to determine what the origin of HLSCs is in the basic research point of view.
REFERENCES [1] Fox IJ, Chowdhury JR. Hepatocyte transplantation. Am J Transplant 2004;4:7. [2] Goldstein MJ, Salame E, Kapur S, et al. Analysis of failure in living donor liver transplantation: differential outcomes in children and adults. World J Surg 2003;27:356. [3] Strom SC, Fisher RA, Thompson MT, et al. Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation 1997;63:559. [4] Strom SC, Chowdhury JR, Fox LJ. Hepatocyte transplantation for the treatment of human disease. Semin Liver Dis 1999;19:39. [5] Avital I, Inderbitzin D, Aoki T, et al. Isolation, characterization, and transplantation of bone marrow-derived
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