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TRANSLATIONAL AND CLINICAL RESEARCH

Transplantation of Embryonic Stem Cell-Derived Endodermal Cells into Mice with Induced Lethal Liver Damage

^aDepartment of Surgery, Graduate School of Medicine,
^bLaboratory of Embryonic Stem Cell Research, Stem Cell Research Center, and
^cDepartment of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan;
^dGraduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan

Key Words. Embryonic stem cell • Cell transplantation • -Fetoprotein • Hepatocyte differentiation

Correspondence: Iwao Ikai, M.D., Ph.D., 54 Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Telephone: 81-75-751-3242; Fax: 81-75-751-4263; e-mail: ikai{at}kuhp.kyoto-u.ac.jp

Received on March 22, 2007; accepted for publication on September 11, 2007.

First published online in STEM CELLS EXPRESS  September 20, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
ESCs are a potential cell source for cell therapy. However, there is no evidence that cell transplantation using ESC-derived hepatocytes is therapeutically effective. The main objective of this study was to assess the therapeutic efficacy of the transplantation of ESC-derived endodermal cells into a liver injury model. The β-galactosidase-labeled mouse ESCs were differentiated into -fetoprotein (AFP)-producing endodermal cells. AFP-producing cells or ESCs were transplanted into transgenic mice that expressed diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. Selective damage was induced in the recipient hepatocytes by the administration of DT. Although the transplanted AFP-producing cells had repopulated only 3.4% of the total liver mass 7 days after cell transplantation, they replaced 32.8% of the liver by day 35. However, these engrafted cells decreased (18.3% at day 40 and 7.9% at day 50) after the cessation of DT administration, and few donor cells were observed by days 60–90. The survival rate of the AFP-producing cell-transplanted group (66.7%) was significantly higher in comparison with that of the sham-operated group (17.6%). No tumors were detected by day 50 in the AFP-producing cell-transplanted group; however, splenic teratomas did form 60 days or more after transplantation. ESC transplantation had no effect on survival rates; furthermore, there was a high frequency of tumors in the ESC-transplanted group 35 days after transplantation. In conclusion, this study demonstrates, for the first time, that ESC-derived endodermal cells improve the survival rates after transplantation into mice with induced hepatocellular injury.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Pluripotent ESCs have the ability to differentiate into all three germ layers [1, 2]. ESC-derived hepatocytes will potentially be used for drug development and the creation of artificial livers. Moreover, these cells are a possible cell source for cell therapy, as an alternative to liver transplantation. Previous studies have reported that cell transplantation using ESC-derived dopaminergic neurons [3] or insulin-producing cells [4, 5] can treat disease models in animals. Some reports have described the transplantation of mouse ESC-derived hepatic progenitor-like cells into mouse liver disease models, including fumarylacetoacetate hydrolase-deficient –/– mice [6] and major urinary/protein-urokinase type plasminogen activator mice [7], and their engraftment into livers. However, there have been few reports of any actual therapeutic improvements in animal models of liver failure using ESC-derived hepatocyte transplantation [8], and the efficacy of cell therapy using ESC-derived hepatocytes remains unclear.

This laboratory previously reported that -fetoprotein (AFP)-producing cells can be isolated using transgenic mouse ESCs that express a fusion of a hygromycin-resistant gene with an enhanced green fluorescent protein (Hyg/EGFP) under the control of an AFP promoter [9]. In addition, the AFP-producing endodermal cells maturate into hepatocyte-like cells in vitro under coculture conditions with Thy1-positive mesenchymal cells derived from fetal mouse liver.

Saito et al. reported the development of a toxin-receptor-mediated conditional cell knockout (TRECK) method [10]. Using this method, they generated transgenic mice expressing diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. Although wild-type mice, which lack DT receptors, are insensitive to DT, these transgenic mice experience hepatocellular injury as a result of the administration of DT. The extent of the liver damage is dependent on the DT dose. The administration of high doses of DT is lethal to these transgenic mice.

In this study, ESC-derived AFP-producing cells were transplanted into mice with life-threatening liver failure. Selective damage could be induced in recipient hepatocytes (but not to donor cells) in this experimental model. To determine whether ESC-derived hepatocytes are useful for cell therapy, the behavior of the transplanted cells, the outcomes of cell therapy using ESC-derived cells on liver damage, and the tumorigenicity of the transplanted cells in the recipient animal were examined.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Generation of Transgenic ESCs
This laboratory previously generated mouse ESCs that expressed Hyg/EGFP under the control of an AFP promoter (Fig. 1A) [9]. The parental ESCs were derived from C57BL/6 mice. To distinguish between recipient hepatocytes and transplanted ESC-derived cells, a lacZ gene was introduced into the transgenic ESCs. The transgenic vector pCMV SPORT-βgal (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) was transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Linear Puromycin Marker (BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com) was cotransfected. Stable transfected cells were selected in the presence of 1 µg/ml puromycin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) over a period of 2 days. Proper transgene insertion was confirmed by β-galactosidase staining.

View larger version (40K): [in this window] [in a new window]   Figure 1. Illustrations of differentiating ESCs and animal experimental procedures. (A): Graphical display of the transgenic vector that expresses a fusion of Hyg/EGFP under the control of an AFP promoter. (B): Flow diagram illustrating the differentiation of ESCs and the enrichment of AFP-producing cells. (C): The Alb-TR1–2(+/+)/C57BL6 mice received cell transplantation via splenic injection (day 0). The transplanted mice were subsequently administered DT twice a week intraperitoneally until day 35 and were then kept free of DT until day 90. Survival analyses were performed at day 35, and the long-term engraftment and tumorigenicity were assessed until day 90. Abbreviations: EGFP, enhanced green fluorescent protein; AFP, -fetoprotein; LIF, leukemia inhibitory factor; bFGF, basic fibroblast growth factor; HGF, hepatocyte growth factor; Hyg, hygromycin-resistant gene; DT, diphtheria toxin; HBSS, Hanks' balanced salt solution.

Immunohistochemistry of Cultured Cells
The detailed procedures of immunocytochemistry have been described previously [9, 11]. To perform Oct-3/4 immunostaining, mouse anti-Oct-3/4 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) was used as the primary antibody, and horseradish peroxidase-conjugated goat anti-mouse immunoglobulins (Dako, Glostrup, Denmark, http://www.dako.com) were used as the secondary antibody. The samples were colored using Envision kit/horseradish peroxidase/3,3'-diaminobenzidine tetrahydrochloride (DAB) (Dako). To stain with albumin, goat anti-mouse albumin antibody (Bethyl Inc., Montgomery, TX, http://www.bethyl.com) was used as the primary antibody, and Alexa 546-conjugated donkey anti-goat IgG (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) was used as the secondary antibody.

Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted from AFP-producing cells derived from ESCs 10 days after the initiation of differentiation, embryonic day 13.5 fetal mouse whole livers, and 15-week-old wild-type C57BL/6 adult mouse whole livers. We used an RNeasy Mini Kit (Qiagen, Chatsworth, CA, http://www.qiagen.com) and treated with RNase-free DNase (Qiagen). Total RNA (1 µg) was reverse-transcribed into cDNA with oligo(dT) 12–18 primer (Invitrogen) using an Omniscript reverse transcription (RT) kit (Qiagen). Polymerase chain reaction (PCR) used Ex Taq polymerase (Takara Bio, Otsu, Japan, http://www.takara-bio.com) according to the manufacturer's instructions. Primers were generated for the following mouse genes (oligonucleotide sequences are given in parentheses, followed by the annealing temperature and the number of cycles used for the PCR): Oct-3/4 (5'-GAGAACAATGAGAACCTTCAGGAGA, 3'-TTCTGGCGCCGGTTACAGAACCA, 58°C, 25 cycles), AFP (5'-TGCTGCAAATTACCCATGAT, 5'-AAGGTTGGGGTGAGTTCTTG, 60°C, 30 cycles), GATA4 (5'-GACCCTGTATGTAATGCCTGC, 5'-GTTCCA-AGAGTCCTGCTTGG, 60°C, 30 cycles), Foxa2 (5'-AGTGGATCA-TGGACCTCTTCC, 5'-CTTCCTTCAGTGCCAGTTGC, 60°C, 30 cycles), albumin (5'-CGAGAAGCTTGGAGAATATGG, 5'-GTCAGA-GCAGAGAAGCATGG, 60°C, 30 cycles), tyrosine aminotransferase (TAT) (5'-TCCAGGAGTTCTGTGAACAGC, 5'-AGTATATGGTGC-CTGCCTGC, 60°C, 30 cycles), tryptophan 2,3-dioxygenase (TO) (5'-GCTCAAGGTGATAGCTCGGA, 5'-GGAACTCTGCCATCTGTT-CC, 60°C, 30 cycles), glucose-6-phosphatase (G6P) (5'-TGCATTCCT-GTATGGTAGTGG, 5'-GAATGAGAGCTCTTGGCTGG, 60°C, 30 cycles), cytokeratin 19 (CK19) (5'-GTGCCACCATTGACAACTCC, 5'-AATCCACCTCCACACTGACC, 60°C, 30 cycles), Pdx1 (5'-CCA-AAACCGTCGCATGAAGTG, 5'-TCTGGGTCCCAGACCCG, 60°C, 30 cycles), insulin1 (5'-TCAGAGACCATCAGCAAGCA, 5'-TTG-TTCCACTTGTGGGTCCT, 60°C, 30 cycles), insulin2 (5'-TTTGTC-AAGCAGCACCTTTG, 5'-GCTGGGTAGTGGTGGGTCTA, 60°C, 30 cycles), and glyceraldehyde-3-phosphate dehydrogenase (5'-ATT-CAAGGGCACAGTCAAGG, 5'-ATCATAAACATGGGGGCATC, 60°C, 25 cycles).

Flow Cytometry
On day 10, the differentiated ESCs were dissociated with trypsin (Gibco)-EDTA (Dojindo Laboratories, Kumamoto, Japan, http://www.dojindo.com) solution and resuspended in 3% FBS-phosphate-buffered saline (PBS). 7-Aminoactinomysin D (BD Biosciences) was used to exclude any nonviable cells. The cells were analyzed using a FACSVantage SE (BD Biosciences).

Animal Models and Cell Transplantation Procedures
TRECK mice that were homozygous for the albumin enhancer/promoter-driven DT receptor alleles (Alb-TR1–2(+/+)/C57BL6) were generated by mating with heterozygous (Alb-TR1–2(+/–)) C57BL/6Jcl mice. Homozygosity was confirmed by backcrossing for at least three generations. Alb-TR1–2(+/+)/C57BL6 mice were viable and reproduced with normal phenotypes in the absence of DT.

AFP-producing cells were dissociated on day 10 with trypsin-EDTA. The harvested cells were suspended in Hanks' balanced salt solution (HBSS) (Gibco). Viable cells were counted using trypan blue (Gibco). The recipients were 8- to 12-week-old female Alb-TR1–2(+/+)/C57BL6 in all the animal experiments. Under general anesthesia, 2 x 10⁶ cells, suspended in 200 µl of HBSS per mouse, were injected via the inferior splenic poles using 29-gauge needles (Terumo, Tokyo, http://www.terumo.co.jp/English/index.html). The injection sites were ligated to prevent cell leakage and bleeding.

DT was purified as previously described [10]. To induce hepatocellular injury, DT was administered intraperitoneally twice a week from the day of cell transplantation for 5 weeks. Seventy-five ng/kg DT was administered during the first 3 weeks, and 150 ng/kg DT was given during the subsequent 2 weeks. The recipient mice were subsequently kept free of DT until day 90 (Fig. 1C). All experimental procedures using animals were performed in accordance with the Animal Protection Guidelines of Kyoto University.

Histology and Immunohistology
The harvested tissues were fixed in 4% paraformaldehyde (Nacalai Tesque, Inc., Kyoto, Japan, http://www.nacalai.co.jp/en) at 4°C overnight. After a thorough rinsing and soaking in 30% sucrose (Wako Pure Chemical) in PBS, the tissues were embedded in optimum cutting temperature compound (Sakura Finetechnical Co., Ltd., Tokyo, http://www.sakuraus.com), frozen in liquid nitrogen, and then sectioned using a Cryostat (Leica, Nussloch, Germany, http://www.leica.com) into 7-µm-thick sections. Prior to immunostaining, nonspecific binding was blocked with 0.4% bovine serum albumin (Sigma-Aldrich) dissolved in 0.1% saponin (Wako Pure Chemical) in PBS. The sections were incubated with mouse anti-β-galactosidase antibody (Promega, Madison, WI, http://www.promega.com) diluted at 1:200 at 4°C overnight. After washing, the stained sections were incubated with Alexa 488- or Alexa 555-congugated goat anti-mouse IgG (Molecular Probes) diluted at 1:500 for 30 minutes at room temperature. To perform immunostaining for proliferating cell nuclear antigen (PCNA), the liver sections were incubated in HistoVT One (Nacalai Tesque) for 20 minutes at 70°C, and then nonspecific binding was blocked. The sections were incubated with rabbit anti-PCNA antibody (Santa Cruz Biotechnology) diluted at 1:100 at 4°C overnight and then with Alexa 546-congugated goat anti-rabbit IgG (Molecular Probes) diluted at 1:500 for 30 minutes at room temperature. The stained sections were covered with Vectashield mounting medium with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Immunostaining for albumin and AFP were performed as previously described [12]. H&E staining was performed according to a standard protocol.

Detection of β-Galactosidase (5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside Staining)
The tissues were frozen as described above and sliced into 20-µm-thick sections. The sections were washed with PBS and then incubated at 37°C overnight in staining solution that contained 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) (Wako Pure Chemical), 5 mM potassium ferricyanide (Wako Pure Chemical), 5 mM potassium ferrocyanide (Wako Pure Chemical), 2 mM MgCl₂ (Wako Pure Chemical), 0.02% Nonidet P-40 (Sigma-Aldrich), 0.01% sodium deoxycholate (Wako Pure Chemical), and 40 mM Hepes (Sigma-Aldrich). Nuclear counterstaining was performed with Nuclear Fast Red (Vector Laboratories). The X-gal-stained area was measured using ImageJ software (developed at the NIH, Bethesda, MD). Histological samples were collected from three individual mice, and at least three randomly selected sections were measured from each mouse.

For whole tissue staining, fixed tissues were washed by soaking in PBS with 2 mM MgCl₂, 0.02% Nonidet P-40, and 0.01% sodium deoxycholate at 4°C overnight. The tissue specimens were then moved into the X-gal staining solution mentioned above and incubated at 37°C for 6 hours.

Survival Analysis
Thirty-five Alb-TR1–2(+/+)/C57BL6 mice were divided into two groups: one group (n = 18) received cell transplantations of ESC-derived AFP-producing cells, and the other group (n = 17) received splenic injections of 200 µl of HBSS as sham operations. In addition, 25 other Alb-TR1–2(+/+)/C57BL6 mice were divided into two groups: one group (n = 13) received undifferentiated ESCs, and the other group (n = 12) was sham-operated. The observation period was 35 days. Survival analyses were performed by Kaplan-Meier analyses with the log-rank tests, and p < .05 was considered to be statistically significant.

	RESULTS

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ESC-Derived AFP-Producing Cells
One clone of β-galactosidase-labeled transgenic ESCs was obtained by puromycin selection (Fig. 2A). These cells grew normally in comparison with the parental ESCs. Alkaline phosphatase staining (data not shown) and Oct-3/4 immunostaining (Fig. 2C) demonstrated these cells to be cultured in an undifferentiated state. No EGFP expression was detected by either a fluorescent microscope (Fig. 2B) or a flow cytometer (Fig. 2G) in the undifferentiated state. X-gal staining (Fig. 2D) and immunostaining for β-galactosidase (Fig. 2E) demonstrated that these cells expressed β-galactosidase. To induce differentiation into AFP-producing cells, the transgenic ESCs were transferred to serum- and feeder layer-free conditions. In this study, AFP-producing cells were selected by hygromycin instead of using a cell sorter. This procedure enabled us to obtain AFP-producing cells that expressed EGFP at a purity of 90.2% ± 2.5% (mean ± SD; n = 3) 10 days after the initiation of differentiation (Fig. 2G). Therefore, these hygromycin-resistant cells could be used as the AFP-producing cells for the subsequent cell transplantation experiments. An increase of approximately fivefold in the cell number was obtained as AFP-producing, hygromycin-resistant cells from the initial number of original undifferentiated ESCs. Less than 10% of the hygromycin-resistant cells were stained with albumin (Fig. 2F). RT-PCR revealed that the expression pattern of AFP-producing cells was similar to that of fetal liver cells in early hepatic markers (AFP, GATA4, Foxa2, and albumin) and late hepatic markers (TAT, TO, and G6P). However, the expressions of AFP and albumin were weaker in the AFP-producing cells than in the fetal liver cells, and the AFP-producing cells expressed Oct-3/4 mRNA. Moreover, AFP-producing cells expressed CK19 (which is one of cholangiocyte markers) and Pdx1 (a pancreatic marker) (Fig. 2H).

View larger version (47K): [in this window] [in a new window]   Figure 2. The β-galactosidase-labeled ESCs that express hygromycin-resistant gene/EGFP under the control of an AFP promoter. Shown are phase contrast (A) and fluorescent (B) images. These cells stained with Oct-3/4 (C) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (D) and were immunologically positive for β-galactosidase (E). (F): ESCs at day 10 after the initiation of differentiation. Red fluorescence indicates albumin, and blue fluorescence represents DAPI. Original magnifications, x400 (A–E), x200 (F). (G): Flow cytometric analyses of the transgenic ESCs. Ten days after the initiation of differentiation, the proportion of EGFP-positive cells reached 90.2% ± 2.5% of the total viable cells. (H): Reverse transcription-polymerase chain reaction analysis of the RNA samples extracted from 10 days after differentiation of APC-ES, embryonic day 13.5 FL, and AL. Abbreviations: 7-AAD, 7-aminoactinomysin D; AFP, -fetoprotein; AL, adult mouse whole livers; APC-ES, -fetoprotein-producing cells derived from ESCs; CK19, cytokeratin 19; EGFP, enhanced green fluorescent protein; FL, fetal mouse whole livers; G6P, glucose-6-phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; -RT, without reverse transcription; TAT, tyrosine aminotransferase; TO, tryptophan 2,3-dioxygenase.

Seven days after the AFP-producing cell transplantation, X-gal staining showed that the transplanted cells were engrafted and clustered around the intrahepatic portal veins (Fig. 3F, 3G), occasionally causing hepatic infarctions (Fig. 3H). The proportion of the X-gal-stained area was 3.4% ± 0.5%. Samples were immunostained for albumin, AFP, and β-galactosidase to characterize the transplanted cells in the recipient livers. Large numbers of the transplanted cells expressed β-galactosidase and were immunologically positive for albumin (Fig. 4A) and negative for AFP (Fig. 4B), indicating a commitment to the hepatocyte lineage. However, an immunohistological analysis of serial sections revealed that a small number of donor cells were negative for albumin and positive for AFP (Fig. 4C, 4D). These cells were morphologically small, and they were centrally located in the clusters of the transplanted cells.

View larger version (103K): [in this window] [in a new window]   Figure 3. 5-Bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) staining of the liver specimens from -fetoprotein-producing cell-transplanted mice. Shown is X-gal staining of the intact liver lobes (A–E). The numbers displayed on the lower right indicate the days after cell transplantation. Open arrowheads indicate the infarct regions on the surface of the recipient liver. (F–H): The liver sections 7 days after cell transplantation. (I–K): Liver sections at day 35. Original magnifications, x7 (A, B, D, E), x32 (C), x40 (F, I–K), x100 (G, H). Abbreviations: CV, central vein; N, necrotic region; P, portal vein.

View larger version (129K): [in this window] [in a new window]   Figure 4. Immunohistological analyses of AFP-producing cell-transplanted mice livers. The specimens were obtained on day 7 (A–D) and on day 35 (E–L) after cell transplantation. (A, C): Green fluorescence represents albumin, and red fluorescence indicates β-gal. (B, D): Green fluorescence represents β-gal, and red fluorescence indicates AFP. Blue fluorescence indicates DAPI. (C) and (D) were obtained from serial sections. Open arrowheads indicate the donor cell-derived albumin–/AFP+ cells that are located at the center of the cluster of donor cell-derived albumin+/AFP– cells that are surrounded by closed arrowheads. (E–H): Green fluorescence represents albumin, and red fluorescence indicates β-gal. Closed arrowheads indicate the cluster of donor cell-derived albumin-positive cells, and closed arrows show binuclear cells. (I): H&E staining. (J): Green fluorescence represents transfected EGFP expression, and red represents AFP. (K): β-gal (green) and AFP (red). (L): Section stained with β-gal (green), PCNA (red), and DAPI (blue). Closed arrowheads indicate the pink PCNA-positive nuclei. Original magnifications, x100 (A, E, J), x200 (B, F, I, K, L), x400 (C, D, G–H). Abbreviations: AFP, -fetoprotein; DAPI, 4,6-diamidino-2-phenylindole; β-gal, β-galactosidase; PCNA, proliferating cell nuclear antigen.

Amelioration of Liver Damage by AFP-Producing Cell Transplantation
To investigate whether cell transplantation would ameliorate liver damage, AFP-producing cells or ESCs were transplanted into Alb-TR1–2(+/+)/C57BL6 mice, and their survival rates were compared with those of sham-operated mice, respectively. The survival rate of the AFP-producing cell-transplanted group (12 of 18; 66.7%) was significantly higher (p = .006) than that of the sham-operated group (3 of 17; 17.6%) (Fig. 5). However, there was no significant difference between the survival rates of the ESC-transplanted group (4 of 13; 30.8%) and the sham-operated group (3 of 12; 25.0%) (p = .86).

View larger version (23K): [in this window] [in a new window]   Figure 5. Survival curves after transplantation of AFP-producing cells. Twelve mice out of a total of 18 (66.7%) were alive until 35 days after AFP-producing cell transplantation, whereas three mice out of 17 (17.6%) were alive in the sham-operated group. The solid line represents the AFP-producing cell-transplanted group, and the dashed line represents the sham-operated group. *, p < .01. Abbreviation: AFP, -fetoprotein.

To examine the degree of long-term incorporation and the tumorigenicity of the AFP-producing cells, the recipient mice were sacrificed and their organs were harvested 40 days (n = 2), 50 days (n = 3), 60 days (n = 3), and 90 days (n = 5) after AFP-producing cell transplantation. On day 40, H&E staining showed that liver damage still existed in the recipient livers (Fig. 7A). The X-gal stained donor cells occupied 18.3% of the livers (Fig. 7E). On day 50, although the engrafted donor cells still had repopulated 7.9% of the recipient livers, they were scattered in the livers and did not form any clusters (Fig. 7B, 7F). Histological analyses revealed that the structure of the hepatic lobules was almost normal 60 days and 90 days after the AFP-producing cell transplantation (Fig. 7C, 7D), thus indicating that the recipient livers had recovered from hepatic failure. X-gal staining revealed few donor cells in the recipient livers (Fig. 7G, 7H). To assess tumorigenicity, the abdominal and thoracic cavities were searched for tumors. Splenic tumors were observed in one of three mice at day 60 and two of the five mice at day 90 (Fig. 7I), whereas no tumors were found in any other organs, including the liver, peritoneum, and lungs. Histological analyses revealed that these tumors exhibited differentiation into all three germ layers, including squamous epithelium (Fig. 7J), muscle (Fig. 7K), and intestinal epithelium (Fig. 7L), thus indicating that these tumors are teratomas.

View larger version (94K): [in this window] [in a new window]   Figure 6. Organs harvested from the ESC-transplanted mice 35 days after transplantation. (A): The liver lobe stained with 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal). (B): A small, pale T protruded from the hepatic edge. This T was stained with X-gal staining (C). (D): A T was detected at the mesentery. The inset shows that this T also stained with X-gal. (E, F): Splenic Ts. *, Splenic inferior pole. Original magnification, x7. Abbreviation: T, tumor.

View larger version (136K): [in this window] [in a new window]   Figure 7. Long-term engraftment and tumorigenicity of the AFP-producing cell-transplanted mice. H&E staining of recipient livers obtained 40 days (A), 50 days (B), 60 days (C), and 90 days (D) after transplantation. 5-Bromo-4-chloro-3-indolyl-β-D-galactoside staining of liver sections of transplanted mice liver at day 40 (E), day 50 (F), day 60 (G), and day 90 (H). (I): Splenic Ts at day 90. *, splenic inferior pole. H&E staining analyses show that these Ts have all three germ layer components, including squamous epithelium (J), smooth muscle (K), and intestinal epithelium (L). Original magnifications, x100 (A–H), x7 (I), x400 (J–L). Abbreviations: CV, central vein; P, portal vein; T, tumor.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

There were two modifications to the experimental procedures in this study from those used in previous reports [9, 10]. The first modification was the use of homozygous TRECK mice (Alb-TR1–2(+/+)/C57BL6). This change reduced the individual differences in liver damage induced by DT. Second, the AFP-producing cells were enriched using hygromycin selection in this study, whereas in previous studies, AFP-producing cells were isolated using flow cytometry [9]. Although EGFP-positive cells could be isolated with more than 95% purity, it was difficult to obtain sufficient viable cell counts for cell transplantation using a flow cytometer. Approximately 90% of the differentiated cells after hygromycin selection expressed EGFP, indicating that they were AFP-producing cells. The characteristics of the remaining 10% of cells were not well identified. Although we could not detect any Oct-3/4-positive cells in hygromycin-resistant cells (data not shown), the Oct-3/4 mRNA expression was observed in them. It therefore appeared that the population of the hygromycin-resistant cells included a small number of undifferentiated ESCs and that undifferentiated ESCs were not completely depleted by hygromycin selection. RT-PCR showed that AFP-producing cells expressed early hepatic markers, a cholangiocyte marker, and a pancreatic marker. This suggested that AFP-producing cells were cell mixtures containing not only cells that underwent differentiation into hepatocytes but also cells that underwent differentiation into cholangiocytes and into pancreatic cells.

It is commonly assumed that an adult mouse liver contains approximately 5 x 10⁷ hepatocytes [23]. In this experiment, 2 x 10⁶ AFP-producing cells were transplanted per mouse, corresponding to 4% of the total number of hepatocytes. The transplanted AFP-producing cells were engrafted in the recipient livers and formed hepatic infarctions with intrahepatic portal vein emboli [24, 25]. Most of the transplanted cells were albumin-positive and AFP-negative by day 7 after cell transplantation, whereas only a small proportion of the AFP-producing cells had been positive for albumin at the beginning of cell transplantation. However, a small number of engrafted cells, located at the center of the donor cell clusters, remained albumin-negative and AFP-positive and showed characteristics of either immature hepatocytes or hepatic progenitor cells. These albumin–/AFP+ cells were thought to be in the process of hepatic differentiation to the albumin+/AFP– cells. At this time, the donor cells had replaced roughly 3.4% of the recipient livers. By contrast, a vast majority of the donor cells were albumin-positive and AFP-negative at day 35 and resembled mature hepatocytes in morphology. Approximately 20% of the engrafted donor cells expressed PCNA, thus indicating that these cells had a proliferative capacity. Indeed, the donor cells expanded and repopulated more than 30% of the liver tissue by day 35.

The two possible mechanisms for hepatic regeneration after liver injury are hepatocyte-driven regeneration and stem/progenitor cell-dependent regeneration [23, 26]. Mature hepatocytes have an enormous proliferative potential [27–29]. Existing hepatocytes divide and proliferate quickly after a liver injury such as a partial hepatectomy, resulting in the compensatory growth of the residual liver tissue. Oval cells are thought to be hepatic progenitor cells [30–32], and they usually proliferate and differentiate into hepatocytes only when the replication of mature hepatocytes is delayed or entirely blocked [23, 26]. As a potential mechanism for liver repopulation by ESC-derived AFP-producing cells, it was suggested that the transplanted AFP-producing cells rapidly committed to hepatocyte differentiation after cell transplantation, and these ESC-derived hepatocytes then replicated and expanded into the necrotic spaces in the recipient hepatic tissue.

Undifferentiated ESC transplantation did not, however, improve the survival rate. Regarding the experimental data from the AFP-producing cell transplantation and the ESC transplantation, it appears that more hepatic-differentiated ESCs might be more suitable for cell transplantation. Previous publications described the coculture system to differentiate ESCs to mature hepatocyte-like cells in vitro [9]. Although these cells were intended for grafting, it was technically difficult to do so. Our coculture system required fetal liver mesenchymal cells as the primary cultures, and quite a lot of fetal mice were needed to obtain a sufficient population for cell transplantation. If this speculation about the suitable cells for transplantation is correct, it will therefore be necessary to establish an easier and more effective culture method to induce ESCs to differentiate and maturate into hepatocytes in vitro for subsequent therapeutic use.

The recipient livers were harvested periodically until day 90 after cell transplantation to observe the long-term engraftments. The recipient livers were kept damaged for approximately 1 week after the cessation of DT administration. As the recipient livers recovered from hepatic damage in histological analyses, the engrafted donor cells decreased, and few donor cells were found in the recipient livers 60–90 days after the AFP-producing cell transplantation. No inflammatory cell infiltration was observed surrounding the donor cell clusters that indicated the host rejection against donor cells. These results suggested that recipient liver regeneration after the cessation of DT administration was driven by the recipient-derived hepatocytes rather than the transplanted cells. Therefore, the ESC-derived hepatocytes might have had a limited potential for liver repopulation, and they consequently disappeared from recipient livers after the cessation of liver damage. In addition, splenic teratomas were detected in one of three mice at day 60 and in two of five mice at day 90. Taking together with the RT-PCR results, the population of transplanted cells was thus suggested to contain a small number of undifferentiated ESCs [33]. Therefore, the removal of undifferentiated ESCs from transplanted cells would be needed to exclude the risk of teratoma formation. The splenic injection sites were the primary locations for neoplasms derived from both undifferentiated ESCs and AFP-producing cells. This observation suggests that a splenectomy would be helpful to reduce the long-term risk of tumor formation. These findings indicate that to improve the efficacy and safety of cell transplantation using ESCs, it might be necessary to obtain more differentiated endodermal cells as donor cells, isolate lineage-specific cells and remove undifferentiated ESCs more strictly, and thereafter refine the transplantation procedures. However, additional experiments will be needed to address these issues.

Some studies have reported that transplanted bone marrow cells were found in the livers of both human and mouse recipients [34, 35]. This laboratory has also previously transplanted bone marrow cells into recipient mice and subsequently identified donor-derived endothelial cells, Kupffer cells [36], and hepatic stellate cells [37] in the liver. In this study, almost all of the AFP-producing cells differentiated into hepatocytes. Apparently, this was because the donor cells were endodermal cells, and DT administration caused cell damage only to the recipient hepatocytes in this animal model. Therefore, the donor cells would commit to the endodermal lineage and differentiate into hepatocytes, instead of other endodermal cell types, such as cholangiocytes. It has been demonstrated that hepatocytes generated from transplanted hematopoietic stem cells are the products of cellular fusion [38, 39]. In this study, it was impossible to eliminate the possibility that the transplanted ESC-derived cells fused to the recipient hepatocytes. However, fused cells would possess DT receptors on the cell surface and would consequently become sensitive to DT. Fused cells would therefore not survive even if cell fusion had occurred in the recipient livers.

	CONCLUSION

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In conclusion, this study successfully demonstrated that ESC-derived endodermal cells could be engrafted in murine hepatic injury models and subsequently differentiate into hepatocyte-like cells. Furthermore, they could improve the survival rate of the recipient mice in the short term. However, the problem of long-term tumorigenicity remains unsolved. This study illustrates the potential therapeutic effect of cell therapies using ESCs for the treatment of liver diseases, even if the risk of teratoma formation needs to be resolved.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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This work was supported in part by grants from the Scientific Research Fund of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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